Leishmania donovani triose phosphate isomerase: a potential vaccine target against visceral leishmaniasis

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9–97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine

Network Inference Algorithms Elucidate Nrf2 Regulation of Mouse Lung Oxidative Stress

A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein–protein and protein–DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as novel Nrf2 targets include Atf1, Srxn1, Prnp, Sod2, Als2, Nfkbib, and Ppp1r15b. Furthermore, microarray and quantitative RT-PCR experiments following cigarette-smoke-induced oxidative stress in Nrf2+/+ and Nrf2−/− mouse lung affirmed many of the predictions made. Several new potential feed-forward regulatory loops involving Nrf2, Nqo1, Srxn1, Prdx1, Als2, Atf1, Sod1, and Park7 were predicted. This work shows the promise of network inference algorithms operating on high-throughput gene expression data in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease

Prevalence, Distribution and Functional Significance of the −237C to T Polymorphism in the IL-12Rβ2 Promoter in Indian Tuberculosis Patients

Cytokine/cytokine receptor gene polymorphisms related to structure/expression could impact immune response. Hence, the −237 polymorphic site in the 5′ promoter region of the IL-12Rβ2 (SNP ID: rs11810249) gene associated with the AP-4 transcription motif GAGCTG, was examined. Amplicons encompassing the polymorphism were generated from 46 pulmonary tuberculosis patients, 35 family contacts and 28 miscellaneous volunteers and sequenced. The C allele predominated among patients, (93.4%, 43/46), and in all volunteers and contacts screened, but the T allele was exclusively limited to patients, (6.5%, 3/46). The functional impact of this polymorphism on transcriptional activity was assessed by Luciferase-reporter and electrophoretic mobility shift assays (EMSA). Luciferase-reporter assays showed a significant reduction in transcriptional efficiency with T compared to C allele. The reduction in transcriptional efficiency with the T allele construct (pGIL-12Rb2-T), in U-87MG, THP-1 and Jurkat cell lines, were 53, 37.6, and 49.8% respectively, compared to the C allele construct (pGIL-12Rb2-C). Similarly, densitometric analysis of the EMSA assay showed reduced binding of the AP-4 transcription factor, to T compared to the C nucleotide probe. Reduced mRNA expression in all patients (3/3) harboring the T allele was seen, whereas individuals with the C allele exhibited high mRNA expression (17/25; 68%, p = 0.05). These observations were in agreement with the in vitro assessment of the promoter activity by Luciferase-reporter and EMSA assays. The reduced expression of IL-12Rβ2 transcripts in 8 patients despite having the C allele was attributed to the predominant over expression of the suppressors (IL-4 and GATA-3) and reduced expression of enhancers (IFN-α) of IL-12Rβ2 transcripts. The 17 high IL-12Rβ2 mRNA expressers had significantly elevated IFN-α mRNA levels compared to low expressers and volunteers. Notwithstanding the presence of high levels of IL-12Rβ2 mRNA in these patients elevated IFN-α expression could modulate their immune responses to Mycobacterium tuberculosis

Rapid Prediction of Microbial Load on Intact Mango Surface Using Spectroscopy

Agriculture industries are continuously in search of new user friendly techniques for evaluating overall quality(microbial and biochemical) of fruits as per quarantine requirements. In the current study, the potential of visibleand near-infrared (NIR) spectroscopy in the wavelength range of 299-1100 nm and 900-1700 nm was evaluatedto determine total microbial population on the surface of seven major cultivars of mangoes collected from fourstates of India. NIR models were developed based on multiple-linear regression (MLR) and partial least square(PLS) regression employing pre-processing technologies (baseline correction, smoothening, multiplicative scattercorrection (MSC) and second-order derivatives). Wavelength range of 299-1100 nm was found to be more suitablefor determination of microbial load on the mango surface as compared to wavelength range of 900-1700 nm.PLS models were found to be the best with multiple correlation coefficients of 0.66 and 0.56, for calibration andvalidation, respectively, in the wavelength range 504.80-533.17 nm. The standard errors of calibration, predictionand differences in them were low, which demonstrated the potential of NIRS to predict microbial load on the surfaceof mango non-destructively in the wavelength range of 504.806-533.176 nm

Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening

Objectives: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening. Methods: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites - both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates - were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs. Results: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for ∼12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports. Conclusions: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection

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Not AvailableIn this study antibacterial activity of pomegranate peel (PPE) was evaluated against bacteria isolated from poultry meat. The bacteria were identified using 16S rRNA gene and DNA sequencing. Results of molecular characterization showed that the bacteria isolated were having 100% homology with the Pseudomonas stutzeri strain CTSP36 and further analysis showed that sample sequence clustered with the P. stutzeri strain CTSP36. Antibacterial activity of PPE was demonstrated by clear zone of inhibition in plates inoculated with extract. The diameter of inhibition zones were significantly (p<0.05) higher in PPE as compared to standard antibiotic discs used (tetracycline, vancomycin and streptomycin). Results of broth dilution assay also revealed that PPE at 1%, 5% and 10% were effective in inhibiting bacterial growth in test plates. Further, a decrease in the growth of bacterial cells and a gradual decline in protein content of bacterial cells were also observed when bacterial culture was grown with different concentration of PPE along with a control. These results showed the potential application of pomegranate peel extract as antibacterial agent against P. stutzeri.Not Availabl

Intravitam Diagnosis of Rabies in Patients with Acute Encephalitis: A Study of Two Cases

Rabies is one of the oldest known zoonotic diseases. Rhabdovirus, an RNA virus belonging to the genus Lyssavirus and family Rhabdoviridae, causes rabies. Rabies diagnosis is challenging as the rabies virus remains confined to neurons after the initial animal bite. It largely remains immune-evasive until the infection reaches the central nervous system. The bottleneck in rabies diagnosis remains the non-availability of technical expertise and failure to collect an appropriate sample. The laboratory confirmation of rabies in both antemortem and postmortem samples is important. The samples were tested for anti-rabies antibodies using quantitative ELISA. In this report, two case studies are presented to demonstrate the suitability of ELISA for the intra vitam diagnosis of rabies using cerebrospinal fluid (CSF) as a diagnostic sample. The interpretation of serology results for both vaccinated and unvaccinated individuals has been discussed in detail, which has helped to confirm the antemortem diagnosis of rabies. In this report, we observed that ELISA can be a viable alternative for anti-rabies antibody detection in CSF and can be used as a viable alternative to more technically challenging tests, such as Rapid Fluorescent Focus Inhibition Test (RFFFIT) and Immunofluorescence Assays (IFA)

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Not AvailableThe multitude of agronomic merits of a traditionally tall and photosensitive japonica rice landrace C14-8 has enabled its popularity in the tropical Andaman and Nicobar Islands, India. However, we noticed distinct variation for grain husk colour in this culture. Field evaluation of four grain husk color selections over 4 years across three major rice growing islands revealed signifcant variation for agro-morphological traits studied. In the overall population, harvest index was identifed as the potent selectable trait for indirect selection. Through AMMI stability analysis, the environmental, G×E interaction and genotype efects were recorded as 24.4%, 12.5% and 12.3%, respectively. The highest positive genotypic index was recorded at C14-8-11-108 (0.39) followed by C14-8-11-113 (0.29) and C14-8-11-91 (0.20) which also out-yielded the original population by about 20% across years thus indicating the consistency and favorability of these selections under marginal ecosystem.The fndings of this paper will be useful for the breeding and conservation perspectives of such unique germplasm having climatically adaptive traits under marginal ecosystems.Not Availabl

Intra-varietal stability performance of popular rice landrace ‘C14-8’ in the Andaman Islands

The multitude of agronomic merits of a traditionally tall and photosensitive japonica rice landrace C14-8 has enabled its popularity in the tropical Andaman and Nicobar Islands, India. However, we noticed distinct variation for grain husk colour in this culture. Field evaluation of four grain husk color selections over 4 years across three major rice growing islands revealed signifcant variation for agro-morphological traits studied. In the overall population, harvest index was identifed as the potent selectable trait for indirect selection. Through AMMI stability analysis, the environmental, G×E interaction and genotype efects were recorded as 24.4%, 12.5% and 12.3%, respectively. The highest positive genotypic index was recorded at C14- 8-11-108 (0.39) followed by C14-8-11-113 (0.29) and C14-8-11-91 (0.20) which also out-yielded the original population by about 20% across years thus indicating the consistency and favorability of these selections under marginal ecosystem. The fndings of this paper will be useful for the breeding and conservation perspectives of such unique germplasm having climatically adaptive traits under marginal ecosystems