Tobacco Smoking and Its Association with Illicit Drug Use among Young Men Aged 15-24 Years Living in Urban Slums of Bangladesh

Background

Tobacco smoking (TS) and illicit drug use (IDU) are of public health concerns especially in developing countries, including Bangladesh. This paper aims to (i) identify the determinants of TS and IDU, and (ii) examine the association of TS with IDU among young slum dwellers in Bangladesh.

Methodology/Principal Findings

Data on a total of 1,576 young slum dwellers aged 15–24 years were extracted for analysis from the 2006 Urban Health Survey (UHS), which covered a nationally representative sample of 13,819 adult men aged 15–59 years from slums, non-slums and district municipalities of six administrative regions in Bangladesh. Methods used include frequency run, Chi-square test of association and multivariable logistic regression. The overall prevalence of TS in the target group was 42.3%, of which 41.4% smoked cigarettes and 3.1% smoked bidis. The regression model for TS showed that age, marital status, education, duration of living in slums, and those with sexually transmitted infections were significantly (p<0.001 to p<0.05) associated with TS. The overall prevalence of IDU was 9.1%, dominated by those who had drug injections (3.2%), and smoked ganja (2.8%) and tari (1.6%). In the regression model for IDU, the significant (p<0.01 to p<0.10) predictors were education, duration of living in slums, and whether infected by sexually transmitted diseases. The multivariable logistic regression (controlling for other variables) revealed significantly (p<0.001) higher likelihood of IDU (OR = 9.59, 95% CI = 5.81–15.82) among users of any form of TS. The likelihood of IDU increased significantly (p<0.001) with increased use of cigarettes.

Conclusions/Significance

Certain groups of youth are more vulnerable to TS and IDU. Therefore, tobacco and drug control efforts should target these groups to reduce the consequences of risky lifestyles through information, education and communication (IEC) programs.     

Proteomic Profiling of SupT1 Cells Reveal Modulation of Host Proteins by HIV-1 Nef Variants

Nef is an accessory viral protein that promotes HIV-1 replication, facilitating alterations in cellular pathways via multiple protein-protein interactions. The advent of proteomics has expanded the focus on better identification of novel molecular pathways regulating disease progression. In this study, nef was sequenced from randomly selected patients, however, sequence variability identified did not elicited any specific mutation that could have segregated HIV-1 patients in different stages of disease progression. To explore the difference in Nef functionality based on sequence variability we used proteomics approach. Proteomic profiling was done to compare the effect of Nef variants in host cell protein expression. 2DGE in control and Nef transfected SupT1 cells demonstrated several differentially expressed proteins. Fourteen protein spots were detected with more than 1.5 fold difference. Significant down regulation was seen in six unique protein spots in the Nef treated cells. Proteins were identified as Cyclophilin A, EIF5A-1 isoform B, Rho GDI 1 isoform a, VDAC1, OTUB1 and α-enolase isoform 1 (ENO1) through LC-MS/MS. The differential expression of the 6 proteins was analyzed by Real time PCR, Western blotting and Immunofluorescence studies with two Nef variants (RP14 and RP01) in SupT1 cells. There was contrasting difference between the effect of these Nef variants upon the expression of these six proteins. Downregulation of α-enolase (ENO1), VDAC1 and OTUB1 was more significant by Nef RP01 whereas Cyclophilin A and RhoGDI were found to be more downregulated by Nef RP14. This difference in Nef variants upon host protein expression was also studied through a site directed mutant of Nef RP01 _{(55AAAAAAA61)} and the effect was found to be reversed. Deciphering the role of these proteins mediated by Nef variants will open a new avenue of research in understanding Nef mediated pathogenesis. Overall study determines modulation of cellular protein expression in T cells by HIV-1 Nef variants.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: IDENTIFICATION OF ALTERED METABOLIC PATHWAYS IN DHCR7 and SC5D DEFICIENCY*

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.Smith-Lemli-Opitz syndrome (SLOS¹; Online Mendelian Inheritance in Man 270400) is a multiple malformation syndrome with cognitive and behavioral deficiencies due to an inborn error of cholesterol synthesis. Typical findings in SLOS include dysmorphic facial features, limb defects, genital anomalies, growth retardation, cognitive disabilities, behavioral problems, and autistic features (for a review, see Ref. 1). The incidence of SLOS has been estimated to be on the order of 1/20,000–1/70,000 (1). SLOS is an autosomal recessive disorder caused by mutation of the 7-dehydrocholesterol reductase gene (DHCR7) (2–4). DHCR7 catalyzes the final step in the Kandutsch-Russel cholesterol biosynthetic pathway. Impaired DHCR7 activity results in increased 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (Fig. 1A). Lathosterolosis is a rare “SLOS-like” malformation syndrome due to mutations of lathosterol 5-desaturase (SC5D) (5–7). SC5D catalyzes the conversion of lathosterol to 7DHC. Thus, in lathosterolosis, like SLOS, there is a deficiency of cholesterol. However, the accumulating precursor sterol is lathosterol rather than 7DHC (Fig. 1A). Because of its rarity and the fact that all known cases of lathosterolosis were ascertained due to similarity with SLOS, the phenotypic spectrum of lathosterolosis has not been defined.Open in a separate windowFig. 1.Representative 2-DE maps of SLOS and lathosterolosis mouse brain proteins. A, SLOS and lathosterolosis are inborn errors of cholesterol synthesis. SLOS is caused by mutations in the DHCR7 gene. DHCR7 catalyzes the final step in cholesterol synthesis. Lathosterolosis is caused by mutations of the SC5D gene. Cholesterol levels are decreased in both SLOS and lathosterolosis, but the accumulating precursor sterol differs. In SLOS, 7DHC accumulates, whereas in lathosterolosis, the accumulating sterol is lathosterol. B, representative 2-DE maps of control (Dhcr7^+/+ and Sc5d^+/+), Dhcr7^{Δ3–5/Δ3–5}, and Sc5d^−/− mouse brain proteins. Eighty micrograms of the pooled protein sample from Dhcr7^+/+, Dhcr7^{Δ3–5/Δ3–5}, Sc5d^+/+, and Sc5d^−/− embryonic mouse brain tissues were separated on a pH 3–10 nonlinear IPG strip followed by electrophoretic separation on a 12% SDS-polyacrylamide gel. Acidic pH is to the left, and increased molecular mass is at the top. Compared with Dhcr7^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Dhcr7^{Δ3–5/Δ3–5} mouse brains are marked in Dhcr7^+/+ and Dhcr7^{Δ3–5/Δ3–5} mouse brain 2-DE maps, respectively. Compared with Sc5d^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Sc5d^−/− mouse brains are marked in Sc5d^+/+ and Sc5d^−/− mouse brain 2-DE maps, respectively. Supplemental Table 2 provides detailed information on the differentially expressed protein spots.Although the genetic and biochemical causes of SLOS are defined, the pathophysiological mechanisms contributing to specific malformations have not been delineated. The classic paradigm for the pathogenesis of an inborn error of metabolism includes the accumulation of a toxic precursor and/or deficiency of an essential product. In the case of SLOS, the observed defects are postulated to be caused, either singly or in combination, by cholesterol deficiency or the accumulation of 7DHC (8, 9).Cholesterol is an essential lipid with multiple critical functions. In addition to being a structural lipid in membranes and myelin, cholesterol is the precursor for bile acid, steroid hormone, neuroactive steroid, and oxysterol synthesis. In cellular membranes, cholesterol rafts are microdomains that function in receptor-mediated signal transduction. Functional defects in IgE receptor-mediated mast cell degranulation and cytokine production (10), N-methyl-d-aspartate receptor function (11), and serotonin 1A receptor ligand binding (12, 13) have been reported in SLOS. The altered sterol composition in SLOS affects the physiochemical properties and function of lipid rafts. Membrane domains incorporating 7DHC differ from those containing only cholesterol in protein composition (14), packing (15), and stability (16–18). Substitution of 7DHC for cholesterol also decreases membrane bending rigidity (19). In addition, model membranes mimicking SLOS membranes have been reported to exhibit atypical membrane organization (20) and curvature (19). These alterations may have functional consequences. Depletion of cholesterol from hippocampal membranes and replenishment with 7-dehydrocholesterol does not restore ligand binding activity of the serotonin 1A receptor despite the recovery of the overall membrane order (12). Cholesterol is also necessary for maturation and function of the hedgehog family of morphogens during embryonic development, and several mechanisms by which sonic hedgehog signaling might be impaired in SLOS have been proposed (21–23).To understand the pathophysiological processes underlying cognitive defects found in SLOS, we need to consider the potential detrimental effects of decreased cholesterol/functional sterol levels versus the potential toxic effects of increased 7DHC. To give insight into pathological effects due to cholesterol deficiency and precursor accumulation, we have produced mouse models deficient in either 7-dehydrocholesterol reductase (11) or lathosterol reductase (6) activity (Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/−, respectively). Although the two models are similar in many respects, significant differences exist. Dhcr7 pups have relatively few physical malformations other than a low frequency of cleft palate but die during the 1st day of life due to failure to feed (11). In contrast Sc5d mutant embryos are stillborn and have multiple developmental malformations (6). In addition, although secretory granule formation is altered in both models, consistent with differing physiochemical properties of the two precursor sterols, the specific changes differ between the two models (19). For these reasons, a comparison of the two models will provide insight into common mechanisms that are likely due to cholesterol/sterol deficiency and syndrome-specific mechanisms that are due to specific effects of one of the two precursors.We now report the use of two-dimensional electrophoresis (2-DE) mass spectrometry proteomics analysis to identify proteins with altered expression in brain tissue from both Dhcr7 and Sc5d mutants with the goal of identifying novel pathophysiological mechanisms contributing to the neurological deficits in these two inborn errors of cholesterol synthesis. Because our focus was on identifying processes that could contribute to abnormal neurological development, our analysis was focused on brain tissue from E18.5 embryos. This embryonic age was selected because the biochemical defect increases with embryonic age (6, 11), and it is the latest time point for which we could obtain viable tissue for both mutants. Western blot analysis was used to validate selected individual proteins and pathways. Functional annotation suggested that alterations in mevalonate metabolism, glycolysis, oxidative stress, apoptosis, protein biosynthesis, intracellular trafficking, and cytoskeleton may contribute to the pathology of inborn errors of cholesterol synthesis. In addition, our data are consistent with the hypothesis that both cholesterol deficiency and increased precursor sterol levels contribute to SLOS and lathosterolosis pathology.     

Genetic variability of Cotton leaf curl betasatellite in Northern India

Cotton is an important crop and its production is affected by various disease pathogens. Monopartite begomovirus associated betasatellites cause Cotton leaf curl disease (CLCuD) in Northern India. In order to access the occurrence and genetic variability of Cotton leaf curl betasatellites, an extensive field survey was conducted in states of Rajasthan, Punjab and Haryana. We selected the betasatellite sequence for analysis as they are reported as important for disease severity and sequence variability. Based on the field observations, the disease incidence ranged from 30% to 80% during the survey. Full genome and DNA β were amplified from various samples while no amplicon was obtained in some samples. The nucleotide sequence homology ranged from 90.0% to 98.7% with Cotton leaf curl virus (CLCuV), 55.2–55.5% with Bhendi yellow vein mosaic virus, 55.8% with Okra leaf curl virus and 51.70% with Tomato leaf curl virus isolates. The lowest similarity (47.8%) was found in CLCuV-Sudan isolate. Phylogenetic analysis showed that analyzed isolates formed a close cluster with various CLCuV isolates reported earlier. The analysis results show sequence variation in Cotton leaf curl betasatellite which could be the result of recombination. The results obtained by genome amplification and sequence variability indicate that some new variants are circulating and causing leaf curl disease in Rajasthan, Punjab and Haryana.Abbreviations: CLCuD, Cotton leaf curl disease; CLCuV, Cotton leaf curl virus; PCR, polymerase chain reaction; SCR, satellite conserved region     

Synthesis and Biological Activity of 5-Azacytosine Nucleosides Derived from 4-Thio-2-Deoxy-L-threo-Pentofuranose and 4-Thio-2-Deoxy-D-erythro-Pentofuranose

Abstract

1-O-Acetyl-2-deoxy-3,5-di-O-toluoyl-4-thio-_d-erythro-pentofuranose and 2-deoxy-1,3,5-tri-O-acetyl-4-thio-_l-threo-pentofuranose were coupled with 5-azacytosine to obtain α and β anomers of nucleosides.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Dengue virus 2 capsid protein chaperones the strand displacement of 5′-3′ cyclization sequences

By virtue of its chaperone activity, the capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements. However, the role of DENV2C during the interaction of RNA elements involved in stabilizing the 5′-3′ panhandle structure of DENV RNA is still unclear. Therefore, we determined how DENV2C affects structural functionality of the capsid-coding region hairpin element (cHP) during annealing and strand displacement of the 9-nt cyclization sequence (5CS) and its complementary 3CS. cHP has two distinct functions: a role in translation start codon selection and a role in RNA synthesis. Our results showed that cHP impedes annealing between 5CS and 3CS. Although DENV2C does not modulate structural functionality of cHP, it accelerates annealing and specifically promotes strand displacement of 3CS during 5′-3′ panhandle formation. Furthermore, DENV2C exerts its chaperone activity by favouring one of the active conformations of cHP. Based on our results, we propose mechanisms for annealing and strand displacement involving cHP. Thus, our results provide mechanistic insights into how DENV2C regulates RNA synthesis by modulating essential RNA elements in the capsid-coding region, that in turn allow for DENV replication.