Analysis of Potential Transcriptional Regulators of the Universal Stress Protein A (UspA) Gene of Escherchia coli

The universal stress protein A (UspA) of E. coli is induced by a wide variety of stressful conditions and appears to be important in regulating the cell\u27s overall stress response. However, the exact mechanism of uspA induction remains unknown. The purpose of this project was to identify potential regulators of the uspA gene. Regulators tested included the LexA repressor of the SOS regulon, and the sigma factors RpoH and RpoS. Sigma factors initiate transcription by recognizing target promoters and allowing RNA polymerase to bind. Most sigma factors are involved in allowing the cell to respond to a particular stressful condition, such as membrane stress or nutrient starvation. Using a bioluminescent reporter system in which the uspA promoter is fused to the lux operon from Vibrio fischeri, the level of uspA transcription under different stress conditions was quantified. When stressed with 5 mJ/m² UV radiation, lexA-independent mutants showed, on average, a six-fold increase in uspA induction compared to the wildtype strain. The lexA-independent strain encodes a mutant form of the LexA repressor that cannot be cleaved by RecA and thus remains bound to the promoters of genes in the SOS regulon. The increased transcription levels of uspA in the lexA-independent strain suggest that uspA is not a member of the SOS regulon. Similarly, when ethanol stressed, uspA transcription in the rpoH mutant strain showed a four-fold increase over the rpoH wildtype strain, suggesting that RpoH is not involved in initiating uspA transcription. Conversely, uspA transcription in rpoS mutants was reduced by almost 70% during stationary phase compared to the rpoS wildtype strain. This suggests that either the RpoS protein itself or the product of an RpoS-upregulated gene is important in initiating uspA transcription

Cellular Localization and Antigenic Characterization of Crimean-Congo Hemorrhagic Fever Virus Glycoproteins

Crimean-Congo hemorrhagic fever virus (CCHFV), a member of the genus Nairovirus of the family Bunyaviridae, causes severe disease with high rates of mortality in humans. The CCHFV M RNA segment encodes the virus glycoproteins G(N) and G(C). To understand the processing and intracellular localization of the CCHFV glycoproteins as well as their neutralization and protection determinants, we produced and characterized monoclonal antibodies (MAbs) specific for both G(N) and G(C). Using these MAbs, we found that G(N) predominantly colocalized with a Golgi marker when expressed alone or with G(C), while G(C) was transported to the Golgi apparatus only in the presence of G(N). Both proteins remained endo-β-N-acetylglucosaminidase H sensitive, indicating that the CCHFV glycoproteins are most likely targeted to the cis Golgi apparatus. Golgi targeting information partly resides within the G(N) ectodomain, because a soluble version of G(N) lacking its transmembrane and cytoplasmic domains also localized to the Golgi apparatus. Coexpression of soluble versions of G(N) and G(C) also resulted in localization of soluble G(C) to the Golgi apparatus, indicating that the ectodomains of these proteins are sufficient for the interactions needed for Golgi targeting. Finally, the mucin-like and P35 domains, located at the N terminus of the G(N) precursor protein and removed posttranslationally by endoproteolysis, were required for Golgi targeting of G(N) when it was expressed alone but were dispensable when G(C) was coexpressed. In neutralization assays on SW-13 cells, MAbs to G(C), but not to G(N), prevented CCHFV infection. However, only a subset of G(C) MAbs protected mice in passive-immunization experiments, while some nonneutralizing G(N) MAbs efficiently protected animals from a lethal CCHFV challenge. Thus, neutralization of CCHFV likely depends not only on the properties of the antibody, but on host cell factors as well. In addition, nonneutralizing antibody-dependent mechanisms, such as antibody-dependent cell-mediated cytotoxicity, may be involved in the in vivo protection seen with the MAbs to G(C)

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline