A novel switch region regulates H-ras membrane orientation and signal output

The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and medium-throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix alpha4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP- or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP- or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the beta2-beta3 loop and helix alpha5 are involved in a novel conformational switch that operates through helix alpha4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.     

Cloning, expression and characterization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus

CYP102s represent a family of natural self-sufficient fusions of cytochrome P450 and cytochrome P450 reductase found in some bacteria. One member of this family, named CYP102A1 or more traditionally P450BM-3, has been widely studied as a model of human P450 cytochromes. Remarkable detail of P450 structure and function has been revealed using this highly efficient enzyme. The recent rapid expansion of microbial genome sequences has revealed many relatives of CYP102A1, but to date only two from Bacillus subtilis have been characterized. We report here the cloning and expression of CYP102A5, a new member of this family that is very closely related to CYP102A4 from Bacillus anthracis. Characterization of the substrate specificity of CYP102A5 shows that it, like the other CYP102s, will metabolize saturated and unsaturated fatty acids as well as N-acylamino acids. CYP102A5 catalyzes very fast substrate oxidation, showing one of the highest turnover rates for any P450 monooxygenase studied so far. It does so with more specificity than other CYP102s, yielding primarily ω-1 and ω-2 hydroxylated products. Measurement of the rate of electron transfer through the reductase domain reveals that it is significantly faster in CYP102A5 than in CYP102A1, providing a likely explanation for the increased monooxygenation rate. The availability of this new, very fast fusion P450 will provide a great tool for comparative structure-function studies between CYP102A5 and the other characterized CYP102s.     

Stunting Is Associated with Food Diversity while Wasting with Food Insecurity among Underfive Children in East and West Gojjam Zones of Amhara Region,Ethiopia

Background

Food insecurity has detrimental effects in protecting child undernutrition.This study sought to determine the level of child undernutrition and its association with food insecurity.

Methods

A community based comparative cross-sectional study design involving multistage sampling technique was implemented from 24^th of May to 20^th of July 2013. Using two population proportion formula, a total of 4110 randomly selected households were included in the study. Availability of the productive safety net programme was used for grouping the study areas. A multiple linear regression model was used to assess the association between food insecurity and child malnutrition. Clustering effects of localities were controlled during analysis.

Results

Stunting (37.5%), underweight (22.0%) and wasting (17.1%) were observed in East Gojjam zone, while 38.3% stunting, 22.5% underweight, and 18.6% wasting for the West Gojjam zone. Food insecurity was significantly associated with wasting (β = - 0.108, P < 0.05).Food diversity and number of meals the child ate per day significantly associated with stunting (β = 0.039, P < 0.01) and underweight (β = 0.035, P < 0.05) respectively. Residential area was the significant predictor of all indices.

Conclusion

The magnitude of child undernutrition was found to be very high in the study areas. Food insecurity was the significant determinant of wasting. Food diversity and number of meals the child ate per day were the significant determinants of stunting and underweight respectively. Child nutrition intervention strategies should take into account food security, dietary diversity, and carefully specified with regard to residential locations. Addressing food insecurity is of paramount importance.     

Retention in HIV Care and Predictors of Attrition from Care among HIV-Infected Adults Receiving Combination Anti-Retroviral Therapy in Addis Ababa

Background

Patient retention in chronic HIV care is a major challenge following the rapid expansion of combination antiretroviral therapy (cART) in Ethiopia.

Objective

To describe the proportion of patients who are retained in HIV care and characterize predictors of attrition among HIV-infected adults receiving cART in Addis Ababa.

Method

A retrospective analysis was conducted among 836 treatment naïve patients, who started cART between May 2009 and April 2012. Patients were randomly selected from ten health-care facilities, and their current status in HIV care was determined based on routinely available data in the medical records. Patients lost to follow-up (LTFU) were traced by telephone. Kaplan-Meier technique was used to estimate survival probabilities of retention and Cox proportional hazards regression was performed to identify the predictors of attrition.

Results

Based on individual patient data from the medical records, nearly 80% (95%CI: 76.7, 82.1) of the patients were retained in care in the first 3 and half years of antiretroviral therapy. After successfully tracing more than half of the LTFU patients, the updated one year retention in care estimate became 86% (95% CI: 83.41%, 88.17%). In the multivariate Cox regression analyses, severe immune deficiency at enrolment in care/or at cART initiation and ‘bed-ridden’ or ‘ambulatory’ functional status at the start of cART predicted attrition.

Conclusion

Retention in HIV care in Addis Ababa is comparable with or even better than previous findings from other resource-limited as well as EU/USA settings. However, measures to detect and enroll patients in HIV care as early as possible are still necessary.     

Childhood Diarrhea Exhibits Spatiotemporal Variation in Northwest Ethiopia: A SaTScan Spatial Statistical Analysis

Background

Childhood diarrhea continues to be a public health problem in developing countries, including Ethiopia. Detecting clusters and trends of childhood diarrhea is important to designing effective interventions. Therefore, this study aimed to investigate spatiotemporal clustering and seasonal variability of childhood diarrhea in northwest Ethiopia.

Methods

Retrospective record review of childhood diarrhea was conducted using quarterly reported data to the district health office for the seven years period beginning July 1, 2007. Thirty three districts were included and geo-coded in this study. Spatial, temporal and space-time scan spatial statistics were employed to identify clusters of childhood diarrhea. Smoothing using a moving average was applied to visualize the trends and seasonal pattern of childhood diarrhea. Statistical analyses were performed using Excel^® and SaTScan programs. The maps were plotted using ArcGIS 10.0.

Results

Childhood diarrhea in northwest Ethiopia exhibits statistical evidence of spatial, temporal, and spatiotemporal clustering, with seasonal patterns and decreasing temporal trends observed in the study area. A most likely purely spatial cluster was found in the East Gojjam administrative zone of Gozamin district (LLR = 7123.89, p <0.001). The most likely spatiotemporal cluster was detected in all districts of East Gojjam zone and a few districts of the West Gojjam zone (LLR = 24929.90, p<0.001), appearing from July 1, 2009 to June 30, 2011. One high risk period from July 1, 2008 to June 30, 2010 (LLR = 9655.86, p = 0.001) was observed in all districts. Peak childhood diarrhea cases showed a seasonal trend, occurring more frequently from January to March and April to June.

Conclusion

Childhood diarrhea did not occur at random. It has spatiotemporal variation and seasonal patterns with a decreasing temporal trend. Accounting for the spatiotemporal variation identified in the study areas is advised for the prevention and control of diarrhea.     

Synthesis and structure-activity relationship of N-alkyl Gly-boro-Pro inhibitors of DPP4, FAP, and DPP7

The structure-activity relationship of various N-alkyl Gly-boro-Pro derivatives against three dipeptidyl peptidases (DPPs) was studied. In a series of N-cycloalkyl analogs, DPP4 and fibroblast activation protein-alpha (FAP) optimally preferred N-cycloheptyl whereas DPP7 tolerated even larger cycloalkyl rings. Gly alpha-carbon derivatization of N-cyclohexyl or N-(2-adamantyl) Gly-boro-Pro resulted in a significant decrease in potency against all the three DPPs.     

Modification of Astrocyte Metabolism as an Approach to the Treatment of Epilepsy: Triheptanoin and Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-Carnitine

Epilepsy is a severe neurological disorder characterized by altered electrical activity in the brain. Important pathophysiological mechanisms include disturbed metabolism and homeostasis of major excitatory and inhibitory neurotransmitters, glutamate and GABA. Current drug treatments are largely aimed at decreasing neuronal excitability and thereby preventing the occurrence of seizures. However, many patients are refractory to treatment and side effects are frequent. Temporal lobe epilepsy (TLE) is the most common type of drug-resistant epilepsy in adults. In rodents, the pilocarpine-status epilepticus model reflects the pathology and chronic spontaneous seizures of TLE and the pentylenetetrazole kindling model exhibits chronic induced limbic seizures. Accumulating evidence from studies on TLE points to alterations in astrocytes and neurons as key metabolic changes. The present review describes interventions which alleviate these disturbances in astrocyte–neuronal interactions by supporting mitochondrial metabolism. The compounds discussed are the endogenous transport molecule acetyl-l-carnitine and the triglyceride of heptanoate, triheptanoin. Both provide acetyl moieties for oxidation in the tricarboxylic acid cycle whereas heptanoate is also provides propionyl-CoA, which after carboxylation can produce succinyl-CoA, resulting in anaplerosis—the refilling of the tricarboxylic acid cycle.     

Concordance of ompA types in children re-infected with ocular Chlamydia trachomatis following mass azithromycin treatment for trachoma

BackgroundThe chlamydial major outer membrane protein, encoded by the ompA gene, is a primary target for chlamydial vaccine research. However, human studies of ompA-specific immunity are limited, and prior studies have been limited in differentiating re-infection from persistent infection. The purpose of this study was to assess whether children living in trachoma-endemic communities with re-infections of ocular chlamydia were more likely to be infected with a different or similar genovar.Methodology and findingsThe study included 21 communities from a trachoma-hyperendemic area of Ethiopia that had been treated with a mass azithromycin distribution for trachoma. Conjunctival swabbing was offered to all children younger than 5 years of age at baseline (i.e., pre-treatment), and then at follow-up visits 2 and 6 months later. Swabs were subjected to polymerase chain reaction (PCR) to detect C. trachomatis. A random sample of 359 PCR-positive swabs, stratified by study visit and study community, was chosen for ompA sequencing. In addition, ompA sequencing was performed on all swabs of 24 children who experienced chlamydial re-infection (i.e., positive chlamydial test before treatment, negative test 2 months following mass distribution of azithromycin, and again a positive test 6 months post-treatment). ompA sequencing was successful for 351 of 359 swabs of the random sample and 44 of 48 swabs of the re-infection sample. In the random sample, ompA types clustered within households more than would be expected by chance. Among the 21 re-infected children with complete ompA data, 14 had the same ompA type before and after treatment.ConclusionThe high frequency of ompA concordance suggests incomplete genovar-specific protective immunity and the need for multiple antigens as vaccine targets.     

Identification of a Tumor Specific,Active-Site Mutation in Casein Kinase 1α by Chemical Proteomics

We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS² platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.     

Visceral Leishmaniasis in Ethiopia: An Evolving Disease

Visceral leishmaniasis (also known as kala-azar) is classified as one of the most neglected tropical diseases. It is becoming a growing health problem in Ethiopia, with endemic areas that are continually spreading. The annual burden of visceral leishmaniasis (VL) in Ethiopia is estimated to be between 4,500 and 5,000 cases, and the population at risk is more than 3.2 million. There has been a change in the epidemiology of VL in Ethiopia. Over the last decades, almost all cases and outbreaks of VL were reported from arid and semi-arid parts of the country; however, recent reports indicated the introduction of this disease into the highlands. Migration of labourers to and from endemic areas, climatic and environmental changes, and impaired immunity due to HIV/AIDS and malnutrition resulted in the change of VL epidemiology. HIV spurs the spread of VL by increasing the risk of progression from asymptomatic infection towards full VL. Conversely, VL accelerates the onset of AIDS. In Ethiopia, VL epidemiology remains complex because of the diversity of risk factors involved, and its control is becoming an increasing challenge. This paper reviews the changes in epidemiology of VL in Ethiopia and discusses some of the possible explanations for these changes. The prospects for novel approaches to VL control are discussed, as are the current and future challenges facing Ethiopia''s public health development program.