Transcriptional initiation of a small RNA, not R-loop stability, dictates the frequency of pilin antigenic variation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the sole causative agent of gonorrhea, constitutively undergoes diversification of the Type IV pilus. Gene conversion occurs between one of the several donor silent copies located in distinct loci and the recipient pilE gene, encoding the major pilin subunit of the pilus. A guanine quadruplex (G4) DNA structure and a cis-acting sRNA (G4-sRNA) are located upstream of the pilE gene and both are required for pilin antigenic variation (Av). We show that the reduced sRNA transcription lowers pilin Av frequencies. Extended transcriptional elongation is not required for Av, since limiting the transcript to 32 nt allows for normal Av frequencies. Using chromatin immunoprecipitation (ChIP) assays, we show that cellular G4s are less abundant when sRNA transcription is lower. In addition, using ChIP, we demonstrate that the G4-sRNA forms a stable RNA:DNA hybrid (R-loop) with its template strand. However, modulating R-loop levels by controlling RNase HI expression does not alter G4 abundance quantified through ChIP. Since pilin Av frequencies were not altered when modulating R-loop levels by controlling RNase HI expression, we conclude that transcription of the sRNA is necessary, but stable R-loops are not required to promote pilin Av.

Optical control of the antigen translocation by synthetic photo-conditional viral inhibitors.

The immune system makes use of major histocompatibility complex class I (MHC I) molecules to present peptides to other immune cells, which can evoke an immune response. Within this process of antigen presentation, the MHC I peptide loading complex, consisting of a transporter associated with antigen processing TAP, MHC I, and chaperones, is key to the initiation of immune response by shuttling peptides from the cytosol into the ER lumen. However, it is still enigmatic how the flux of antigens is precisely coordinated in time and space, limiting our understanding of antigen presentation pathways. Here, we report on the development of a synthetic viral TAP inhibitor that can be cleaved by light. This photo-conditional inhibitor shows temporal blockade of TAP-mediated antigen translocation, which is unleashed upon illumination. The recovery of TAP activity was monitored at single-cell resolution both in human immune cell lines and primary cells. The development of a photo-conditional TAP inhibitor thus expands the repertoire of chemical intervention tools for immunological processes.

A comparison of sampling approaches for monitoring schoolwide inclusion program fidelity.

Including students with disabilities requires schoolwide interventions that are implemented with fidelity (adherence). Collection of fidelity data may become problematic when multiple evidence-based treatments exist in one setting. To address concerns around efficiency of data collection, this study hypothesized that the three sampling approaches (proportional, consensus, stratified random) provided similar levels of agreement with an expert rater, thus warranting the differentiation in their applications based on the needs of the evaluators. Three high schools were randomly assigned to one of three sampling approaches (i.e. proportional, consensus, stratified random) to complete a fidelity measure for the implementation of a schoolwide inclusion approach. Based on the Median Test, each sampling approach did not differ significantly from the scores of an expert rater. The results indicate that schools may have some choice in the methods they use to sample their staff around the measurement of fidelity of implementation.

Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies.

Accurate labeling of endogenous proteins for advanced light microscopy in living cells remains challenging. Nanobodies have been widely used for antigen labeling, visualization of subcellular protein localization and interactions. To facilitate an expanded application, we present a scalable and high-throughput strategy to simultaneously target multiple endogenous proteins in living cells with micro- to nanometer resolution. For intracellular protein labeling, we advanced nanobodies by site-specific and stoichiometric attachment of bright organic fluorophores. Their fast and fine-tuned intracellular transfer by microfluidic cell squeezing enabled high-throughput delivery with less than 10% dead cells. This strategy allowed for the dual-color imaging of distinct endogenous cellular structures, and culminated in super-resolution imaging of native protein networks in genetically non-modified living cells. The simultaneous delivery of multiple engineered nanobodies does not only offer exciting prospects for multiplexed imaging of endogenous protein, but also holds potential for visualizing native cellular structures with unprecedented accuracy.

Modeling blast waves, gas and particles dispersion in urban and hilly ground areas.

The numerical simulation of shock and blast waves as well as particles dispersion in highly heterogeneous media such as cities, urban places, industrial plants and part of countries is addressed. Examples of phenomena under study are chemical gas products dispersion from damaged vessels, gas dispersion in urban places under explosion conditions, shock wave propagation in urban environment. A three-dimensional simulation multiphase flow code (HI2LO) is developed in this aim. To simplify the consideration of complex geometries, a heterogeneous discrete formulation is developed. When dealing with large scale domains, such as countries, the topography is considered with the help of elevation data. Meteorological conditions are also considered, in particular regarding complex temperature and wind profiles. Heat and mass transfers on sub-scale objects, such as buildings, trees and other obstacles are considered as well. Particles motion is addressed through a new turbulence model involving a single parameter to describe accurately plumes. Validations against experiments in basic situations are presented as well as examples of industrial and environmental computations.

Adenoma detection rate is necessary but insufficient for distinguishing high versus low endoscopist performance.

BACKGROUND: Endoscopist quality is benchmarked by the adenoma detection rate (ADR)-the proportion of cases with 1 or more adenomas removed. However, the ADR rewards the same credit for 1 versus more than 1 adenoma. OBJECTIVE: We evaluated whether 2 endoscopist groups could have a similar ADR but detect significantly different total adenomas. DESIGN: We retrospectively measured the ADR and multiple measures of total adenoma yield, including a metric called ADR-Plus, the mean number of incremental adenomas after the first. We plotted ADR versus ADR-Plus to create 4 adenoma detection patterns: (1) optimal (↑ADR/↑ADR-Plus); (2) one and done (↑ADR/↓ADR-Plus); (3) all or none (↓ADR/↑ADR-Plus); (4) none and done (↓ADR/↓ADR-Plus). SETTING: Tertiary-care teaching hospital and 3 nonteaching facilities servicing the same patient pool. PATIENTS: A total of 3318 VA patients who underwent screening between 2005 and 2009. MAIN OUTCOME MEASUREMENTS: ADR, mean total adenomas detected, advanced adenomas detected, ADR-Plus. RESULTS: The ADR was 28.8% and 25.7% in the teaching (n = 1218) and nonteaching groups (n = 2100), respectively (P = .052). Although ADRs were relatively similar, the teaching site achieved 23.5%, 28.7%, and 29.5% higher mean total adenomas, advanced adenomas, and ADR-Plus versus nonteaching sites (P < .001). By coupling ADR with ADR-Plus, we identified more teaching endoscopists as optimal (57.1% vs 8.3%; P = .02), and more nonteaching endoscopists in the none and done category (42% vs 0%; P = .047). LIMITATIONS: External generalizability, nonrandomized study. CONCLUSION: We found minimal ADR differences between the 2 endoscopist groups, but substantial differences in total adenomas; the ADR missed this difference. Coupling the ADR with other total adenoma metrics (eg, ADR-Plus) provides a more comprehensive assessment of adenoma clearance; implementing both would better distinguish high- from low-performing endoscopists.

Ghrelin in neuroendocrine tumors.

Ghrelin is a 28 amino acid peptide, primarily produced by the oxyntic mucosa X/A like neuroendocrine cells in the stomach. It is also found in the small intestine, hypothalamus, pituitary gland, pancreas, heart, adipose tissue, and immune system. In gastrointestinal neuroendocrine tumors (NETs) ghrelin release has been well documented. Ghrelin is a brain-gut circuit peptide with an important role in the physiological regulation of appetite, response to hunger and starvation, metabolic and endocrine functions as energy expenditure, gastric motility and acid secretion, insulin secretion and glucose homeostasis, as well as in the potential connection to the central nervous system. Recently, there has been a significant interest in the biological effects of ghrelin in NETs. In this article, we present a comprehensive review of ghrelin's expression and a brief summary of ghrelin's physiological role in NETs patients with carcinoids, type A chronic atrophic gastritis (CAG), with or without MEN-1, and with and without liver metastases. We hope, with the research reviewed here, to offer compelling evidence of the potential significance of ghrelin in NETs, as well as to provide a useful guide to the future work in this area.

Management of hepatitis B in special patient populations.

Hepatitis B virus is a common cause of acute liver failure. It can be especially problematic in patients coinfected with hepatitis C, hepatitis D or human immunodeficiency virus. In addition, immunosuppression-associated hepatitis B reactivation is being increasingly recognized following chemotherapy, biologic therapy, and organ transplantation. This article highlights treatment options in these special populations.

Comparative efficacy of rabeprazole and pantoprazole in the control of nocturnal Acid output and intragastric acidity.

BACKGROUND/AIMS: Nocturnal reflux is a largely undiagnosed and unmanaged condition predisposing to multiple esophageal complications. We evaluated the effects of rabeprazole and pantoprazole on nocturnal intragastric pH and gastric acid output during Day 1 of therapy following the consumption of standard meals. METHODS: The study had a double-blinded, randomized, two-way crossover design, and involved 15 patients with a history of mild reflux. Following an overnight fast, patients were given either rabeprazole (20 mg) or pantoprazole (40 mg) prior to the first of three standard Western meals. They then underwent overnight continuous intragastric pH monitoring and gastric acid output measurement. The drug effect was analyzed using a two-treatment, two-period crossover mixed model. RESULTS: The percentage of time during which the mean intragastric pH was greater than 4.0 and gastric acid output was less than 2.0 was higher for oral rabeprazole (p<0.05). The inhibition of acid output was greater for rabeprazole at almost all time points. Furthermore, the mean time-matched pH values differed significantly over the first 8.3 hours (p<0.05). CONCLUSIONS: On day 1, oral rabeprazole inhibited acid output to a greater extent and for a longer period than pantoprazole, and the intragastric pH was significantly higher for rabeprazole than for pantoprazole over the first 8.3 hours.

Comparative Efficacy of Rabeprazole and Pantoprazole in the Control of Nocturnal Acid Output and Intragastric Acidity

BACKGROUND/AIMS: Nocturnal reflux is a largely undiagnosed and unmanaged condition predisposing to multiple esophageal complications. We evaluated the effects of rabeprazole and pantoprazole on nocturnal intragastric pH and gastric acid output during Day 1 of therapy following the consumption of standard meals. METHODS: The study had a double-blinded, randomized, two-way crossover design, and involved 15 patients with a history of mild reflux. Following an overnight fast, patients were given either rabeprazole (20 mg) or pantoprazole (40 mg) prior to the first of three standard Western meals. They then underwent overnight continuous intragastric pH monitoring and gastric acid output measurement. The drug effect was analyzed using a two-treatment, two-period crossover mixed model. RESULTS: The percentage of time during which the mean intragastric pH was greater than 4.0 and gastric acid output was less than 2.0 was higher for oral rabeprazole (p<0.05). The inhibition of acid output was greater for rabeprazole at almost all time points. Furthermore, the mean time-matched pH values differed significantly over the first 8.3 hours (p<0.05). CONCLUSIONS: On day 1, oral rabeprazole inhibited acid output to a greater extent and for a longer period than pantoprazole, and the intragastric pH was significantly higher for rabeprazole than for pantoprazole over the first 8.3 hours.

Elevated serum ghrelin exerts an orexigenic effect that may maintain body mass index in patients with metastatic neuroendocrine tumors.

Ghrelin is a potent orexigenic peptide principally produced in the stomach by a distinct population of neuroendocrine cells in the oxyntic mucosa of the fundus. Exogenous ghrelin given as an intravenous infusion has been shown to increase caloric intake in patients with cancer cachexia. In this study, we hypothesized that elevated endogenous ghrelin, produced by increased neuroendocrine cell tumor burden, also exerts an orexigenic effect helping to maintain body mass index. To evaluate the effect of elevated endogenous ghrelin, 35 patients with neuroendocrine tumors were enrolled, assigning them to one of two groups depending on the presence of hepatic metastases. Following an overnight fast, serum was collected and sent for ghrelin measurement by an outside laboratory. The two groups were well matched for all other relevant clinical variables including subtype of tumor, primary location of tumor and tumor treatment history. Nearly all patients with hepatic metastases had elevated levels of ghrelin compared to the standard reference range given for matched controls. The presence of hepatic metastases was associated with significantly elevated ghrelin levels (p<0.05) and a greater mean body mass index. In addition, we report a positive correlation between serum ghrelin and total tumor surface area and between serum ghrelin and body mass index, suggesting that elevated endogenous ghrelin may be sufficient to overcome any partial ghrelin resistance typically seen in cancer cachexia. These results support the possibility that ghrelin is co-released from neuroendocrine tumors and exerts an orexigenic effect in these patients, helping to maintain their body mass index despite widely disseminated disease.

Validation of a new endoscopic technique to assess acid output in Zollinger-Ellison syndrome.

BACKGROUND & AIMS: To safely manage the hypersecretory state in patients with Zollinger-Ellison syndrome, both upper endoscopy and gastric analysis are required to titrate optimal medical therapy. Conventional gastric analysis requires more than 1 hour to perform and results in significant patient dissatisfaction. In this study, we have validated endoscopic gastric analysis as a novel technique that can effectively replace conventional gastric analysis. METHODS: In a prospective, cross-over study, 12 patients with Zollinger-Ellison syndrome underwent gastric analysis, first by the conventional method and then by an endoscopic technique performed on the same day. Acid concentration was determined by titration, volume output was recorded, and acid output was calculated using standard methodologies and formulas. Agreement was assessed following the Bland-Altman method. To assess repeatability, both techniques were repeated on the same day in a subset of patients. RESULTS: Excellent agreement was reported between acid output (95% limits of agreement, -1.27 to 1.61 mEq/h) and acid concentration (95% limits of agreement, -0.01 to 0.01 mEq/mL), although poor agreement was observed between volume output measured. Endoscopic gastric analysis showed greater reproducibility regarding acid and volume output measured. CONCLUSIONS: We introduce a new, rapid, reproducible, and accurate endoscopic technique to measure acid output in patients with Zollinger-Ellison syndrome who require both annual endoscopy and gastric analysis. The data presented here suggest that endoscopic gastric analysis would be equally effective in determining acid output in other hypersecretory states. Additional analysis of cost effectiveness is needed to evaluate its use as a screening tool in select populations.

Cyto-reduction of neuroendocrine tumours using Sandostatin LAR in combination with Infergen: results of a case series.

Historically, limited trials evaluating biotherapy in treating metastatic neuroendocrine tumours have yielded mixed results. In this study, the efficacy of a novel combination therapy featuring longacting Sandostatin LAR plus alpha-interferon was evaluated. In a prospective case series, 12 patients with unresectable metastatic neuroendocrine tumours refractory to treatment initiated therapy with Infergen and Sandostatin LAR. Radiological response was followed serially at 3-month intervals. A biochemical response was considered significant if marker levels decreased by > or = 50% compared with baseline. Inhibition of tumour growth lasting for greater than 3 months (mean response 22.6+/-17.7 months) was seen in eight patients. Complete tumour regression was observed in one patient, lasting for 40 months; three patients exhibited partial tumour regression (mean response 29.3+/-24.0 months), and four patients maintained a stable tumour response (mean response 13.3+/-9.2 months). Four patients showed no response to therapy (mean response 5.0+/-6.0 months). All enrolled patients are alive currently. The biochemical response seen in seven patients did not correlate with the radiological response. These results suggest that the novel combination of longacting Sandostatin LAR with an alpha-interferon may be at least as effective as either combination therapy with short-acting octreotide or monotherapy with Sandostatin LAR.

Single pilus motor forces exceed 100 pN.

Force production by type IV pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 +/- 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the pilus by a cooperative conformational change.