Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Statistical and methodological challenges for disaster preparedness and medical needs assessment in Rio Grande Valley of Texas

In recent years, disaster preparedness through assessment of medical and special needs persons (MSNP) has taken a center place in public eye in effect of frequent natural disasters such as hurricanes, storm surge or tsunami due to climate change and increased human activity on our planet. Statistical methods complex survey design and analysis have equally gained significance as a consequence. However, there exist many challenges still, to infer such assessments over the target population for policy level advocacy and implementation. ^ Objective. This study discusses the use of some of the statistical methods for disaster preparedness and medical needs assessment to facilitate local and state governments for its policy level decision making and logistic support to avoid any loss of life and property in future calamities. ^ Methods. In order to obtain precise and unbiased estimates for Medical Special Needs Persons (MSNP) and disaster preparedness for evacuation in Rio Grande Valley (RGV) of Texas, a stratified and cluster-randomized multi-stage sampling design was implemented. US School of Public Health, Brownsville surveyed 3088 households in three counties namely Cameron, Hidalgo, and Willacy. Multiple statistical methods were implemented and estimates were obtained taking into count probability of selection and clustering effects. Statistical methods for data analysis discussed were Multivariate Linear Regression (MLR), Survey Linear Regression (Svy-Reg), Generalized Estimation Equation (GEE) and Multilevel Mixed Models (MLM) all with and without sampling weights. ^ Results. Estimated population for RGV was 1,146,796. There were 51.5% female, 90% Hispanic, 73% married, 56% unemployed and 37% with their personal transport. 40% people attained education up to elementary school, another 42% reaching high school and only 18% went to college. Median household income is less than $15,000/year. MSNP estimated to be 44,196 (3.98%) [95% CI: 39,029; 51,123]. All statistical models are in concordance with MSNP estimates ranging from 44,000 to 48,000. MSNP estimates for statistical methods are: MLR (47,707; 95% CI: 42,462; 52,999), MLR with weights (45,882; 95% CI: 39,792; 51,972), Bootstrap Regression (47,730; 95% CI: 41,629; 53,785), GEE (47,649; 95% CI: 41,629; 53,670), GEE with weights (45,076; 95% CI: 39,029; 51,123), Svy-Reg (44,196; 95% CI: 40,004; 48,390) and MLM (46,513; 95% CI: 39,869; 53,157). ^ Conclusion. RGV is a flood zone, most susceptible to hurricanes and other natural disasters. People in the region are mostly Hispanic, under-educated with least income levels in the U.S. In case of any disaster people in large are incapacitated with only 37% have their personal transport to take care of MSNP. Local and state government’s intervention in terms of planning, preparation and support for evacuation is necessary in any such disaster to avoid loss of precious human life. ^ Key words: Complex Surveys, statistical methods, multilevel models, cluster randomized, sampling weights, raking, survey regression, generalized estimation equations (GEE), random effects, Intracluster correlation coefficient (ICC).^

The epidemiology of left ventricular outflow tract obstruction defects in Texas, 2002-2008: An update and assessment of effect heterogeneity

Left ventricular outflow tract (LVOT) defects are an important group of congenital heart defects (CHDs) because of their associated mortality and long-term complications. LVOT defects include aortic valve stenosis (AVS), coarctation of aorta (CoA), and hypoplastic left heart syndrome (HLHS). Despite their clinical significance, their etiology is not completely understood. Even though the individual component phenotypes (AVS, CoA, and HLHS) may have different etiologies, they are often "lumped" together in epidemiological studies. Though "lumping" of component phenotypes may improve the power to detect associations, it may also lead to ambiguous findings if these defects are etiologically distinct. This is due to potential for effect heterogeneity across component phenotypes. ^ This study had two aims: (1) to identify the association between various risk factors and both the component (i.e., split) and composite (i.e., lumped) LVOT phenotypes, and (2) to assess the effect heterogeneity of risk factors across component phenotypes of LVOT defects. ^ This study was a secondary data analysis. Primary data were obtained from the Texas Birth Defect Registry (TBDR). TBDR uses an active surveillance method to ascertain birth defects in Texas. All cases of non complex LVOT defects which met our inclusion criteria during the period of 2002–2008 were included in the study. The comparison groups included all unaffected live births for the same period (2002–2008). Data from vital statistics were used to evaluate associations. Statistical associations between selected risk factors and LVOT defects was determined by calculating crude and adjusted prevalence ratio using Poisson regression analysis. Effect heterogeneity was evaluated using polytomous logistic regression. ^ There were a total of 2,353 cases of LVOT defects among 2,730,035 live births during the study period. There were a total of 1,311 definite cases of non-complex LVOT defects for analysis after excluding "complex" cardiac cases and cases associated with syndromes (n=168). Among infant characteristics, males were at a significantly higher risk of developing LVOT defects compared to females. Among maternal characteristics, significant associations were seen with maternal age > 40 years (compared to maternal age 20–24 years) and maternal residence in Texas-Mexico border (compared to non-border residence). Among birth characteristics, significant associations were seen with preterm birth and small for gestation age LVOT defects. ^ When evaluating effect heterogeneity, the following variables had significantly different effects among the component LVOT defect phenotypes: infant sex, plurality, maternal age, maternal race/ethnicity, and Texas-Mexico border residence. ^ This study found significant associations between various demographic factors and LVOT defects. While many findings from this study were consistent with results from previous studies, we also identified new factors associated with LVOT defects. Additionally, this study was the first to assess effect heterogeneity across LVOT defect component phenotypes. These findings contribute to a growing body of literature on characteristics associated with LVOT defects. ^