Assessing Differential Expression in Two-Color Microarrays: A Resampling-Based Empirical Bayes Approach

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth''s ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth''s parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth''s parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.     

Photosynthetic Electron Transport System Promotes Synthesis of Au-Nanoparticles

In this communication, a novel, green, efficient and economically viable light mediated protocol for generation of Au-nanoparticles using most vital organelle, chloroplasts, of the plant system is portrayed. Thylakoids/chloroplasts isolated from Potamogeton nodosus (an aquatic plant) and Spinacia oleracea (a terrestrial plant) turned Au³⁺ solutions purple in presence of light of 600 µmol m⁻² s⁻¹ photon flux density (PFD) and the purple coloration intensified with time. UV-Vis spectra of these purple colored solutions showed absorption peak at ∼545 nm which is known to arise due to surface plasmon oscillations specific to Au-nanoparticles. However, thylakoids/chloroplasts did not alter color of Au³⁺ solutions in dark. These results clearly demonstrated that photosynthetic electron transport can reduce Au³⁺ to Au⁰ which nucleate to form Au-nanoparticles in presence of light. Transmission electron microscopic studies revealed that Au-nanoparticles generated by light driven photosynthetic electron transport system of thylakoids/chloroplasts were in range of 5–20 nm. Selected area electron diffraction and powder X-ray diffraction indicated crystalline nature of these nanoparticles. Energy dispersive X-ray confirmed that these nanoparticles were composed of Au. To confirm the potential of light driven photosynthetic electron transport in generation of Au-nanoparticles, thylakoids/chloroplasts were tested for their efficacy to generate Au-nanoparticles in presence of light of PFD ranging from 60 to 600 µmol m⁻² s⁻¹. The capacity of thylakoids/chloroplasts to generate Au-nanoparticles increased remarkably with increase in PFD, which further clearly demonstrated potential of light driven photosynthetic electron transport in reduction of Au³⁺ to Au⁰ to form nanoparticles. The light driven donation of electrons to metal ions by thylakoids/chloroplasts can be exploited for large scale production of nanoparticles.     

Extraction and optimization of exopolysaccharide from Lactobacillus sp. using response surface methodology and artificial neural networks

Abstract

The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.     

TcVac3 Induced Control of Trypanosoma cruzi Infection and Chronic Myocarditis in Mice

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8⁺T cell response with type-1 cytokine (IFN-γ⁺TNF-α>IL-4⁺IL-10) and cytolytic effector (CD8⁺CD107a⁺IFN-γ⁺Perforin⁺) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ⁺CD8⁺T cells, a predominance of immunoregulatory IL-10⁺/CD4⁺T and IL10⁺/CD8⁺T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8⁺T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.     

Effect of aberrant promoter methylation of FHIT and RASSF1A genes on susceptibility to cervical cancer in a North Indian population

As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.     

Optimization of media components for enhanced arachidonic acid production by <Emphasis Type="Italic">Mortierella alpina</Emphasis> under submerged cultivation

Mortierella alpina, an oleaginous zygomycete is a potent producer of arachidonic acid, the pharmaceutically and nutraceutically important polyunsaturated fatty acid of the n-6 series. It serves a wide variety of purposes, from being a purely structural element in phospholipids to being involved in signal transduction, and as a substrate for a host of derivatives involved in second messenger function. Arachidonic acid has applications in diverse areas including infant and geriatric nutrition. In the present study, the interactive effects of four major media constituents on arachidonic acid production were investigated by applying a central composite rotatable design (CCRD) and response surface methodology (RSM). The independent variables, which were selected byconcentrations of glucose, corn solids, KH₂PO₄, and KNO₃ influenced the production of biomass, total lipid, and arachidonic acid by M. alpina. A second-order polynomial was fitted by multiple regression analysis of the experimental data. The optimum conditions (glucose 10.0 g/L, corn solids 5.0 g/L, KH₂PO₄ 1.0 g/L, and KNO₃ 1.0 g/L) resulted in maximum production of arachidonic acid (1.39 g/L) and the corresponding biomass and total lipid concentrations were 12.49 and 5.87 g/L, respectively.     

Studies on filarial GST as a target for antifilarial drug development-in silico and in vitro inhibition of filarial GST by substituted 1,4-naphthoquinones

Eleven 1,4-naphthoquinone analogues with different amino substitutions at position 3 of the quinone ring earlier reported for macrofilaricidal activity were selected and screened against purified cytosolic GST isolated from the bovine filarial worm Setaria digitata and IC₅₀ values were determined. Of the 11 compounds tested, 8 showed good inhibition against S. digitata GST. The IC₅₀ values of the most effective macrofilaricidal compounds—11 [2-(4-methylpiperazin-1-yl)naphthalene-1,4-dione] and 9 {2-[(1,3-dimethylbutyl)amino]naphthalene -1,4-dione}—were 0.872 and 0.994 mM, respectively. Compounds 9 and 11 were further studied for type of enzyme inhibition and found to exhibit competitive and uncompetitive inhibition kinetics, respectively, with respect to substrate GSH. All 11 compounds were in agreement with Lipinski’s rule of five and passed through the FAFDrugs ADME/tox filter. Molecular docking was carried out using the modeled 3D structure of wbGST PDB ID:1SFM as receptor and substituted naphthoquinones as ligands using AutoDock 4.0. The binding energy of nine compounds varied from −9.15 to −6.58 Kcal mol⁻¹, whereas compounds 8 and 10 did not show any binding to the receptor. Among the compounds studied, compound 7 {2-[3-(diethylamino) propyl]aminonaphthalene-1,4-dione} showed maximum affinity towards wbGST as it exhibited the lowest binding energy, followed by compounds 11 and 9. However compound 7 was not macrofilaricidal while 11 and 9 exhibited macrofilaricidal activity. The results of in silico and in vitro studies with the synthesized 1,4 -naphthoquinone analogues on filarial GST and in vitro macrofilaricidal activity against adult bovine filarial worm S. digitata open up a promising biochemical target for antifilarial drug development.