A Novel Transgenic Mouse Line for Tracing MicroRNA-155-5p Activity In Vivo

MicroRNA-155 (miR-155) plays significant role in various physiological processes involving both innate and adaptive immunity. miR-155 expression level changes dynamically during various immune responses. However, current approaches for miR-155 detection at the RNA level do not precisely reflect the real-time activity. Herein, we generated a transgenic mouse line (R26-DTR-155T) for determination of miR-155-5p activity in vivo by inserting miR-155-5p target sequence downstream of a reporter transgene comprising Diphtheria Toxin Receptor and TagBlue fluorescence protein. Using this approach, R26-DTR-155T mice were able to measure variation in levels of miR-155-5p activity in specific cell types of interest. The DTR expression levels were inversely correlated with the endogenous miR-155 expression pattern as detected by quantitative RT-PCR. Our data demonstrate a novel transgenic mouse line which could be useful for tracing miR-155-5p activity in specific cell types through measurement of miR-155-5p activity at single cell level.     

Structural and active site analysis of plasmepsins of Plasmodium falciparum: potential anti-malarial targets

Comparative protein modeling, active site analysis and binding site specificity for the homologous series of plasmepsins (PM's), present in food vacuole of Plasmodium falciparum, are carried out. Four loops (L1, L2, L3 and L4), which show maximum structural deviations irrespective of type of inhibitor, have been identified. Comparison of the crystal structures of ligand complexes reveal that residues belonging to these loops have negligible coulomb and VDW interactions with the inhibitor but play major role in determining the openness of the binding cavity. The coulomb and VDW interactions between the PMII subsite pockets and inhibitors, which play a major role in determining the inhibition constants, are delineated. Besides small displacements, the catalytic residues D32 of PMII undergoes rotation around the Cgamma-Cbeta single bond to assist catalysis whereas side chain conformational deviations are not observed in D214 on plasmepsin activation. The mutant S79D of PMII (and the corresponding residues of PMI and PMIV) which helps in recognizing and cleaving substrates containing lysine at P1 position is surrounded by highly polar atmosphere stabilized by lysine. However, in PMIII significantly lower polar atmosphere around the mutant A78S/A78D is observed. Large buried side chain area of residues located at M15 and I289 of PMII (and corresponding residues of PMI and PMIV) corroborates well with increase in specificity constant for hydrophobic substrates.     

Side-chain conformation angles of amino acids: effect of temperature factor cut-off

The paper presents the analysis of the side-chain conformation angles of amino acids in 90% non-identical protein structures. The analysis has been carried out using 113,699 residues, which is higher compared to the previous studies. In the present study, one more quality check, namely, temperature factor cut-off, has been introduced in addition to resolution and R-factor cut-offs. Due to this, the present calculation reveals the approximate values for the minimum and the maximum of the three-rotameric states of chi1. In addition, the conformation angles chi2 and chi3 have been addressed with the improved data set. The results reported here could be of use in protein modeling.     

Mineral content of water and food in fluorotic villages and prevalence of dental fluorosis

The fluoride (F), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and phosphorus (P) content in potable water and food samples (cereals, pulses, and vegetables) from endemic and nonendemic villages for fluorosis were analyzed. It was found that the F content in water was significantly higher (p<0.01) in endemic villages (4.20±1.6 ppm) than control villages (0.63±0.15 ppm), whereas the Ca, Cu, and Mg contents were found to be significantly lower (p<0.05) in endemic villages compared to control villages. However, there was no significant difference in Zn and P contents between the villages. Foods (cereals, pulses, and vegetables) grown in endemic villages contained significantly higher (p<0.01) fluoride content as compared to control villages. There was no significant difference in Ca, Mg, P, and Zn contents in food grown in endemic and control villages. Copper content in cereals (p<0.05), pulses (p<0.01), and vegetables (p<0.01) in endemic villages was found significantly higher as compared to control villages. The overall prevalence of dental fluorosis in six endemic villages was 97.4% in boys and 96% in girls, which was significantly higher (p<0.01) than that of control villages, where it was 10.5% in boys and 8.3% in girls. The prevalence of dental fluorosis was positively correlated (r=0.125, p<0.01) to fluoride and negatively correlated to Ca and Cu content in drinking water in endemic villages.     

The HDAC9-associated risk locus promotes coronary artery disease by governing TWIST1

Genome wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of common disorders. However, since the large majority of these risk SNPs reside outside gene-coding regions, GWAS generally provide no information about causal mechanisms regarding the specific gene(s) that are affected or the tissue(s) in which these candidate gene(s) exert their effect. The ‘gold standard’ method for understanding causal genes and their mechanisms of action are laborious basic science studies often involving sophisticated knockin or knockout mouse lines, however, these types of studies are impractical as a high-throughput means to understand the many risk variants that cause complex diseases like coronary artery disease (CAD). As a solution, we developed a streamlined, data-driven informatics pipeline to gain mechanistic insights on complex genetic loci. The pipeline begins by understanding the SNPs in a given locus in terms of their relative location and linkage disequilibrium relationships, and then identifies nearby expression quantitative trait loci (eQTLs) to determine their relative independence and the likely tissues that mediate their disease-causal effects. The pipeline then seeks to understand associations with other disease-relevant genes, disease sub-phenotypes, potential causality (Mendelian randomization), and the regulatory and functional involvement of these genes in gene regulatory co-expression networks (GRNs). Here, we applied this pipeline to understand a cluster of SNPs associated with CAD within and immediately adjacent to the gene encoding HDAC9. Our pipeline demonstrated, and validated, that this locus is causal for CAD by modulation of TWIST1 expression levels in the arterial wall, and by also governing a GRN related to metabolic function in skeletal muscle. Our results reconciled numerous prior studies, and also provided clear evidence that this locus does not govern HDAC9 expression, structure or function. This pipeline should be considered as a powerful and efficient way to understand GWAS risk loci in a manner that better reflects the highly complex nature of genetic risk associated with common disorders.     

Oligomeric viral proteins: small in size,large in presence

Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states.     

Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification

Probiotics are the healthy living bacteria when administered in adequate amounts confers health benefits in the host. The main objective of present study was to screen the bacteria for potential probiotic characters and enzyme production. The probiotic characters like tolerance to low pH, bile salts, antibiotic sensitivity, hydrophobicity and auto-aggregation properties were evaluated. Among all isolates Lactobacillus fermentum and Lactobacillus sp G3_4_1TO2 showed maximum potential probiotic characters and produced amylase enzyme by observing the halo zone around the colonies with the diameter 0.9?mm and 1.23?mm. Lactobacillus sp G3_4_1TO2 produced maximum amylase when compared with Lb. fermentum. The protein yield was 55.4% with the specific activity of 88.9 U/mg and obtained 40.8% purification fold. The molecular weight of amylase enzyme determined by SDS PAGE was 95,000?Da. From the present study it was considered that Lactobacillus sp G3_4_1TO2 was a potential probiotic bacteria producing maximum amylase enzyme.