Spatio-temporal diversity of biological aerosols over Northeast India: a metagenomic approach.

Northeast India is considered as one of the major biodiversity hotspots in the world, but the region is underexplored for their microbial biodiversity. Extensive characterization of biological aerosol (bioaerosol) samples collected from various locations of Northeast India was carried out for all four seasons in a year. These were characterized in terms of their constituents, such as pollens, fungal spores, animal debris, and non-biological components, and particulate matters, such as inhalable, thoracic, and alveolic, and finally, the bacterial diversity was determined by DNA-based metagenomic approach. The non-biological (non-viable) component of aerosols is found to vary from 30 to 89% in the pre-monsoon season, which coexists with pollens (4-20%), animal debris (1-24%), and fungal spores (1-17%). The highest number of culturable microbial populations in terms of CFU count was observed in the pre-monsoon samples (i.e., 125.24-632.45 CFU/m3), and the lowest CFU was observed in monsoon season (i.e., 20.83-319.0 CFU/m3). The metagenomic approach with the samples collected during pre-monsoon season showed a total of bacterial 184 OTUs (operational taxonomic units) with 28,028 abundance count comprising 7 major phylum, 6 classes, 10 orders, 15 families, 13 genus, and 8 species of bacteria. The species-level distribution clearly shows the presence of Gammaproteobacteria (52%) most abundantly, followed by Bacilli (21%), Alphaproteobacteria (14%), Betaproteobacteria (5%), Flavobacteria (5%), and Sphingobacteria (3%). It is the first report from the entire Northeast India to uncover spatio-temporal distribution of biological components and bacterial diversity in aerosol samples through a DNA-based metagenomic approach.

Therapeutic Potential of Curcumin as an Antimycobacterial Agent.

Curcumin is the principal curcuminoid obtained from the plant Curcuma longa and has been extensively studied for its biological and chemical properties. Curcumin displays a vast range of pharmacological properties, including antimicrobial, anti-inflammatory, antioxidant, and antitumor activity. Specifically, curcumin has been linked to the improvement of the outcome of tuberculosis. There are many reviews on the pharmacological effects of curcumin; however, reviews of the antitubercular activity are comparatively scarcer. In this review, we attempt to discuss the different aspects of the research on the antitubercular activity of curcumin. These include antimycobacterial activity, modulation of the host immune response, and enhancement of BCG vaccine efficacy. Recent advances in the antimycobacterial activity of curcumin synthetic derivatives, the role of computer aided drug design in identifying curcumin targets, the hepatoprotective role of curcumin, and the dosage and toxicology of curcumin will be discussed. While growing evidence supports the use of curcumin and its derivatives for tuberculosis therapy, further preclinical and clinical investigations are of pivotal importance before recommending the use of curcumin formulations in public health.

Accumulating Impact of Smoking and Co-morbidities on Severity and Mortality of COVID-19 Infection: A Systematic Review and Meta-analysis.

Background: High prevalence, severity, and formidable morbidity have marked the recent emergence of the novel coronavirus disease (COVID-19) pandemic. The significant association with the pre-existing co-morbid conditions has increased the disease burden of this global health emergency, pushing the patients, healthcare workers and facilities to the verge of complete disruption. Methods: Meta-analysis of pooled data was undertaken to assess the cumulative risk assessment of multiple co-morbid conditions associated with severe COVID-19. PubMed, Scopus, and Google Scholar were searched from January 1st to June 27th 2020 to generate a well-ordered, analytical, and critical review. The exercise began with keying in requisite keywords, followed by inclusion and exclusion criteria, data extraction, and quality evaluation. The final statistical meta-analysis of the risk factors of critical/severe and non-critical COVID-19 infection was carried out on Microsoft Excel (Ver. 2013), MedCalc (Ver.19.3), and RevMan software (Ver.5.3). Results: We investigated 19 eligible studies, comprising 12037 COVID-19 disease patients, representing the People's Republic of China (PRC), USA, and Europe. 18.2% (n = 2200) of total patients had critical/severe COVID-19 disease. The pooled analysis showed a significant association of COVID-19 disease severity risk with cardiovascular disease (RR: 3.11, p < 0.001), followed by diabetes (RR: 2.06, p < 0.001), hypertension (RR: 1.54, p < 0.001), and smoking (RR: 1.52, p < 006). Conclusion: The review involved a sample size of 12037 COVID-19 patients across a wide geographical distribution. The reviewed reports have focussed on the association of individual risk assessment of co-morbid conditions with the heightened risk of COVID-19 disease. The present meta-analysis of cumulative risk assessment of co-morbidity from cardiovascular disease, diabetes, hypertension, and smoking signals a novel interpretation of inherent risk factors exacerbating COVID-19 disease severity. Consequently, there exists a definite window of opportunity for increasing survival of COVID-19 patients (with high risk and co-morbid conditions) by timely identification and implementation of appropriately suitable treatment modalities.

Exploring rotavirus proteome to identify potential B- and T-cell epitope using computational immunoinformatics.

Rotavirus is the most common cause of acute gastroenteritis in infants and children worldwide. The functional correlation of B- and T-cells to long-lasting immunity against rotavirus infection in the literature is limited. In this work, a series of computational immuno-informatics approaches were applied and identified 28 linear B-cells, 26 conformational B-cell, 44 TC cell and 40 TH cell binding epitopes for structural and non-structural proteins of rotavirus. Further selection of putative B and T cell epitopes in the multi-epitope vaccine construct was carried out based on immunogenicity, conservancy, allergenicity and the helical content of predicted epitopes. An in-silico vaccine constructs was developed using an N-terminal adjuvant (RGD motif) followed by TC and TH cell epitopes and B-cell epitope with an appropriate linker. Multi-threading models of multi-epitope vaccine construct with B- and T-cell epitopes were generated and molecular dynamics simulation was performed to determine the stability of designed vaccine. Codon optimized multi-epitope vaccine antigens was expressed and affinity purified using the E. coli expression system. Further the T cell epitope presentation assay using the recombinant multi-epitope constructs and the T cell epitope predicted and identified in this study have not been investigated. Multi-epitope vaccine construct encompassing predicted B- and T-cell epitopes may help to generate long-term immune responses against rotavirus. The computational findings reported in this study may provide information in developing epitope-based vaccine and diagnostic assay for rotavirus-led diarrhea in children's.

Essential role of the ESX-3 associated eccD3 locus in maintaining the cell wall integrity of Mycobacterium smegmatis.

Mycobacterial pathogens have evolved a unique secretory apparatus called the Type VII secretion system (T7SS) which comprises of five gene clusters designated as ESX1, ESX2, ESX3, ESX4, and ESX5. Of these the ESX3 T7SS plays an important role in the regulatory uptake of iron from the environment, thereby enabling the bacteria to establish successful infection in the host. However, ESX3 secretion system is conserved among all the mycobacterial species including the fast-growing nonpathogenic species M. smegmatis. Although the function of ESX3 T7SS is known to be absolutely critical for establishing infection by M. tuberculosis, its conserved nature in all the pathogenic and nonpathogenic mycobacterial species intrigues to explore the additional functional roles in Mycobacterium species through which potent targets for drugs can be identified and developed. In the present study, we investigated the possible role of EccD3, a transmembrane protein of the ESX3 T7SS in M. smegmatis by deleting the entire eccD3 gene by efficient allelic exchange method. The preliminary investigations through the creation of knockout mutant of the eccD3 gene indicate that this secretory apparatus has an important role in maintaining the cell wall integrity which was evident from the abnormal colony morphology, lack of biofilm formation and difference in cell wall permeability.

Anti-Proliferative Activities of Vasicinone on Lung Carcinoma Cells Mediated via Activation of Both Mitochondria-Dependent and Independent Pathways.

Vasicinone, a quinazoline alkaloid from Adhatoda vasica Nees. is well known for its bronchodilator activity. However its anti-proliferative activities is yet to be elucidated. Here-in we investigated the anti-proliferative effect of vasicinone and its underlying mechanism against A549 lung carcinoma cells. The A549 cells upon treatment with various doses of vasicinone (10, 30, 50, 70 µM) for 72 h showed significant decrease in cell viability. Vasicinone treatment also showed DNA fragmentation, LDH leakage, and disruption of mitochondrial potential, and lower wound healing ability in A549 cells. The Annexin V/PI staining showed disrupted plasma membrane integrity and permeability of PI in treated cells. Moreover vasicinone treatment also lead to down regulation of Bcl-2, Fas death receptor and up regulation of PARP, BAD and cytochrome c, suggesting the anti-proliferative nature of vasicinone which mediated apoptosis through both Fas death receptors as well as Bcl-2 regulated signaling. Furthermore, our preliminary studies with vasicinone treatment also showed to lower the ROS levels in A549 cells and have potential free radical scavenging (DPPH, Hydroxyl) activity and ferric reducing power in cell free systems. Thus combining all, vasicinone may be used to develop a new therapeutic agent against oxidative stress induced lung cancer.

Identification of novel human renin inhibitors through a combined approach of pharmacophore modelling, molecular DFT analysis and in silico screening.

Renin is an aspartyl protease of the renin-angiotensin system (RAS) and the first enzyme of the biochemical pathway for the generation of angiotensin II - a potent vasoconstrictor involved in the maintenance of cardiovascular homeostasis and the regulation of blood pressure. High enzymatic specificity of renin and its involvement in the catalysis of the rate-limiting step of the RAS hormone system qualify it as a good target for inhibition of hypertension and other associated diseases. Ligand-based pharmacophore model (Hypo1) was generated from a training set of 24 compounds with renin inhibitory activity. The best hypothesis consisted of one Hydrogen Bond Acceptor (HBA), three Hydrophobic Aliphatic (HY-Al) and one Ring Aromatic (AR) features. This well-validated pharmacophore hypothesis (correlation coefficient 0.95) was further utilized as a 3D query to screen database compounds, which included structures from two natural product repositories. These screened compounds were further analyzed for drug-likeness and ADMET studies. The compounds which satisfied the qualifying criteria were then subjected to molecular docking and Density Functional Theory (DFT) analysis in order to discern their atomic level interactions at the active site of the 3D structure of rennin. The pharmacophore-based modelling that has been used to generate the novel findings of the present study would be an avant-garde approach towards the development of potent inhibitors of renin.

Vermicompost and farmyard manure improves food quality, antioxidant and antibacterial potential of Cajanus cajan (L. Mill sp.) leaves.

BACKGROUND: Pigeon pea (Cajanus cajan) leaves are a good source of nutrition and health benefitting phenolic compounds. However, its importance has not yet been effectively addressed. Recently, a 2-year field experiment was attempted in an alluvial soil to understand the role of various organic and inorganic fertilisers and their combinations not only on soil quality, but also on production of foremost phenolic compounds and imparting antioxidant and antibacterial properties in C. cajan under vermicompost treatments. RESULTS: Notable enhancements in crude protein, soluble carbohydrate, ash content and total flavonoid content were recorded in Cajanus leaves under vermicompost treatments. We detected a significant rise in carlinoside content in C. cajan leaves, which is known to reduce bilirubin concentration in hepatitis affected human blood. Farmyard manure treatments resulted in a high crude fibre content coupled with a substantially high concentration of total phenols, and chlorophyll. In addition, incorporation of vermicompost with or without inorganic fertiliser in the soil had a significant impact on antioxidant and antibacterial properties of C. cajan leaves. Above and beyond, farmyard manure and vermicompost positively influenced the physico-chemical health of the soil. CONCLUSION: The present nutrient management scheme based on organic input not only induced a higher yield of C. cajan endowed with improved antioxidant and antibacterial properties, but also enhanced the production of various phenolic compounds. © 2016 Society of Chemical Industry.

Novel butyrylcholinesterase inhibitors through pharmacophore modeling, virtual screening and DFT-based approaches along-with design of bioisosterism-based analogues.

Ligand and structure-based pharmacophore models were used to identify the important chemical features of butyrylcholinesterase (BChE) inhibitors. A training set of 16 known structurally diverse compounds with a wide range of inhibitory activity against BChE was used to develop a quantitative ligand-based pharmacophore (Hypo1) model to identify novel BChE inhibitors in virtual screening databases. A structure-based pharmacophore hypothesis (Phar1) was also developed with the ligand-binding site of BChE in consideration. Further, the models were validated using test set, Fisher's Randomization and Leave-one-out validation methods. Well-validated pharmacophore hypotheses were further used as 3D queries in virtual screening and 430 compounds were finally selected for molecular docking analysis. Subsequently, ADMET, DFT and chemical similarity search were employed to narrow down on seven compounds as potential drug candidates. Analogues of the best hit were further developed through a bioisosterism-guided approach to further generate a library of potential BChE inhibitors.

3D pharmacophore-based virtual screening, docking and density functional theory approach towards the discovery of novel human epidermal growth factor receptor-2 (HER2) inhibitors.

Human epidermal growth factor receptor 2 (HER2) is one of the four members of the epidermal growth factor receptor (EGFR) family and is expressed to facilitate cellular proliferation across various tissue types. Therapies targeting HER2, which is a transmembrane glycoprotein with tyrosine kinase activity, offer promising prospects especially in breast and gastric/gastroesophageal cancer patients. Persistence of both primary and acquired resistance to various routine drugs/antibodies is a disappointing outcome in the treatment of many HER2 positive cancer patients and is a challenge that requires formulation of new and improved strategies to overcome the same. Identification of novel HER2 inhibitors with improved therapeutics index was performed with a highly correlating (r=0.975) ligand-based pharmacophore model (Hypo1) in this study. Hypo1 was generated from a training set of 22 compounds with HER2 inhibitory activity and this well-validated hypothesis was subsequently used as a 3D query to screen compounds in a total of four databases of which two were natural product databases. Further, these compounds were analyzed for compliance with Veber's drug-likeness rule and optimum ADMET parameters. The selected compounds were then subjected to molecular docking and Density Functional Theory (DFT) analysis to discern their molecular interactions at the active site of HER2. The findings thus presented would be an important starting point towards the development of novel HER2 inhibitors using well-validated computational techniques.

Comparative analysis of codon usage bias in Crenarchaea and Euryarchaea genome reveals differential preference of synonymous codons to encode highly expressed ribosomal and RNA polymerase proteins.

The present study was undertaken to investigate the pattern of optimal codon usage in Archaea. Comparative analysis was executed to understand the pattern of codon usage bias between the high expression genes (HEG) and the whole genomes in two Archaeal phyla, Crenarchaea and Euryarchaea. The G+C% of the HEG was found to be less in comparison to the genome G+C% in Crenarchaea, whereas reverse was the case in Euryarchaea. The preponderance of U/A ending codons that code for HEG in Crenarchaea was in sharp contrast to the C/G ended ones in Euryarchaea. The analysis revealed prevalence of Uending codons even within theWWY(nucleotide ambiguity code) families in Crenarchaea vis-à-vis Euryarchaea, bacteria and Eukarya. No plausible interpretation of the observed disparity could be made either in the context of tRNA gene composition or genome G+C%. The results in this study attested that the preferential biasness for codons in HEG of Crenarchaea might be different from Euryarchaea. The main highlights are (i) varied CUB in the HEG and in the whole genomes in Euryarchaea and Crenarchaea. (ii) Crenarchaea was found to have some unusual optimal codons (OCs) compared to other organisms. (iii) G+C% (and GC3) of the HEG were different from the genome G+C% in the two phyla. (iv) Genome G+C% and tRNA gene number failed to explain CUB in Crenarchaea. (v) Translational selection is possibly responsible for A+T rich OCs in Crenarchaea.

Discrepancy among the synonymous codons with respect to their selection as optimal codon in bacteria.

The different triplets encoding the same amino acid, termed as synonymous codons, are not equally abundant in a genome. Factors such as G + C% and tRNA are known to influence their abundance in a genome. However, the order of the nucleotide in each codon per se might also be another factor impacting on its abundance values. Of the synonymous codons for specific amino acids, some are preferentially used in the high expression genes that are referred to as the 'optimal codons' (OCs). In this study, we compared OCs of the 18 amino acids in 221 species of bacteria. It is observed that there is amino acid specific influence for the selection of OCs. There is also influence of phylogeny in the choice of OCs for some amino acids such as Glu, Gln, Lys and Leu. The phenomenon of codon bias is also supported by the comparative studies of the abundance values of the synonymous codons with same G + C. It is likely that the order of the nucleotides in the triplet codon is also perhaps involved in the phenomenon of codon usage bias in organisms.

Ectopic expression of amaranth seed storage albumin modulates photoassimilate transport and nutrient acquisition in sweetpotato.

Storage proteins in plants, because of high nutrient value, have been a subject of intensive investigation. These proteins are synthesized de novo in the cytoplasm and transported to the storage organelles where they serve as reservoir of energy and supplement of nitrogen during rapid growth and development. Sweetpotato is the seventh most important food crop worldwide, and has a significant contribution to the source of nutrition, albeit with low protein content. To determine the behaviour of seed storage proteins in non-native system, a seed albumin, AmA1, was overexpressed in sweetpotato with an additional aim of improving nutritional quality of tuber proteins. Introduction of AmA1 imparted an increase in protein and amino acid contents as well as the phytophenols. The proteometabolomics analysis revealed a rebalancing of the proteome, with no significant effects on the global metabolome profile of the transgenic tubers. Additionally, the slower degradation of starch and cellulose in transgenic tubers, led to increased post-harvest durability. Present study provides a new insight into the role of a seed storage protein in the modulation of photoassimilate movement and nutrient acquisition.

Comparison of proteomic and metabolomic profiles of two contrasting ecotypes of sweetpotato (Ipomoea batata L.).

UNLABELLED: Sweetpotato has long been acknowledged as a significant contributor of global caloric needs, which continues to be of remarkable economic value. It is an important staple and emergency food in many countries and its annual world production hovers to about 130 million tons. The tubers act as sink and compete for the available photoassimilates eventually leading to the acquisition of nutrients and phytochemicals. Differential display of genes or gene-products, and metabolites causes differences in nutritive value of closely related ecotypes. To better understand the molecular basis for differential nutrient availability and phytochemicals, and exploit the natural genetic variation(s), we aimed at developing proteometabolic profiles of two contrasting ecotypes of sweetpotato. Proteomic analyses led to the identification of 1541 and 1201 proteins in orange fleshed and white fleshed sweetpotato ecotypes, respectively, presumably associated with binding, followed by catalytic, transferase, hydrolase, kinase and transporter activities. Furthermore, metabolome profiling revealed 148 and 126 metabolites in cv. OFSP and WFSP, respectively. This study would provide a basis for future comparative proteometabolomic efforts for sweetpotato, in particular and tuber crops in general. The results would expand our understanding of the proteome as well as metabolome and give new insights into how ecotype-specific traits are developed. BIOLOGICAL SIGNIFICANCE: Sweetpotato, the potato of the tropics, is the seventh most important crop worldwide in terms of production for food and additional industrial resources. Over 95% of the global sweet potato is produced in developing countries where it is considered as emergency food. It is also a vegetable, a snack food and confectionery item in most countries. It greatly contributes as a phytochemical source of nutrition and can produce more edible energy per hectare per day than wheat or rice. The adaptability to a wide range of agroecological conditions with least growth requirements makes it a preferred tuber crop of high commercial significance. Despite its nutritional merits, it has always remained outside the realm of large-scale functional genomics. Therefore, this study was aimed at constructing the proteomics and metabolomics shared resource for sweetpotato. These data are particularly significant, at least partially due to the fact that the currently available information about sweet potato is under-represented.

An In Silico Approach for Identification of Potential Anti-Mycobacterial Targets of Vasicine and Related Chemical Compounds.

Tuberculosis (TB) is known to mankind as one of the most pervasive and persistent of diseases since the early days of civilization. The growing resistance of the causative pathogen Mycobacterium tuberculosis to the standard drug regimen for TB poses further difficulty in its treatment and control. Screening of novel plant-derived compounds with promising anti-tubercular activity has been cited as a prospective route for new anti-tubercular drug discovery and design. Justicia adhatoda L. is a perennial evergreen shrub which is widely mentioned in scientific literature on account of its potent anti-mycobacterial properties. In the present study, we have employed a series of computational methodologies to reveal the probable molecular interactions of vasicine, the principal alkaloid of Justicia adhatoda L., and two of its close natural derivatives- vasicinone and deoxyvasicine, with certain biological targets in M. tuberculosis. Targets were identified from literature and through a reverse Pharmacophore-based approach. Subsequent comparative molecular docking to identify the best ligand-target interactions revealed Antigen 85C of M. tuberculosis as the most potent biological target of vasicine on the basis of optimum molecular docking values. A chemogenomics approach was also employed to validate the molecular interactions between the same class of chemical compounds as vasicine and Antigen 85C. Further, a library of structural analogs of vasicine was created by bioiosterism-based drug design to identify structural analogs with better inhibitory potential against Antigen 85C.

Cigarette smoke compounds induce cellular redox imbalance, activate NF-κB, and increase TNF-α/CRP secretion: a possible pathway in the pathogenesis of COPD.

Cigarette smoke has always been considered as a risk factor for chronic obstructive pulmonary diseases (COPD). In this study, we have examined the effect of ten individual cigarette smoke compounds (nicotine, benzo[a]pyrene, naphthalene, formaldehyde, ammonia, acrylic acid, toluene, benzene, m-xylene, and hexamine) on glutathione S transferase (GST) activity, an important Phase II metabolic enzyme and their possible role in inflammatory pathophysiology leading to COPD. Lower Glutathione (GSH) levels and GST activity and higher CRP, TNF-α, and IL-6 levels were observed in COPD patients compared to age and gender-matched controls. Using human recombinant GST and plasma as well as erythrocytes collected from normal subjects this study demonstrates that out of the ten compounds, nicotine (5 mg mL-1), benzo[a]pyrene (10 ng mL-1), naphthalene (250 µg mL-1), and formaldehyde (5 pg mL-1) caused a significant decrease in recombinant, plasma, and erythrocyte GST activity. Further cell culture studies show that exposure to nicotine, benzo[a]pyrene, naphthalene, and formaldehyde caused a significant decrease in GSH levels and GST activity and its protein expression and an increase in intracellular ROS production in THP-1 monocytes. Interestingly, treatment with benzo[a]pyrene and naphthalene significantly up regulated the phosphorylation of the p65 subunit of NF-κB and increased the secretion of TNF-α and CRP compared to control. This study suggests the potential role of benzo[a]pyrene and naphthalene in the activation of the inflammatory signaling pathway leading to cigarette smoke-induced COPD.

Comparative analysis of phytochemicals and nutrient availability in two contrasting cultivars of sweet potato (Ipomoea batatas L.).

Sweet potato ranks as the world's seventh most important food crop, and has major contribution to energy and phytochemical source of nutrition. To unravel the molecular basis for differential nutrient availability, and to exploit the natural genetic variation(s) of sweet potato, a series of physiochemical and proteomics experiment was conducted using two contrasting cultivars, an orange-fleshed sweet potato (OFSP) and a white-fleshed sweet potato (WFSP). Phytochemical screening revealed high percentage of carbohydrate, reducing sugar and phenolics in WFSP, whereas OFSP showed increased levels of total protein, flavonoids, anthocyanins, and carotenoids. The rate of starch and cellulose degradation was found to be less in OFSP during storage, indicating tight regulation of gene(s) responsible for starch-degradation. Comparative proteomics displayed a cultivar-dependent expression of proteins along with evolutionarily conserved proteins. These results suggest that cultivar-specific expression of proteins and/or their interacting partners might play a crucial role for nutrient acquisition in sweet potato.

A polyphenol rescues lipid induced insulin resistance in skeletal muscle cells and adipocytes.

Skeletal muscle and adipose tissues are known to be two important insulin target sites. Therefore, lipid induced insulin resistance in these tissues greatly contributes in the development of type 2 diabetes (T2D). Ferulic acid (FRL) purified from the leaves of Hibiscus mutabilis, showed impressive effects in preventing saturated fatty acid (SFA) induced defects in skeletal muscle cells. Impairment of insulin signaling molecules by SFA was significantly waived by FRL. SFA markedly reduced insulin receptor ß (IRß) in skeletal muscle cells, this was affected due to the defects in high mobility group A1 (HMGA1) protein obtruded by phospho-PKCε and that adversely affects IRß mRNA expression. FRL blocked PKCε activation and thereby permitted HMGA1 to activate IRß promoter which improved IR expression deficiency. In high fat diet (HFD) fed diabetic rats, FRL reduced blood glucose level and enhanced lipid uptake activity of adipocytes isolated from adipose tissue. Importantly, FRL suppressed fetuin-A (FetA) gene expression, that reduced circulatory FetA level and since FetA is involved in adipose tissue inflammation, a significant attenuation of proinflammatory cytokines occurred. Collectively, FRL exhibited certain unique features for preventing lipid induced insulin resistance and therefore promises a better therapeutic choice for T2D.

Selection on GGU and CGU codons in the high expression genes in bacteria.

The fourfold degenerate site (FDS) in coding sequences is important for studying the effect of any selection pressure on codon usage bias (CUB) because nucleotide substitution per se is not under any such pressure at the site due to the unaltered amino acid sequence in a protein. We estimated the frequency variation of nucleotides at the FDS across the eight family boxes (FBs) defined as Um(g), the unevenness measure of a gene g. The study was made in 545 species of bacteria. In many bacteria, the Um(g) correlated strongly with Nc'-a measure of the CUB. Analysis of the strongly correlated bacteria revealed that the U-ending codons (GGU, CGU) were preferred to the G-ending codons (GGG, CGG) in Gly and Arg FBs even in the genomes with G+C % higher than 65.0. Further evidence suggested that these codons can be used as a good indicator of selection pressure on CUB in genomes with higher G+C %.

Constraint on di-nucleotides by codon usage bias in bacterial genomes.

It has been reported earlier that the relative di-nucleotide frequency (RDF) in different parts of a genome is similar while the frequency is variable among different genomes. So RDF is termed as genome signature in bacteria. It is not known if the constancy in RDF is governed by genome wide mutational bias or by selection. Here we did comparative analysis of RDF between the inter-genic and the coding sequences in seventeen bacterial genomes, whose gene expression data was available. The constraint on di-nucleotides was found to be higher in the coding sequences than that in the inter-genic regions and the constraint at the 2nd codon position was more than that in the 3rd position within a genome. Further analysis revealed that the constraint on di-nucleotides at the 2nd codon position is greater in the high expression genes (HEG) than that in the whole genomes as well as in the low expression genes (LEG). We analyzed RDF at the 2nd and the 3rd codon positions in simulated coding sequences that were computationally generated by keeping the codon usage bias (CUB) according to genome G+C composition and the sequence of amino acids unaltered. In the simulated coding sequences, the constraint observed was significantly low and no significant difference was observed between the HEG and the LEG in terms of di-nucleotide constraint. This indicated that the greater constraint on di-nucleotides in the HEG was due to the stronger selection on CUB in these genes in comparison to the LEG within a genome. Further, we did comparative analyses of the RDF in the HEG rpoB and rpoC of 199 bacteria, which revealed a common pattern of constraints on di-nucleotides at the 2nd codon position across these bacteria. To validate the role of CUB on di-nucleotide constraint, we analyzed RDF at the 2nd and the 3rd codon positions in simulated rpoB/rpoC sequences. The analysis revealed that selection on CUB is an important attribute for the constraint on di-nucleotides at these positions in bacterial genomes. We believe that this study has come with major findings of the role of CUB on di-nucleotide constraint in bacterial genomes.