Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis

Rel_Mtb, a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel_Mtb has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel _Mtb gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel_Mtb and its truncated derivatives were expressed from an arabinose inducible promoter (P _BAD ), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188–430 residues) containing fragment are sufficient for Rel_Mtb (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel_Mtb by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.     

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.     

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K⁺ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca²⁺ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca²⁺ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.     

Prevalence of exclusive breastfeeding and associated factors among mothers in rural Bangladesh: a cross-sectional study

Background

Exclusive breastfeeding (EBF) means that the infant receives only breast milk for the first six months of life after birth. In Bangladesh, the prevalence of EBF remained largely unchanged for nearly two decades and was 43% in 2007. However, in 2011, a prevalence of 64% was reported, an increase by 21 percentage points. The reasons for this large change remain speculative at this point. Thus to investigate the issue further, this study was conducted. The objective was to assess the prevalence of EBF and associated factors among mothers having children aged 0–6 months in rural Bangladesh.

Methods

A cross-sectional study was conducted in Mirzapur Upazilla (sub district) among 121 mothers of infants aged 0–6 months. Eligible mothers were identified and randomly selected using the demographic surveillance system’s computerized database that is updated weekly. A semi-structured questionnaire was used for interviews that inquired information on socio-demographic characteristics, obstetric, health service, breastfeeding related factors (initiation of breastfeeding, prelacteal feeding and colostrum feeding) and economic factors. EBF prevalence was calculated using 24 hour recall method. In multivariate analysis, a logistic regression model was developed using stepwise modeling to analyze the factors associated with EBF.

Results

The prevalence of EBF in the last 24 hours preceding the survey was 36%. Bivariate and multivariate analysis revealed no significant association between EBF and its possible predictors at 0.05 level of alpha. However, there was some evidence of an association between EBF and having a caesarean delivery (OR?=?0.47, 95% CI: 0.21, 1.06). In multivariate analysis, type of delivery: caesarean (AOR?=?0.45, 95% CI: 0.19, 1.03) and wealth quintile: richer (AOR?=?2.40, 95% CI: 0.94, 6.16) also showed some evidence of an association with EBF.

Conclusion

The prevalence of EBF in Mirzapur (36%) is lower than the national figure (64%). Prelacteal feeding was not uncommon. These findings suggest that there is a need for breastfeeding support provided by health services. Hence, promotion of EBF during the first six months of life needs to be addressed and future breastfeeding promotion programmes should give special attention to those women who are not practicing EBF.

     

Encapsulation of Sorbitan Ester-Based Organogels in Alginate Microparticles

Leaching of the internal apolar phase from the biopolymeric microparticles during storage is a great concern as it undoes the beneficial effects of encapsulation. In this paper, a novel formulation was prepared by encapsulating the sunflower oil-based organogels in alginate microparticles. Salicylic acid and metronidazole were used as the model drugs. The microparticles were prepared by double emulsion methodology. Physico-chemical characterization of the microparticles was done by microscopy, FTIR, XRD, and DSC studies. Oil leaching studies, biocompatibility, mucoadhesivity, in vitro drug release, and the antimicrobial efficiency of the microparticles were also performed. The microparticles were found to be spherical in shape. Gelation of the sunflower oil prevented leaching of the internal phase from the microparticles. Release of drugs from the microparticles followed Fickian kinetics and non-Fickian kinetics in gastric and intestinal environments, respectively. Microparticles showed good antimicrobial activity against both Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. The results suggested that the developed formulations hold promise to carry oils without leakage of the internal phase. Encapsulation of organogels within the microparticles has improved the drug entrapment efficiency and improved characteristics for controlled delivery applications.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0147-2) contains supplementary material, which is available to authorized users.KEY WORDS: alginate, drug delivery, leaching, microparticles, organogels     

Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding,Organization of Active Site Residues and Stereopreferences

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.     

Pathological Changes in Pulmonary Circulation in Carbon Tetrachloride (ccl4)-Induced Cirrhotic Mice

Rationale

Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease.

Objective

We investigated the effects of CCl₄-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH.

Methods And Results

Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl₄ 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl₄-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl₄ did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery.

Conclusion

Collectively, our data demonstrate that chronic CCl₄ treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.     

Autonomic Cardiovascular Responses in Acclimatized Lowlanders on Prolonged Stay at High Altitude: A Longitudinal Follow Up Study

Acute exposure to hypobaric hypoxia at high altitude is reported to cause sympathetic dominance that may contribute to the pathophysiology of high altitude illnesses. The effect of prolonged stay at high altitude on autonomic functions, however, remains to be explored. Thus, the present study aimed at investigating the effect of high altitude on autonomic neural control of cardiovascular responses by monitoring heart rate variability (HRV) during chronic hypobaric hypoxia. Baseline electrocardiography (ECG) data was acquired from the volunteers at mean sea level (MSL) (<250 m) in Rajasthan. Following induction of the study population to high altitude (4500–4800 m) in Ladakh region, ECG data was acquired from the volunteers after 6 months (ALL 6) and 18 months of induction (ALL 18). Out of 159 volunteers who underwent complete investigation during acquisition of baseline data, we have only included the data of 104 volunteers who constantly stayed at high altitude for 18 months to complete the final follow up after 18 months. HRV parameters, physiological indices and biochemical changes in serum were investigated. Our results show sympathetic hyperactivation along with compromise in parasympathetic activity in ALL 6 and ALL 18 when compared to baseline data. Reduction of sympathetic activity and increased parasympathetic response was however observed in ALL 18 when compared to ALL 6. Our findings suggest that autonomic response is regulated by two distinct mechanisms in the ALL 6 and ALL 18. While the autonomic alterations in the ALL 6 group could be attributed to increased sympathetic activity resulting from increased plasma catecholamine concentration, the sympathetic activity in ALL 18 group is associated with increased concentration of serum coronary risk factors and elevated homocysteine. These findings have important clinical implications in assessment of susceptibility to cardio-vascular risks in acclimatized lowlanders staying for prolonged duration at high altitude.