In a propensity-matched analysis of off- versus on-clamp robot-assisted partial nephrectomy, we reported no difference in functional outcomes. Off-clamp partial nephrectomy was associated with a significantly higher need for blood transfusion and conversion to radical nephrectomy.

S84 UP-27 Uptake of a second transurethral resection of the bladder tumor in T1 bladder cancer in Ontario: An interrupted time series analysis involving 15 years of observation Marian S. Wettstein1,2,3,4, Nancy N. Baxter2,3, Rinku Sutradhar2,3, Muhammad M. Mamdani2,3, Song Pham3, Syed R. Qadri1, Kathy Li1, Ning Liu3, van der Kwast Theodorus5, Thomas Hermanns4, Girish S. Kulkarni1,2,3 1Division of Urology, Department of Surgery, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON, Canada; 2Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, ON, Canada; 3ICES, Toronto, ON, Canada; 4Department of Urology, University Hospital of Zurich, University of Zurich, Zurich, Switzerland; 5Department of Pathology, Princess Margaret Cancer Centre, University Health Network, University of Toronto, Toronto, ON, Canada Introduction: A second transurethral resection of the bladder tumor (reTUR) within 2–6 weeks after initial resection is thought to have diagnostic, therapeutic, and prognostic benefits in T1 bladder cancer. However, little is known about the real-world uptake of this guideline-endorsed intervention. We aimed to: 1) measure reTUR rates over time; 2) investigate if a guideline revision (April 2008) explicitly endorsing reTUR within 2–6 weeks in all T1 bladder cancer patients led to an increase in reTUR rates; and 3) investigate the uptake among different groups of surgeons. Methods: Province-wide bladder cancer pathology reports (January 2001 to December 2015; Ontario, Canada) were manually abstracted and linked with health administrative data to: 1) identify primary cases of T1 bladder cancer and 2) ascertain whether these patients received reTUR. The resulting patients were then aggregated into quarterly time series and investigated by descriptive analysis, ARIMA modeling, and Poisson regression analysis. Results: A cohort of 7373 patients was aggregated into a time series. We observed a linear increase in reTUR rates from 8.4% in 2001 to 28.3% in 2015. An actual effect of the guideline revision in April 2008 on reTUR rates could not be detected (p=0.41). However, we observed a rather heterogeneous uptake behavior among different groups of surgeons. Specifically, female surgeons, more junior surgeons, high-volume surgeons, Canadian graduates, and surgeons without an academic affiliation were all independently more likely to perform reTUR (all p<0.05 in adjusted analysis). Conclusions: ReTUR rates in primary T1 bladder cancer increased between 2001 and 2015 in Ontario regardless of the guideline revision in April 2008. Our study demonstrates that the uptake of this guideline-endorsed intervention varies among different groups of surgeons and therefore warrants further research to identify barriers to change that can be addressed by tailored interventions.

Abstract Background Indigenous patients with inflammatory bowel disease (IBD) have expressed concerns about barriers to access IBD care. The limited evidence of IBD among Indigenous people highlights the need for studies evaluating access to IBD care in this population. Aims We aimed to compare health care utilization between First Nations (FNs) and individuals from the general population (GP) diagnosed with IBD in Saskatchewan (SK). Methods A population-based retrospective cohort study was conducted using administrative health databases of SK from 1998 to 2017 fiscal years. As a patient-oriented research initiative, outcomes of interest were chosen in collaboration with Indigenous patients and family advocates. A validated algorithm requiring multiple health care contacts was applied to identify incident IBD cases. The self-declared FN status variable was used to divide IBD cases between FNs and the general population (GP). To balance the groups, 1:5 age and sex matching was applied. Cox-proportional models were used to estimate hazard ratios (HRs) and 95% confidence intervals (95%CI). Stratified analysis was completed for those diagnosed before and after 2008 (pre- and post-biologic eras). Results A matched cohort with 696 IBD incident cases was created (FN=116, GP=580). Comparing health care utilization of FNs and individuals from the GP with IBD, there were no statistically significant differences in outpatient gastroenterology visits (FNs=81.0%, GP=83.6%), colonoscopies (FNs=91.4%, GP=86.9%), and surgeries for IBD (FNs=31.0%, GP=33.5%). We observed differences in prescription claims for any medication for IBD (FNs=79.3%, GP=89.3%) and 5-aminosalicylic acid (5-ASA) claims (FNs=75.9%, GP=81.4%). The HRs adjusted by rural/urban residence and diagnostic type showed differences in prescription claims for any IBD medication (HR=0.52, 95%CI 0.41–0.65) and 5-ASA (HR=0.57, 95%CI 0.45–0.72). In the pre-biologic era, FNs had a lower risk of having a prescription claim for any IBD medication (HR=0.32, 95%CI 0.23–0.45) and 5-ASA (HR=0.33, 95%CI 0.24–0.47), respectively. These differences were not significant in the post-biologic era. Conclusions Our study identified an inverse association between FN status and having prescription medication claims for IBD in SK. We considered multiple confounding variables when evaluating this association but could not control by disease severity. Thus, this association might reflect a barrier to access IBD medications or that FNs with IBD might present a milder disease. Further studies should continue evaluating access to IBD care, medication use, and disease severity among FNs living with IBD. Funding Agencies Saskatchewan Centre for Patient-Oriented Research (SCPOR), Saskatchewan Health Research Foundation (SHRF), and College of Medicine, University of Saskatchewan.

Gold nanoparticles are promising drug delivery agents with the potential to deliver chemotherapeutic agents to tumour sites. The highly cytotoxic maytansinoid tubulin inhibitor DM1 has been attached to gold nanoparticles and shows tumour growth inhibition in mouse models of hepatocellular carcinoma. Attempting to improve the stability of the gold-cytotoxin bond led to the design and synthesis of novel maytansinoids with improved potency in cell viability assays and improved in vivo tolerability compared to the DM1 analogues. These novel maytansines may also have applications in other methods of drug delivery, for example as the cytotoxic component of antibody drug conjugates.

OBJECTIVE To evaluate the effect of obesity and overweight on surgical, functional and survival outcomes in patients with large kidney masses after minimally invasive surgery (MIS). MATERIAL AND METHODS Within a multi-center, multi-national dataset, patients diagnosed with a ≥cT2 renal mass and treated with minimally invasive (laparoscopic or robotic) kidney surgery (radical or partial nephrectomy) during the period 2003-2017 were abstracted. They were stratified according to the body mass index (BMI) classes as normal-weight (18.5-24.9 kg/m2), overweight (25.0-29.9 kg/m2) and obese (≥30.0 kg/m2). Mixed models and Cox proportional hazard regression tested differences in complication rates, eGFR change over time, overall mortality (OM) and disease recurrence (DR) rates. RESULTS Of 812 patients, 30.6% were normal-weight, 42.7% were overweight and 26.8% obese. Overweight (OR 0.82, 95% CI: 0.51-1.51, p=0.406) and obese patients (0.81, 95% CI: 0.44-1.47, p=0.490) experienced similar complication rates than normal weight. Moreover, no statistically significant differences in eGFR were found for overweight (p=0.129) or obese (p=0.166) patients compared to normal-weight. However, higher OM rates were recorded in overweight (HR 3.59, 95%CI: 1.03-12.51, p=0.044) as well as in obese patients (HR 7.83, 95%CI: 2.20-27.83, p=0.002). Similarly, higher DR rates were recorded in obese (HR 2.76, 95%CI: 1.40-5.44, p=0.002). CONCLUSIONS Obese and overweight patients do not experience higher complication rates or worse eGFR after minimally invasive kidney surgery, which therefore can be deemed feasible and safe also in these subset of patients. Nevertheless, obese and overweight patients seem to carry a higher risk of OM, and therefore they should undergo a strict follow-up after surgery.

Purpose : To detect efflux pump activity (EPA) and screening a suspected efflux pump inhibitor (EPI) [1- (3-(trifluoromethyl)benzyl]-piperazine (TFMBP)], which could help in reducing multi-drug resistance (MDR). Methods : Eighteen isolates, viz, 14 S. aureus, 2 S. lentus, 1 S. xylosus and 1 Micrococcus species from various hospital infections of admitted patients were screened for antibiotics susceptibility to 11 classes of antibiotics including oxacillin and β-lactamase production. Efflux pump activity (EPA) was determined by minimum inhibitory concentration (MIC) technique in the presence and absence of TFMBP, the isolates were also screened for MDR genes. Results: All the isolates were resistant to ampicillin (10 μg) and penicillin (10 μg), but sensitive to bacitracin (10 μg). Majority of the isolates were MDR 12/18 (66.7 %), 10 (55.6 %) were inducible β- lactamase producers and 3 (16.7 %) were intrinsic β-lactamase producers. Seven (38.9 %) were resistant to oxacillin and also produced carbapenemase enzyme. Eight (66.7 %) of the 12 MDR isolates gave evidence of EPA with TFMBP. However, no MDR genes were detected. Conclusion : Staphylococcus and Micrococcus species exhibit EPA in antibiotic resistance while a suitable EPI such as TFMBP when combined with specific antibiotics could help combat this menace. Keywords : [1-(3-(Trifluoromethyl)benzyl]-piperazine, Efflux pump activity, Oxacillin resistant S. aureus , Multidrug resistant, Carbapenemase

"Determination of Fiberglass Lengths: Sample Prepara tion and Automatic Image Analysis’, L. C. Sawyer, reprinted from Polymer Engineering and Science, vol. 19, No. 5, Apr. 1979, pp. 377-382. "Toughening Behavior in SiC-Whisker-Reinforced Alumina”, Paul F. Becher and George C. Wei, Commu nications of the American Ceramic Society, Dec. 1984, pp. 267-269. "Whisker-Reinforced Ceramic Matrix Composites”, J. 11 Patent Number:

INTRODUCTION Mycobacterium tuberculosis (Mtb) causes a large global burden of disease, with a high mortality rate in healthy and immuno-compromised patients. A number of molecular targets have been identified for treatment of this disease, including the Mtb proteasome. The Mtb proteasome enhances Mtb survival during nitrosative and oxidative stress in the latent, non-replicative phase. Therefore, Mtb proteasome inhibition could help to combat Mtb infections that do not respond to current therapies. OBJECTIVE To develop and validate a novel biochemical assay to assess Mtb proteasome activity in the presence of organic and aqueous plant test extracts. METHOD Fluorescence (photoluminescence) and luminescence (chemiluminescence) assays were investigated as potential methods to determine the robustness and repeatability for use in screening natural product extracts for Mtb proteasome inhibitors. RESULTS The fluorescence assay, used widely for Mtb proteasome activity assays, was subject to interference due to the natural fluorescence of compounds in many of the extracts; there is little interference with the luminescence approach. As proof of principle, we used the luminescence assay to screen a small set of plant test extracts. CONCLUSIONS Luminescence is the more suitable assay for assay of plant natural product extracts. The sensitivities of the luminescence and fluorescence assays are comparable. A Z'-factor of 0.58 for the luminescence assay makes it suitable for medium-to-high throughput screening efforts.

Biliatresone is an electrophilic isoflavone isolated from Dysphania species plants that has been causatively linked to naturally occurring outbreaks of a biliary atresia (BA)‐like disease in livestock. Biliatresone has selective toxicity for extrahepatic cholangiocytes (EHCs) in zebrafish larvae. To better understand its mechanism of toxicity, we performed transcriptional profiling of liver cells isolated from zebrafish larvae at the earliest stage of biliatresone‐mediated biliary injury, with subsequent comparison of biliary and hepatocyte gene expression profiles. Transcripts encoded by genes involved in redox stress response, particularly those involved in glutathione (GSH) metabolism, were among the most prominently up‐regulated in both cholangiocytes and hepatocytes of biliatresone‐treated larvae. Consistent with these findings, hepatic GSH was depleted at the onset of biliary injury, and in situ mapping of the hepatic GSH redox potential using a redox‐sensitive green fluorescent protein biosensor showed that it was significantly more oxidized in EHCs both before and after treatment with biliatresone. Pharmacological and genetic manipulation of GSH redox homeostasis confirmed the importance of GSH in modulating biliatresone‐induced injury given that GSH depletion sensitized both EHCs and the otherwise resistant intrahepatic cholangiocytes to the toxin, whereas replenishing GSH level by N‐acetylcysteine administration or activation of nuclear factor erythroid 2‐like 2 (Nrf2), a transcriptional regulator of GSH synthesis, inhibited EHC injury. Conclusion: These findings strongly support redox stress as a critical contributing factor in biliatresone‐induced cholangiocyte injury, and suggest that variations in intrinsic stress responses underlie the susceptibility profile. Insufficient antioxidant capacity of EHCs may be critical to early pathogenesis of human BA. (Hepatology 2016;64:894‐907)

In our previous work, we identified a natural toxin, biliatresone, from Dysphania glomulifera and D. littoralis, endemic plants associated with outbreaks of biliary atresia in Australian neonatal livestock. Biliatresone is a very rare isoflavonoid with an α-methylene ketone between two phenyls, 1,2-diaryl-2-propenone, along with methylenedioxy, dimethoxyl, and hydroxyl functional groups, that causes extrahepatic biliary toxicity in zebrafish. The toxic core of biliatresone is a methylene in the α-position relative to the ketone of 1,2-diaryl-2-propenone that serves as an electrophilic Michael acceptor. The α-methylene of biliatresone spontaneously conjugated with water and methanol (MeOH), respectively, via Michael addition in a reverse phase high-performance liquid chromatography (RP-HPLC) analysis. We here report the reactivity of biliatresone toward glutathione (GSH), several amino acids, and other thiol- or imidazole-containing biomolecules. LC-MS and HPLC analysis of the conjugation reaction showed the reactivity of biliatresone to be in the order histidine > N-acetyl-d-cysteine (D-NAC) = N-acetyl-l-cysteine (L-NAC) > histamine > glutathione ≥ cysteine ≫ glycine > glutamate > phenylalanine, while serine and adenine had no reactivity due to intramolecular hydrogen bonding in the protic solvents. The reactivity of ethyl vinyl ketone (EVK, 1-penten-3-one), an example of a highly reactive α,ß-unsaturated ketone, toward GSH gave a 6.7-fold lower reaction rate constant than that of biliatresone. The reaction rate constant of synthetic 1,2-diaryl-2-propen-1-one (DP), a core structure of the toxic molecule, was 10-fold and 1.5-fold weaker in potency compared to the reaction rate constants of biliatresone and EVK, respectively. These results demostrated that the methylenedioxy, dimethoxyl, and hydroxyl functional groups of biliatresone contribute to the stronger reactivity of the Michael acceptor α-methylene ketone toward nucleophiles compared to that of DP and EVK.

Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three‐dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α‐smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. Conclusion: Biliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880‐893)

Biliary atresia outbreaks were reported four times in newborn Australian livestock between 1964 and 2013. Epidemiologic data implied that these outbreaks were caused by environmental exposure to an unusual forage plant. We isolated and identified the active toxic compound. Two similar isoflavonoids from Iresine herbstii have been reported. We have extracted I. herbstii, and are isolating the compounds by column chromatography, flash chromatography and HPLC. We confirmed the presence of the compounds, 2′,2,5-trimethoxy-6,7-methylenedioxyisoflavanone (1) and 2′,5-dimethoxy-6,7-methylenedioxyisoflavone (2). Future work will focus on ring opening reactions and testing the toxicities of all compounds in a zebrafish bioassay of biliary function. This is part of our ongoing efforts to determine SAR characteristics of the toxic compound.

Podophyllotoxin (PPT) is a plant natural product that serves as a precursor for the synthesis of many well-known chemotherapeutic drugs. The limited availability and high demand for the source plants of PPT have led to the exploration of alternative sources for this compound. In this study, we utilized the endophytic fungus Phialocephala podophylli (strain PPE7) that we isolated from the rhizomes of Podophyllum peltatum and is known to produce detectable amounts of PPT in broth culture. To date, the complete PPT biosynthetic pathway has yet to be determined in any species. Since fungi are well known for clustering genes that belong to secondary metabolite pathways, use of a fungal system for investigation of the PPT biosynthesis genes may ultimately lead to elucidation of the entire pathway. In this study, we investigated the secoisolariciresinol dehydrogenase (SD) gene that facilitates the dehydrogenation of secoisolariciresinol to form matairesinol, a mid-pathway intermediate product in PPT biosynthesis. We utilized PCR amplification to acquire the complete SD gene sequence in PPE7 and opted to synthesize the P. peltatum SD sequence for expression. Through western blotting, we confirmed the expression of the recombinant SD (PpSD) and verified protein functionality with a bioconversion assay followed by HPLC and LC–MS analyses. Here, we report the identification of the SD gene in PPE7; this is the first report of the SD gene in an endophytic fungus. Additionally, we established the groundwork for the future expression of the complete PPT biosynthetic pathway in the heterologous host Pichia pastoris.

Biliary atresia (BA) is a rapidly progressive and destructive fibrotic disorder of unknown etiology affecting the extrahepatic biliary tree of neonates. Epidemiological studies suggest that an environmental factor, such as a virus or toxin, is the cause of the disease, although none have been definitively established. Several naturally occurring outbreaks of BA in Australian livestock have been associated with the ingestion of unusual plants by pregnant animals during drought conditions. We used a biliary secretion assay in zebrafish to isolate a previously undescribed isoflavonoid, biliatresone, from Dysphania species implicated in a recent BA outbreak. This compound caused selective destruction of the extrahepatic, but not intrahepatic, biliary system of larval zebrafish. A mutation that enhanced biliatresone toxicity mapped to a region of the zebrafish genome that has conserved synteny with an established human BA susceptibility locus. The toxin also caused loss of cilia in neonatal mouse extrahepatic cholangiocytes in culture and disrupted cell polarity and monolayer integrity in cholangiocyte spheroids. Together, these findings provide direct evidence that BA could be initiated by perinatal exposure to an environmental toxin.