Investigating the functional role of a buried interchain aromatic cluster in Escherichia coli GrpE dimer

Aromatic clusters in the core of proteins are often involved in imparting structural stability to proteins. However, their functional importance is not always clear. In this study, we investigate the thermosensing role of a phenylalanine cluster present in the GrpE homodimer. GrpE, which acts as a nucleotide exchange factor for the molecular chaperone DnaK, is well known for its thermosensing activity resulting from temperature-dependent structural changes that allow control of chaperone function. Using mutational analysis, we show that an interchain phenylalanine cluster in a four-helix bundle of the GrpE homodimer assists in the thermosensing ability of the co-chaperone. Substitution of aromatic residues with hydrophobic ones in the core of the four-helix bundle reduces the thermal stability of the bundle and that of a connected coiled-coil domain, which impacts thermosensing. Cell growth assays and SEM images of the mutants show filamentous growth of Escherichia coli cells at 42°C, which corroborates with the defect in thermosensing. Our work suggests that the interchain edge-to-face aromatic cluster is important for the propagation of the structural signal from the coiled-coil domain to the four-helical bundle of GrpE, thus facilitating GrpE-mediated thermosensing in bacteria.     

Immunization of Mice with Components of Pasteurella multocida

Log-phase cells of Pasteurella multocida strain P-1059 were used to prepare isolated culture filtrate, cell wall, and cytoplasmic components. Culture filtrate was further separated by column chromatography. A portion of cytoplasm and culture filtrate was conjugated to ferritin by means of metaxylylene diisocyanate. Cell walls induced more protection in mice than the conjugated or unconjugated cytoplasm or culture filtrate. The cell walls caused edema and erythema when given intradermally in rabbits, whereas cytoplasm and culture filtrate produced dermal necrosis. The first of four chromatographically separated fractions of culture filtrate was possibly more immunogenic in mice than cell walls. This fraction was less reactive intradermally in rabbits than cell walls but more reactive than the other fractions.     

Orally active antimalarials: synthesis and bioevaluation of a new series of steroid-based 1,2,4-trioxanes against multi-drug resistant malaria in mice

A new series of steroid-based 1,2,4-trioxanes 7a-f, 8a-f and 9b-e have been synthesized and evaluated for their antimalarial activity against multi-drug resistant Plasmodium yoelii in Swiss mice by oral route. The biological activity shows a strong dependence on the size and the nature of the steroidal side chain. Pregnane-based trioxanes 8a-f show better activity profile than trioxanes 7a-f and 9b-e, derived from cholesterol and tigogenine, respectively.     

Febrifugine analogues as Leishmania donovani trypanothione reductase inhibitors: binding energy analysis assisted by molecular docking,ADMET and molecular dynamics simulation

Visceral leishmaniasis affects people from 70 countries worldwide, mostly from Indian, African and south American continent. The increasing resistance to antimonial, miltefosine and frequent toxicity of amphotericin B drives an urgent need to develop an antileishmanial drug with excellent efficacy and safety profile. In this study we have docked series of febrifugine analogues (n = 8813) against trypanothione reductase in three sequential docking modes. Extra precision docking resulted into 108 ligands showing better docking score as compared to two reference ligand. Furthermore, 108 febrifugine analogues and reference inhibitor clomipramine were subjected to ADMET, QikProp and molecular mechanics, the generalized born model and solvent accessibility study to ensure the toxicity caused by compounds and binding-free energy, respectively. Two best ligands (FFG7 and FFG2) qualifying above screening parameters were further subjected to molecular dynamics simulation. Conducting these studies, here we confirmed that 6-chloro-3-[3-(3-hydroxy-2-piperidyl)-2-oxo-propyl]-7-(4-pyridyl) quinazolin-4-one can be potential drug candidate to fight against Leishmania donovani parasites.     

Patterns of subspecies genetic diversity among oilseed <Emphasis Type="Italic">Brassica rapa</Emphasis> as revealed by agro-morphological traits and SSR markers

Brassica rapa (2n = 20, AA genome) is an important oil yielding species of the family Brassicaceae and characterized by wide range of genetic and morphological subtypes suitable for cultivation under diverse agro-climatic regions of India. In this study, genetic diversity among three subspecies of B. rapa including yellow sarson, toria and outlier brown sarson was estimated using various agro-morphological traits and simple sequence repeat (SSR) markers. Maximum variability was recorded for siliqua angle (Coefficient of variation = 30.9%), followed by seeds/siliqua (CV = 18.8%), leaf length (CV = 10%) and plant height (CV = 16.8%). Principal component analysis explained more than 50% of the total observed morphological variability for first two components. Of the 107 SSR markers tested, 80 generated reproducible, clear and distinct amplicons of which, 65 (81.25%) were found polymorphic. The number of alleles at each locus ranged from 2 to 7, with an average of 3.03 alleles per marker. A total of 197 alleles were detected at 65 SSR loci with average PIC value of 0.457 and a mean resolving power of 3.04. Neighbor-Joining cluster analysis based on morphological traits and SSR markers separately classified all the 28 genotypes into five major groups. The population structure analysis resulted into three sub-populations with certain extent of admixture among the earlier established taxonomic sub-groups. Twenty-three unique alleles were detected in thirteen B. rapa varieties. The clustering analysis and principal coordinate analysis outlined the genetic relationships among different varieties belonging to the three subspecies of B. rapa. Genetically diverse genotypes as illustrated by score plots and from the clustering patterns brought out the wide range of diversity present among B. rapa genotypes and the underlying options available for selecting parental genotypes for hybridization and developing high yielding cultivars suitable for Indian conditions.     

Mechanism of electron transport during thiosulfate oxidation in an obligately mixotrophic bacterium Thiomonas bhubaneswarensis strain S10 (DSM 18181T)

This study describes the thiosulfate-supported respiratory electron transport activity of Thiomonas bhubaneswarensis strain S10 (DSM 18181^T). Whole-genome sequence analysis revealed the presence of complete sox (sulfur oxidation) gene cluster (soxCDYZAXB) including the sulfur oxygenase reductase (SOR), sulfide quinone reductase (SQR), sulfide dehydrogenase (flavocytochrome c (fcc)), thiosulfate dehydrogenase (Tsd), sulfite dehydrogenase (SorAB), and intracellular sulfur oxidation protein (DsrE/DsrF). In addition, genes encoding respiratory electron transport chain components viz. complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (ubiquinone-cytochrome c reductase), and various types of terminal oxidases (cytochrome c and quinol oxidase) were identified in the genome. Using site-specific electron donors and inhibitors and by analyzing the cytochrome spectra, we identified the shortest thiosulfate-dependent electron transport chain in T. bhubaneswarensis DSM 18181^T. Our results showed that thiosulfate supports the electron transport activity in a bifurcated manner, donating electrons to quinol (bd) and cytochrome c (Caa ₃ ) oxidase; these two sites (quinol oxidase and cytochrome c oxidase) also showed differences in their phosphate esterification potential (oxidative phosphorylation efficiency (P/O)). Further, it was evidenced that the substrate-level phosphorylation is the major contributor to the total energy budget in this bacterium.

     

Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs

The internal isopeptide bonds are amide bonds formed autocatalytically between the side chains of Lys and Asn/Asp residues and have been discovered recently. These bonds are well conserved in Gram-positive bacterial pilin proteins and are also observed over a wide range of Gram-positive bacterial surface proteins. The presence of these bonds confers the pilus subunits with remarkable properties in terms of thermal stability and resistance to proteases. Like pili, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) are also surface proteins found only in Gram-positive bacteria. They specifically interact with the extracellular matrix (ECM) molecules like collagen, fibrinogen, fibronectin, laminin, etc. Many biophysical and biochemical studies have been carried out to characterize the isopeptide bonds in pili proteins from Gram-positive bacteria, but no attempts have been made to study the isopeptide bonds in MSCRAMMs. This short review aims to study the significance of the isopeptide bonds in relation to their function, by analyzing the crystal structures of collagen- and fibrinogen-binding MSCRAMMs. In this analysis, interestingly, we observed that the putative isopeptide bonds are restricted to the collagen-binding MSCRAMMs. Based on analogy with bacterial pilus subunits, we hypothesize that the collagen-binding MSCRAMMs possessing putative isopeptide bonds exhibit similar structural properties, which could help the bacteria in colonizing the host and provide resistance against host–defense mechanisms.     

The effect of human population size on the breeding bird diversity of urban regions

Most studies of the effects of urbanization on avian diversity are carried out in a single urban area. However, urban areas vary in characteristics, such as human population size and biophysical context, that likely influence within-urban area ecological structure and functioning. In this paper, we describe the first direct test of the effect of urban area human population size on bird diversity. We applied simultaneous autoregressive modeling and multi-model inference to North American Breeding Bird Survey and US Census data from 48 urban areas in the conterminous United States to determine the effect of urban area human population size on total breeding bird species richness and abundance in surrounding urban regions, controlling for variation in elevation, air temperature, precipitation, urban area age, human population density, and original habitat type. We hypothesized that increasing urban area human population size is a driver of increasing regional habitat loss and fragmentation and disturbance and therefore would have a negative effect on breeding bird species richness and abundance in regions. We found strong evidence for a negative effect of urban area human population size on species richness and show that urban area human population size is positively correlated with impervious surface cover and air pollutant emissions in urban regions, lending provisional support to our hypothesis. Our results imply that we prioritize urban regions surrounding urban areas with large human population sizes for conservation activities that will benefit birds, such as preserving green spaces. In future, greater emphasis should be placed on the acquisition of high-quality ecological data in multiple urban areas in order to increase our understanding of the structure and functioning of complex urban systems, a frontier in urban ecology.     

Identification of the phosphorylation sites in the survival motor neuron protein by protein kinase A

The survival motor neuron (SMN) protein plays an essential role in the assembly of uridine-rich small nuclear ribonuclear protein complexes. Phosphorylation of SMN can regulate its function, stability, and sub-cellular localization. This study shows that protein kinase A (PKA) phosphorylates SMN both in vitro and in vivo. Bioinformatic analysis predicts 12 potential PKA phosphorylation sites in human SMN. Mass spectrometric analysis of a tryptic digest of SMN after PKA phosphorylation identified five distinct phosphorylation sites in SMN (serines 4, 5, 8, 187 and threonine 85). Mutagenesis of this subset of PKA-phosphorylated sites in SMN affects association of SMN with Gemin2 and Gemin8. This result indicates that phosphorylation of SMN by PKA may play a role in regulation of the in vivo function of SMN.