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The Cry1Ab toxin produced by Bacillus thuringiensis binds to a conserved structural motif in the 12th ectodomain module (EC12) of BT-R1, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm Manduca sexta. Toxin binding transmits a signal into the cells and turns on a multi-step signal transduction pathway, culminating in cell death. Using chromatographically purified Cry1Ab and EC12 proteins, we demonstrated the direct formation of a stable complex between these two proteins in solution and visualized it on a native polyacrylamide gel. Moreover, we generated a fluorescent EC12 probe by converting the 36th residue to cysteine to enable maleimide-mediated conjugation of Alexa-488 fluorescent dye to EC12 by site-directed mutagenesis. In addition, we changed the 44th residue of EC12 to tryptophan, which greatly improved accuracy of protein quantification and traceability. Using the fluorescently labeled EC12 probe for direct and competitive binding assays, we were able to determine binding specificity in solution. These accomplishments will facilitate identification and characterization of the interface sequences for both the Cry1Ab toxin and BT-R1.

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OBJECTIVETo evaluate the risk of deep vein thrombosis (DVT) in men treated with testosterone replacement therapy (TRT) or Clomiphene Citrate (CC) and assess other etiologies for DVT as contributing factors. METHODSRetrospective chart review of 1180 consecutive hypogonadal men who were treated with either TRT or CC. Sixty-four percent had mixed, 16% had primary, and 20% had secondary hypogonadism. RESULTSOf the 1180 men with hypogonadism, 694 were treated with TRT, while 486 were treated with CC. Overall, 10 of 1180 (0.8%) men were diagnosed with a DVT during the treatment, 9 of whom were on TRT and 1 on CC. Of the 10 men diagnosed with DVT while on treatment, 7 (70%) had potential identifiable etiologies for DVT other than treatment for hypogonadism. None of the men were found to be polycythemic at the time of DVT diagnosis. There was a higher incidence of DVT in men treated with TRT than CC, however; the overall percentages of DVT in both treatment groups were relatively low. There was no difference in the percentages of men found to have other identifiable etiologies for DVT besides being on treatment between the TRT and CC groups. There was not a difference in testosterone levels between the TRT and CC groups. CONCLUSIONThe overall rates of DVT for TRT and CC treated patients are relatively low, and the majority of patients with DVT had other identifiable etiologies for DVT. Polycythemia was not found to be a risk factor in the patients diagnosed with DVTs. UROLOGY 124: 127−130, 2019.