The Vibrio pathogenicity island (VPI) in epidemic Vibrio cholerae is an essential virulence gene cluster. Like many pathogenicity islands, the VPI has at its termini a phage-like integrase gene (int), a transposase-like gene (vpiT), and phage-like attachment (att) sites, and is inserted at a tRNA-like locus (ssrA). We report that the VPI precisely excises from the chromosome and that its left and right ends join to form an extrachromosomal circular excision product (pVPI). Two-stage nested PCR analysis and DNA sequencing confirmed the int-attvpiT junction and that the core attP of pVPI is identical to the chromosomal VPI attR site. Excision was independent of toxR and toxT. Excision was independent of recA, suggesting that it is mediated by site-specific recombination. Interestingly, while excision was detected in int and vpiT mutants, excision was abolished in a double (int vpiT) mutant and was restored by plasmids containing genes for either recombinase. Excision results in deletion of A361 in the ssrA locus, which flanks the right junction of the VPI. Since A361 encodes U70 in the critical G ⅐ U base pair in the acceptor stem of the ssrA RNA that is the determinant for aminoacylation with alanine, this deletion might have deleterious effects on ssrA function. Also, vpiT may have undergone interchromosomal translocation or may represent an independent integration event, as it was found downstream of hutA in some isolates. Our results provide new insight into the molecular biology of the VPI, and we propose that the process of excision and circularization is important in the emergence, pathogenesis, and persistence of epidemic V. cholerae.Cholera, attributed to the bacterium Vibrio cholerae, is a diarrheal disease of humans that results in significant morbidity and mortality (25). Available records show that there have been seven worldwide pandemics of cholera (40). The current seventh pandemic began on the island of Sulawesi in Indonesia and then rapidly spread throughout Asia (1, 24). Because of its high death-to-case ratio, transmissibility, and persistence in the environment and its ability to occur in explosive epidemic form, V. cholerae continues to be a public health concern.Only toxigenic strains of V. cholerae that possess two essential virulence gene clusters, i.e., the CTX element, which is a prophage encoding cholera toxin (39, 49), and the Vibrio pathogenicity island (VPI) (26, 30), can cause epidemic cholera. The chromosomal VPI is 41.2 kb in size in both sixthpandemic (classical biotype) and seventh-pandemic (El Tor biotype) strains and encodes 29 potential proteins (26, 27). The VPI encodes proteins with essential roles in virulence, such as toxin-coregulated pili (TCP) (17, 44), and proteins that regulate virulence, such as ToxT, TcpP, and TcpH (4,6,7,14). In addition, the VPI contains several open reading frames with no known or demonstrated function.The VPI has many features that are typical of pathogenicity islands (PAIs) (2, 12, 13). It has a low percent GϩC content (35%) compared to the re...

Endogenous nitric oxide (NO) may contribute to the maintenance of normal pulmonary vasomotor tone, and inhaled NO is used to treat patients with pulmonary hypertension. Because pulmonary vascular tone is regulated by intracellular free Ca2+ concentration and membrane potential, which are controlled by the K+ channel activity in pulmonary artery (PA) smooth muscle cells, we sought to determine whether K+ channels are involved in NO-induced relaxation and, if so, which types of K+ channels are responsible. Authentic NO (approximately 0.3 microM) and sodium nitroprusside (SNP, 10 pM) both produced significant relaxation in isolated PA rings precontracted by increasing extracellular K+ concentration. Further elevation of the K+ concentration from 20 to 60 mM resulted in a significant increase in contraction but caused a marked decline in SNP- and NO-mediated PA relaxation. The dependence of SNP- and NO-induced relaxation on transmembrane K+ gradient suggests that K+ efflux through K+ channels is involved in these effects. Furthermore, 4-aminopyridine (4-AP, 5-10 mM), which blocks voltage-gated K+ channels (K(V)), and charybdotoxin (200 nM), which blocks Ca2+-activated K+ channels (K(Ca)), both significantly inhibited NO- and SNP-induced PA relaxation. The ATP-sensitive K+ channel blocker glibenclamide, however, had no effect on the relaxation response. The blocking of guanylate cyclase diminished, but did not abolish, the NO-induced relaxation, whereas 4-AP further decreased the NO-induced relaxant response in the presence of the guanylate cyclase inhibitor LY-83583. These data suggest that activation of both K(V) channels and K(Ca) channels by guanosine 3',5'-cyclic monophosphate-dependent and -independent pathways is a mechanism, at least in part, by which NO induces PA relaxation.

To develop an individually optimized contrastenhanced (CE) 4D-CT for radiotherapy simulation in pancreatic adenocarcinoma (PDA). Materials/Methods: Ten PDA patients were enrolled and underwent three CT scans: a 4D-CT immediately following a CE 3D-CT, and an individually optimized CE 4D-CT using a test injection to estimate the peak contrast enhancement time and to optimize the delay time. Three physicians contoured the tumor and pancreatic tissues. We compared image quality scores, tumor volume, motion, image noise, tumor-to-pancreas contrast, and contrast-to-noise ratio (CNR) in the three CTs. We also evaluated inter-observer variations in contouring the tumor using simultaneous truth and performance level estimation (STAPLE). Results: The average image quality scores for CE 3D-CT and CE 4D-CT were comparable (4.0 and 3.8, PZ0.47), and both were significantly better than that for 4D-CT (2.6, P<0.001). The tumor-to-pancreas contrast in CE 3D-CT and CE 4D-CT were comparable (15.5 and 16.7 HU, PZ0.71), and the later was significantly higher than that in 4D-CT (9.2 HU, PZ0.03). Image noise in CE 3D-CT (12.5 HU) was significantly lower than that in CE 4D-CT (22.1 HU, P<0.001) and 4D-CT (19.4 HU, PZ0.005). The CNR in CE 3D-CT and CE 4D-CT were comparable (1.4 and 0.8, PZ0.23), and the former was significantly better than that in 4D-CT (0.6, PZ0.04). The average tumor volume was smaller in CE 3D-CT (29.8 cm 3) and CE 4D-CT (22.8 cm 3) than in 4D-CT (42.0 cm 3), though the differences were not statistically significant. The tumor motion was comparable in 4D-CT and CE 4D-CT (7.2 and 6.2 mm, PZ0.23). The inter-observer variations were comparable in CE 3D-CT and CE 4D-CT (Jaccard index 66.0% and 61.9%), and the former was significantly smaller than that of 4D-CT (55.6%, PZ0.047). Conclusion: The CE 4D-CT demonstrated largely comparable characteristics to the CE 3D-CT. It has high potential for simultaneously delineating the tumor and quantifying the tumor motion with a single scan.

Purpose/Objective(s): Adjuvant radiation therapy for pancreatic cancer is controversial. Certain pathologic variables may help further define the subset of patients more likely to experience locoregional failure. Whereas margin positivity and lymph node (LN) involvement have been shown to be prognostic, degree of margin clearance, number and extent of lymphadenectomy, and LN ratio remain areas of active investigation. We investigated the prognostic implications of LN and margin involvement in patients with resected PDA. Materials/Methods: We identified 106 patients with resected stage 1-3 PDA from 2007-13. Resection margins were categorized as positive (tumor at ink), 1 mm or > 1 mm. LN evaluation included total number examined (NE), number of positive nodes (NP), ratio of NP to NE (NR), and presence of periportal lymphadenopathy. Impact of these variables was assessed on disease-free survival (DFS) and overall survival (OS) using multivariate cox proportional hazards modeling. Actuarial estimates for DFS and OS were calculated using Kaplan-Meier methods. Results: Median follow up was 11 months (range 1-77). Resection margins were positive, 1 mm or > 1 mm in 18, 50, and 38 patients, respectively. A total of 48.1% of patients received adjuvant chemotherapy alone, while 42.5% received chemoradiation therapy and 9.4% received no adjuvant therapy. The median number of LNs resected was 19 (range 4-37). Periportal LNs were resected in 25 of the 106 patients and were positive in 25%. Analysis of variance demonstrated that margin status was highly correlated with NP (pZ0.012) and post-resection CA 19-9 (pZ0.017) Median DFS for the entire cohort was 11 months and median OS was 16 months. On univariate analysis, NP (HR 1.34 PZ0.0417), post-resection CA-19-9 above 90 (HR 1.26, pZ0.039) and positive periportal LNs (HR 5.00 pZ0.039) resulted in significantly lower median DFS and OS rates. Multivariate analysis revealed proximity of margin was a predictor of inferior DFS (pZ0.0412, HR Z 0.51), but did not result in a difference in OS. ROC analysis revealed that ! 5 NP to be the most accurate predictor of inferior DFS (pZ0.005). NE was not associated with DFS or OS, yet absolute NP of 5 or more was associated with a significantly worse DFS. The use of adjuvant CRT was associated with improved OS (pZ0.04; HRZ0.29) relative to patients who received chemotherapy alone or no adjuvant therapy. Conclusion: In patients receiving adjuvant CT alone, there was a clinically significant benefit in clearing the surgical margin beyond tumor at ink. Higher NR, having 5 or more NP and presence of periportal adenopathy clearly led to worse clinical outcomes. A more comprehensive evaluation of margins and LN parameters may identify an increasing number of patients at risk for locoregional failure who may benefit from adjuvant CRT. Volume 93 Number 3S Supplement 2015Poster Viewing Session E159