Improved genetically-encoded, FlincG-type fluorescent biosensors for neural cGMP imaging.

Genetically-encoded biosensors are powerful tools for understanding cellular signal transduction mechanisms. In aiming to investigate cGMP signaling in neurones using the EGFP-based fluorescent biosensor, FlincG (fluorescent indicator for cGMP), we encountered weak or non-existent fluorescence after attempted transfection with plasmid DNA, even in HEK293T cells. Adenoviral infection of HEK293T cells with FlincG, however, had previously proved successful. Both constructs were found to harbor a mutation in the EGFP domain and had a tail of 17 amino acids at the C-terminus that differed from the published sequence. These discrepancies were systematically examined, together with mutations found beneficial for the related GCaMP family of Ca(2+) biosensors, in a HEK293T cell line stably expressing both nitric oxide (NO)-activated guanylyl cyclase and phosphodiesterase-5. Restoring the mutated amino acid improved basal fluorescence whereas additional restoration of the correct C-terminal tail resulted in poor cGMP sensing as assessed by superfusion of either 8-bromo-cGMP or NO. Ultimately, two improved FlincGs were identified: one (FlincG2) had the divergent tail and gave moderate basal fluorescence and cGMP response amplitude and the other (FlincG3) had the correct tail, a GCaMP-like mutation in the EGFP region and an N-terminal tag, and was superior in both respects. All variants tested were strongly influenced by pH over the physiological range, in common with other EGFP-based biosensors. Purified FlincG3 protein exhibited a lower cGMP affinity (0.89 μM) than reported for the original FlincG (0.17 μM) but retained rapid kinetics and a 230-fold selectivity over cAMP. Successful expression of FlincG2 or FlincG3 in differentiated N1E-115 neuroblastoma cells and in primary cultures of hippocampal and dorsal root ganglion cells commends them for real-time imaging of cGMP dynamics in neural (and other) cells, and in their subcellular specializations

Evidence for D1 Dopamine Receptor Activation by a Paracrine Signal of Dopamine in Tick Salivary Glands

Ticks that feed on vertebrate hosts use their salivary secretion, which contains various bioactive components, to manipulate the host's responses. The mechanisms controlling the tick salivary gland in this dynamic process are not well understood. We identified the tick D1 receptor activated by dopamine, a potent inducer of the salivary secretion of ticks. Temporal and spatial expression patterns examined by immunohistochemistry and reverse transcription polymerase chain reaction suggest that the dopamine produced in the basal cells of salivary gland acini is secreted into the lumen and activates the D1 receptors on the luminal surface of the cells lining the acini. Therefore, we propose a paracrine function of dopamine that is mediated by the D1 receptor in the salivary gland at an early phase of feeding. The molecular and pharmacological characterization of the D1 receptor in this study provides the foundation for understanding the functions of dopamine in the blood-feeding of ticks

Seroconversion and asymptomatic infections during oseltamivir prophylaxis against Influenza A H1N1 2009

<p>Abstract</p> <p>Background</p> <p>Anti-viral prophylaxis is used to prevent the transmission of influenza. We studied serological confirmation of 2009 Influenza A (H1N1) infections during oseltamivir prophylaxis and after cessation of prophylaxis.</p> <p>Methods</p> <p>Between 22 Jun and 16 Jul 09, we performed a cohort study in 3 outbreaks in the Singapore military where post-exposure oseltamivir ring chemoprophylaxis (75 mg daily for 10 days) was administered. The entire cohort was screened by RT-PCR (with HA gene primers) using nasopharyngeal swabs three times a week. Three blood samples were taken for haemagglutination inhibition testing - at the start of outbreak, 2 weeks after completion of 10 day oseltamivir prophylaxis, and 3 weeks after the pandemic's peak in Singapore. Questionnaires were also administered to collect clinical symptoms.</p> <p>Results</p> <p>237 personnel were included for analysis. The overall infection rate of 2009 Influenza A (H1N1) during the three outbreaks was 11.4% (27/237). This included 11 index cases and 16 personnel (7.1%) who developed four-fold or higher rise in antibody titres during oseltamivir prophylaxis. Of these 16 personnel, 8 (3.5%) were symptomatic while the remaining 8 personnel (3.5%) were asymptomatic and tested negative on PCR. Post-cessation of prophylaxis, an additional 23 (12.1%) seroconverted. There was no significant difference in mean fold-rise in GMT between those who seroconverted during and post-prophylaxis (11.3 vs 11.7, p = 0.888). No allergic, neuropsychiatric or other severe side-effects were noted.</p> <p>Conclusions</p> <p>Post-exposure oseltamivir prophylaxis reduced the rate of infection during outbreaks, and did not substantially increase subsequent infection rates upon cessation. Asymptomatic infections occur during prophylaxis, which may confer protection against future infection. Post-exposure prophylaxis is effective as a measure in mitigating pandemic influenza outbreaks.</p

Effect of Duration and Intermittency of Rifampin on Tuberculosis Treatment Outcomes: A Systematic Review and Meta-Analysis

In a systematic review of randomized controlled trials on tuberculosis treatment, Dick Menzies and colleagues find shorter courses of rifampin to be associated with poorer treatment outcomes

Colorectal cancer screening among African American church members: A qualitative and quantitative study of patient-provider communication

BACKGROUND: A healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a colorectal cancer (CRC) screening test. We sought to determine the relationship between the general quality of self-rated patient-provider communication and the completion of CRC screening. METHODS: A formative study using qualitative data from focus groups and quantitative data from a cross-sectional survey of church members about the quality of their communication with their healthcare provider, their CRC risk knowledge, and whether they had completed CRC screening tests. Focus group participants were a convenience sample of African American church members. Participants for the survey were recruited by telephone from membership lists of 12 African American churches located in rural counties of North Carolina to participate in the WATCH (Wellness for African Americans Through Churches) Project. RESULTS: Focus Groups. Six focus groups (n = 45) were conducted prior to the baseline survey. Discussions focused on CRC knowledge, and perceived barriers/motivators to CRC screening. A theme that emerged during each groups' discussion about CRC screening was the quality of the participants' communication with their health care provider. Survey. Among the 397 participants over age 50, 31% reported CRC screening within the recommended guidelines. Participants who self-rated their communication as good were more likely to have been screened (36%) within the recommended guidelines than were participants with poor communication (17%) (OR = 2.8, 95% CI 1.2, 6.4; p = 0.013). Participants who had adequate CRC knowledge completed CRC screening at a higher rate than those with inadequate knowledge (p = 0.011). The percentage of participants with CRC screening in the recommended guidelines, stratified by communication and knowledge group were: 42% for good communication/adequate knowledge; 27% for good communication/inadequate knowledge; 29% for poor communication/adequate knowledge; and 5% for poor communication/inadequate knowledge. CONCLUSIONS: Participants who rated their patient-provider communication as good were more likely to have completed CRC screening tests than those reporting poor communication. Among participants reporting good communication, knowledge about colorectal cancer was also associated with test completion. Interventions to improve patient-provider communication may be important to increase low rates of CRC screening test completion among African Americans

Effect of Peripheral 5-HT on Glucose and Lipid Metabolism in Wether Sheep

In mice, peripheral 5-HT induces an increase in the plasma concentrations of glucose, insulin and bile acids, and a decrease in plasma triglyceride, NEFA and cholesterol concentrations. However, given the unique characteristics of the metabolism of ruminants relative to monogastric animals, the physiological role of peripheral 5-HT on glucose and lipid metabolism in sheep remains to be established. Therefore, in this study, we investigated the effect of 5-HT on the circulating concentrations of metabolites and insulin using five 5-HT receptor (5HTR) antagonists in sheep. After fasting for 24 h, sheep were intravenously injected with 5-HT, following which-, plasma glucose, insulin, triglyceride and NEFA concentrations were significantly elevated. In contrast, 5-HT did not affect the plasma cholesterol concentration, and it induced a decrease in bile acid concentrations. Increases in plasma glucose and insulin concentrations induced by 5-HT were attenuated by pre-treatment with Methysergide, a 5HTR 1, 2 and 7 antagonist. Additionally, decreased plasma bile acid concentrations induced by 5-HT were blocked by pre-treatment with Ketanserin, a 5HTR 2A antagonist. However, none of the 5HTR antagonists inhibited the increase in plasma triglyceride and NEFA levels induced by 5-HT. On the other hand, mRNA expressions of 5HTR1D and 1E were observed in the liver, pancreas and skeletal muscle. These results suggest that there are a number of differences in the physiological functions of peripheral 5-HT with respect to lipid metabolism between mice and sheep, though its effect on glucose metabolism appears to be similar between these species

"Courting the multinational": Subnational institutional capacity and foreign market insidership

peer-reviewedSignificant contemporary challenges face an internationalizing firm, including the non-ergodic nature of investment, and the liability of outsidership. Recent revisions to the Uppsala internationalization process model reflect these challenges, whereby “insidership” is represented as realized, successful foreign market entry. Drawing upon socio-spatial concepts from international business and economic geography, this paper demonstrates the endogeneity of subnational institutions in shaping foreign market insidership within an advanced economy. Employing a multi-method research design with almost 60 subnational actors, the role and interaction of subnational institutions within the internationalization process are explored. Our findings illustrate how customized coalitions of subnational institutions effectively initiate, negotiate and accelerate insidership of inward investment within the foreign market both prior to and during formal entry. Key aspects of this dynamic include communicating tangible and intangible locational resources, initiating functional and relevant business relationships, and facilitating access to codified and tacit knowledge. This paper embellishes the Uppsala internationalization process model by demonstrating the capacity of subnational institutions to participate actively with foreign market insidership, and in so doing advances understanding of how the risk and uncertainty associated with foreign market entry are currently navigated.ACCEPTEDpeer-reviewe

In the Absence of Sonic Hedgehog, p53 Induces Apoptosis and Inhibits Retinal Cell Proliferation, Cell-Cycle Exit and Differentiation in Zebrafish

Background: Sonic hedgehog (Shh) signaling regulates cell proliferation during vertebrate development via induction of cell-cycle regulator gene expression or activation of other signalling pathways, prevents cell death by an as yet unclear mechanism and is required for differentiation of retinal cell types. Thus, an unsolved question is how the same signalling molecule can regulate such distinct cell processes as proliferation, cell survival and differentiation. Methodology/Principal Findings: Analysis of the zebrafish shh 2/2 mutant revealed that in this context p53 mediates elevated apoptosis during nervous system and retina development and interferes with retinal proliferation and differentiation. While in shh 2/2 mutants there is activation of p53 target genes and p53-mediated apoptosis, an increase in Hedgehog (Hh) signalling by over-expression of dominant-negative Protein Kinase A strongly decreased p53 target gene expression and apoptosis levels in shh 2/2 mutants. Using a novel p53 reporter transgene, I confirm that p53 is active in tissues that require Shh for cell survival. Proliferation assays revealed that loss of p53 can rescue normal cell-cycle exit and the mitotic indices in the shh 2/2 mutant retina at 24, 36 and 48 hpf. Moreover, generation of amacrine cells and photoreceptors was strongly enhanced in the double p53 2/2 shh 2/2 mutant retina suggesting the effect of p53 on retinal differentiation. Conclusions: Loss of Shh signalling leads to the p53-dependent apoptosis in the developing nervous system and retina