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The chemokine receptor CCR5 exhibits an important role for the CD-4 mediated entry of HIV-1. Previous studies revealed that Δ32 mutation on the CCR5 gene results in truncated protein and hence confers protection against HIV-1 infection and AIDS progression, as observed in Caucasian population. However, the status of Δ32 mutation on CCR5 is still unknown in many Nepali ethnic groups though detection of heterozygous CCR5 Δ32 mutation allele has been reported from Chidimar and Thakali ethnic groups. We studied the presence of the Δ32 mutation in 300 blood samples from 11 ethnic groups of Nepal by analyzing PCR product of CCR5 gene region flanking the Δ32 mutation region. The primer set (forward -5’ CTC CCA GGA ATC ATC TTT ACC 3’ and reverse - 5’ TCA TTT CGA CAC CGA AGC AG 3’) flanks the site of the Δ32 deletion region of CCR5 gene. This results in a PCR fragment of 200 base pairs for the CCR5 wild allele and 168 base pairs for a Δ32 deletion mutation allele. All samples were found to exhibit wild type CCR5 gene; but no Δ32 mutation observed. Absence of Δ32 mutation in CCR5 gene indicates that Nepali population is not genetically resistant to HIV infection, if other genes are not considered.

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The intracellular bacterial pathogen, Chlamydia trachomatis, is a tryptophan auxotroph. Therefore, induction of the host tryptophan catabolizing enzyme, indoleamine-2,3-dioxgenase-1 (IDO1), by interferon gamma (IFNγ) is one of the primary protective responses against chlamydial infection. However, despite the presence of a robust IFNγ response, active and replicating C. trachomatis can be detected in cervical secretions of women. We hypothesized that a primary C. trachomatis infection may evade the IFNγ response, and that the protective effect of this cytokine results from its activation of tryptophan catabolism in bystander cells. To test this hypothesis, we developed a novel method to separate a pool of cells exposed to C. trachomatis into pure populations of live infected and bystander cells and applied this technique to distinguish between the effects of IFNγ on infected and bystander cells. Our findings revealed that the protective induction of IDO1 is suppressed specifically within primary infected cells because Chlamydia attenuates the nuclear import of activated STAT1 following IFNγ exposure, without affecting STAT1 levels or phosphorylation. Critically, the IFNγ-mediated induction of IDO1 activity is unhindered in bystander cells. Therefore, the IDO1-mediated tryptophan catabolism is functional in these cells, transforming these bystander cells into inhospitable hosts for a secondary C. trachomatis infection.

Epidemiological surveys conducted in general populations have found that the prevalence of depression is about 9% in the United States. World Health Organization has projected that depression will be leading cause of disease burden by the year 2030. Growing evidence suggests that sedentary lifestyle is an important risk factor of depression among adults. The relationship between television watching/computer use and depression in US adults is still unknown. The objective of this study was to assess the relationship between television watching/computer use and depression. This is a cross-sectional study that used the secondary data from the National Health and Nutritional Examination Survey (NHANES) (2011/2012). Participants were 3201 US adults who were 20 years or more. Self-reported Patient Health Questionnaire-9 [PHQ-9] was used to classify depression level; self-reported hours of watching TV and use of computer/day, and demographic information were obtained from NHANES data set. SAS®9.4was used to perform all statistical analyses and final model selection procedure. Depression was found to be significantly higher among female. Results showed that moderate or severe depression level was associated with higher time spent on TV watching and use of computer (> 6 h/day) (adjusted odds ratio: 2.3, 95% CI: 1.602–3.442). Duration of screen time was significantly associated when all covariates were adjusted. TV watching and computer use can predict the depression level among adults. Prospective studies and measurement of factors such as: work place sitting, social relationship, and family history of depression are warranted., Highlights • Assess the relationship between television watching/computer use and depression • Depression was found to be significantly higher among female. • Moderate or severe depression level was associated with higher screen time.

A striking difference between genital and ocular clinical isolates of Chlamydia trachomatis is that only the former express a functional tryptophan synthase and therefore can synthesize tryptophan by indole salvage. Ocular isolates uniformly cannot use indole due to inactivating mutations within tryptophan synthase, indicating a selection against maintaining this enzyme in the ocular environment. Here, we demonstrate that this selection occurs in two steps. First, specific indole derivatives, produced by the human gut microbiome and present in serum, rapidly induce expression of C. trachomatis tryptophan synthase, even under conditions of tryptophan sufficiency. We demonstrate that these indole derivatives function by acting as de-repressors of C. trachomatis TrpR. Second, trp operon de-repression is profoundly deleterious when infected cells are in an indole-deficient environment, because in the absence of indole, tryptophan synthase deaminates serine to pyruvate and ammonia. We have used biochemical and genetic approaches to demonstrate that expression of wild-type tryptophan synthase is required for the bactericidal production of ammonia. Pertinently, although these indole derivatives de-repress the trpRBA operon of C. trachomatis strains with trpA or trpB mutations, no ammonia is produced, and no deleterious effects are observed. Our studies demonstrate that tryptophan synthase can catalyze the ammonia-generating β-elimination reaction within any live bacterium. Our results also likely explain previous observations demonstrating that the same indole derivatives inhibit the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prognosis of diseased conditions associated with bacterial dysbiosis.

Arsenic is ubiquitous in nature, highly toxic, and is particularly abundant in Southern Asia. While many studies have focused on areas like Bangladesh and West Bengal, India, disadvantaged regions within Nepal have also suffered from arsenic contamination levels, with wells and other water sources possessing arsenic contamination over the recommended WHO and EPA limit of 10 μg/L, some wells reporting levels as high as 500 μg/L. Despite the region's pronounced arsenic concentrations within community water sources, few investigations have been conducted to understand the impact of arsenic contamination on host gut microbiota health. This study aims to examine differential arsenic exposure on the gut microbiome structure within two disadvantaged communities in southern Nepal. Fecal samples (n = 42) were collected from members of the Mahuawa (n = 20) and Ghanashyampur (n = 22) communities in southern Nepal. The 16S rRNA gene was amplified from fecal samples using Illumina-tag PCR and subject to high-throughput sequencing to generate the bacterial community structure of each sample. Bioinformatics analysis and multivariate statistics were conducted to identify if specific fecal bacterial assemblages and predicted functions were correlated with urine arsenic concentration. Our results revealed unique assemblages of arsenic volatilizing and pathogenic bacteria positively correlated with increased arsenic concentration in individuals within the two respective communities. Additionally, we observed that commensal gut bacteria negatively correlated with increased arsenic concentration in the two respective communities. Our study has revealed that arsenic poses a broader human health risk than was previously known. It is influential in shaping the gut microbiome through its enrichment of arsenic volatilizing and pathogenic bacteria and subsequent depletion of gut commensals. This aspect of arsenic has the potential to debilitate healthy humans by contributing to disorders like heart and liver cancers and diabetes, and it has already been shown to contribute to serious diseases and disorders, including skin lesions, gangrene and several types of skin, renal, lung, and liver cancers in disadvantaged areas of the world like Nepal., Microbiology, Public Health; Gut microbiome; Arsenic; Well water; Nepal

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that cannot synthesize several amino acids, including tryptophan. Rather, C. trachomatis acquires these essential metabolites from its human host cell. Chlamydial dependence on host-provided tryptophan underlies a major host defense mechanism against the bacterium; namely, the induction of the host tryptophan-catabolizing enzyme, indoleamine 2,3- dioxygenase (IDO1) by interferon gamma (IFNγ), which leads to eradication of C. trachomatis by tryptophan starvation. For this reason, IFNγ is proposed to be the major host protective cytokine against genital C. trachomatis infections. The protective effect of IFNγ against C. trachomatis can be recapitulated in vitro using epithelial cell-lines such as the cervical carcinoma derived cell-line Hela, the Hela subclone HEp-2, and the cervical carcinoma derived cell-line ME180. Addition of IFNγ to these cells infected with C. trachomatis results in a strong bactericidal or bacteriostatic effect dependent on the concentration of IFNγ administered. Unlike Hela, HEp-2, and ME180, there are other human epithelial, or epithelial-like cell-lines where administration of IFNγ does not affect chlamydial replication, although they express the IFNγ receptor (IFNGR). In this report, we have characterized the mechanisms that underlie this dichotomy using the cell-lines C33A and 293. Akin to Hela, C33A is derived from a human cervical carcinoma, while 293 cells were produced by transfection of adenovirus type 5 DNA into embryonic kidney cells. We demonstrate that although IFNGR is expressed at high levels in C33A cells, its ligation by IFNγ does not result in STAT1 phosphorylation, an essential step for activation of the IDO1 promoter. Our results indicate that although the IFNγ- dependent signaling cascade is intact in 293 cells; the IDO1 promoter is not activated in these cells because it is epigenetically silenced, most likely by DNA methylation. Because polymorphisms in IFNγ, IFNGR, and the IDO1 promoter are known to affect other human infections or diseased states, our results indicate that the effect of allelic differences in these genes and the pathways they activate should be evaluated for their effect on C. trachomatis pathology.

Dengue is an emerging vector borne viral disease in Nepal. The study was conducted to detect dengue virus specific IgM and to analyze demographic distribution of positive cases. This is a cross-sectional study carried out in western region of Nepal from August-October 2007. A total of 116 serum samples were collected from suspected dengue virus infected patients visiting hospitals and investigated by particle agglutination. Out of 116 sera, 12 (10.34%) had a positive dengue virus specific IgM. The positivity in age group below 15 years old was 23.08%, 7.32% in 15-50 years age group and 14.29% above 50 years age group. The ratio of positivity in male and female was 1:1. However, there were no statistical significance in the positivity with age (P=0.27) and sex (P=0.91). Thus, dengue has been established in western region of Nepal and diagnosis of the disease can be done by particle agglutination assay. The particle agglutination should be suggested for the diagnosis of dengue in all health centers of Nepal. Awareness programmes on prevention of dengue and surveillance of the disease should be conducted to reduce morbidity and mortality during possible outbreak of it.DOI: http://dx.doi.org/10.3126/njst.v12i0.6514 Nepal Journal of Science and Technology 12 (2011) 286-289

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium’s capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxygenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.

Background Dengue is an emerging vector borne disease in Nepal and rapid diagnostic test is important for early diagnosis of the disease. Objectives The aim of the study was to evaluate the diagnostic accuracy of commonly used rapid immunochromatographic test kit in Nepal during 2010 dengue epidemic and to assess disease burden of dengue. Methods A total of 131 acute and nonacute serum samples were collected during recent epidemic of dengue in 2010 from clinically suspected Nepalese patients of different hospitals. Rapid immunochromatographic test kit was used for early diagnosis and enzyme immunosorbent was chosen as a reference assay. Results The sensitivity and specificity of rapid test was 70% and 76.54% respectively whereas the prevalence of the disease was 38.17%. The odds ratio for males was 1.8 however; the association with the disease was statistically not significant. Conclusion The diagnostic accuracy of rapid immunochromatographic test for dengue diagnosis was low (k=0.46). So, it should be substituted by highly sensitive test device for prompt diagnosis and health personnel should consider appropriate timing of sample collection for better performance of rapid test. KATHMANDU UNIVERSITY MEDICAL JOURNAL VOL.10 | NO. 1 | ISSUE 37 | JAN - MAR 2012 | 7-10 DOI: http://dx.doi.org/10.3126/kumj.v10i1.6905