Promoter characterization of relZ-bifunctional (pp)pGpp synthetase in mycobacteria.

The second messenger guanosine 3',5'-bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate/guanosine pentaphosphate (pppGpp) ((p)ppGpp) has been shown to be crucial for the survival of mycobacteria under hostile conditions. Unexpectedly, deletion of primary (p)ppGpp synthetase-Rel did not completely diminish (p)ppGpp levels leading to the discovery of novel bifunctional enzyme-RelZ, which displayed guanosine 5'-monophosphate,3'-diphosphate (pGpp), ppGpp, and pppGpp ((pp)pGpp) synthesis and RNAseHII activity. What conditions does it express itself under, and does it work in concert with Rel? The regulation of its transcription and whether the Rel enzyme plays a role in such regulation remain unclear. In this article, we have studied relZ promoter and compared its activity with rel promoter in different growth conditions. We observed that the promoter activity of relZ was constitutive; it is weaker than rel promoter, lies within 200 bp upstream of translation-start site, and it increased under carbon starvation. Furthermore, the promoter activity of relZ was compromised in the rel-knockout strain in the stationary phase. Our study unveils the dynamic regulation of relZ promoter activity by SigA and SigB sigma factors in different growth phases in mycobacteria. Importantly, elucidating the regulatory network of RelZ would enable the development of the targeted interventions for treating mycobacterial infections.

Tale of Twin Bifunctional Second Messenger (p)ppGpp Synthetases and Their Function in Mycobacteria.

M. tuberculosis, an etiological agent of tuberculosis, requires a long treatment regimen due to its ability to respond to stress and persist inside the host. The second messenger (p)ppGpp-mediated stress response plays a critical role in such long-term survival, persistence, and antibiotic tolerance which may also lead to the emergence of multiple drug resistance. In mycobacteria, (pp)pGpp molecules are synthesized predominantly by two bifunctional enzymes-long RSH-Rel and short SAS-RelZ. The long RSH-Rel is a major (p)ppGpp synthetase and hydrolase. How it switches its activity from synthesis to hydrolysis remains unclear. RelMtb mutant has been reported to be defective in biofilm formation, cell wall function, and persister cell formation. The survival of such mutants has also been observed to be compromised in infection models. In M. smegmatis, short SAS-RelZ has RNase HII activity in addition to (pp)Gpp synthesis activity. The RNase HII function of RelZ has been implicated in resolving replication-transcription conflicts by degrading R-loops. However, the mechanism and regulatory aspects of such a regulation remain elusive. In this article, we have discussed (p)ppGpp metabolism and its role in managing the stress response network of mycobacteria, which is responsible for long-term survival inside the host, making it an important therapeutic target.

Insights into established and emerging roles of SR protein family in plants and animals.

Splicing of pre-mRNA is an essential part of eukaryotic gene expression. Serine-/arginine-rich (SR) proteins are highly conserved RNA-binding proteins present in all metazoans and plants. SR proteins are involved in constitutive and alternative splicing, thereby regulating the transcriptome and proteome diversity in the organism. In addition to their role in splicing, SR proteins are also involved in mRNA export, nonsense-mediated mRNA decay, mRNA stability, and translation. Due to their pivotal roles in mRNA metabolism, SR proteins play essential roles in normal growth and development. Hence, any misregulation of this set of proteins causes developmental defects in both plants and animals. SR proteins from the animal kingdom are extensively studied for their canonical and noncanonical functions. Compared with the animal kingdom, plant genomes harbor more SR protein-encoding genes and greater diversity of SR proteins, which are probably evolved for plant-specific functions. Evidence from both plants and animals confirms the essential role of SR proteins as regulators of gene expression influencing cellular processes, developmental stages, and disease conditions. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Solidification of liquid Modified Apple Polysaccharide by its adsorption on solid porous carriers through spray drying and evaluation of its potential as binding agent for tablets.

In the present study application of Modified Apple Polysaccharide (MAP) as tablet binder was evaluated. Liquid MAP was extracted from apple and solidified by adsorbing it on porous surface of Aerosil-200 and trehalose and this dispersion was dried using spray dryer. The concentration of excipients as well as spray drying conditions was optimised by using Box Behnken Design to achieve desirable powder characteristics. The optimised batch of solid MAP was characterized by DSC, PXRD, SEM, and FT-IR studies that confirmed complete adsorption of liquid MAP on the surface of Aerosil-200 and trehalose. This solid MAP was investigated for its binding efficacy for tablet formulation and its binding potential was compared with acacia and polyvinyl pyrrolidone K-30. Mesalamine (model drug) granules containing different concentration of binders were prepared by wet granulation. The granules were evaluated for micromeritic properties and results were found within the pharmacopoeial limits. The prepared tablets were subjected for post compression studies such as hardness, friability, disintegration, dissolution, physical stability, content uniformity and percentage elastic recovery and their results were found good. At 2.5% w/w concentration in tablet, the solid MAP has shown shorter disintegration time and faster dissolution profile as compared to other concentrations used including good physico-mechanical properties.