Probing the amino acids critical for protein oligomerisation and protein–nucleotide interaction in Mycobacterium tuberculosis PII protein through integration of computational and experimental approaches

We investigated the interacting amino acids critical for the stability and ATP binding of Mycobacterium tuberculosis PII protein through a series of site specific mutagenesis experiments. We assessed the effect of mutants using glutaraldehyde crosslinking and size exclusion chromatography and isothermal titration calorimetry. Mutations in the amino acid pair R60–E62 affecting central electrostatic interaction resulted in insoluble proteins. Multiple sequence alignment of PII orthologs displayed a conserved pattern of charged residues at these positions. Mutation of amino acid D97 to a neutral residue was tolerated whereas positive charge was not acceptable. Mutation of R107 alone had no effect on trimer formation. However, the combination of neutral residues both at positions 97 and 107 was not acceptable even with the pair at 60–62 intact. Reversal of charge polarity could partially restore the interaction. The residues including K90, R101 and R103 with potential to form H-bonds to ATP are conserved throughout across numerous orthologs of PII but when mutated to Alanine, they did not show significant differences in the total free energy change of the interaction as examined through isothermal titration calorimetry. The ATP binding pattern showed anti-cooperativity using three-site binding model. We observed compensatory effect in enthalpy and entropy changes and these may represent structural adjustments to accommodate ATP in the cavity even in absence of some interactions to perform the requisite function. In this respect these small differences between the PII orthologs may have evolved to suite species specific physiological niches.     

Salinity-Induced Attenuation in Secondary Metabolites Profile and Herbicidal Potential of Brassica nigra L. on Anagallis arvensis L.

Studies on plant defense mechanisms in stressful conditions predict that plant cope up with increasing stress for survival. Under environmental stress plants interact with problems like competition and survival under resource constraints or utilization of these resources in production of secondary compounds. In this experiment, we examined the costs of defense by evaluating variation in production of secondary compounds of Brassica nigra grown in the saline (B1?=?100 mM NaCl and B2?=?150 mM NaCl) and control (C?=?0 mM) soils and impact of its extracts on weed Anagallis arvensis L. The main allelopathic compounds in Brassica were determined using gas chromatography–mass spectroscopy (GC–MS). The results indicated that A. arvensis was adversely affected by the aqueous extracts of B. nigra. These observations might be related to extracts induced oxidative stress indicated by superoxide (O₂^?) production through nitroblue tetrazolium, cell integrity by Evans blue staining, malondialdehyde estimation by Schiff reagent, lipid peroxidation, lignin deposition, and antioxidant enzyme activities assay. We observed that soil salinity reduced the phytotoxicity of aqueous extract and decreased its potential. The presence of different bioactive compounds improved the natural herbicidal properties of B. nigra and it can be used in various medicinal and agricultural practices.

     

Cloning, In Silico Characterization and Prediction of Three Dimensional Structure of SbDof1, SbDof19, SbDof23 and SbDof24 Proteins from Sorghum [Sorghum bicolor (L.) Moench]

In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.     

Building an efficient intrusion detection system using grasshopper optimization algorithm for anomaly detection

Cluster Computing - Intrusion detection is one of the most crucial activities for security infrastructures in network environments, and it is widely used to detect, identify and track malicious...     

Evidence for Serpentine as a novel antioxidant by a redox sensitive HABP1 overexpressing cell line by inhibiting its nuclear translocation of NF-κB

Herbal antioxidants are gradually gaining importance as dietary supplements considering the growing implications of oxidative stress in most degenerative diseases and aging. Thus, continuous attempts are made to search for novel herbal molecules with antioxidative properties, using chemical methods predominantly with the need arising for cell based assays. We have generated a stable cell line F-HABP07, by constitutively overexpressing human Hyaluronan Binding Protein1 (HABP1) in murine fibroblasts which accumulates in the mitochondria leading to excess ROS generation without any external stimuli. In the present study, we demonstrated the nuclear translocation of p65 subunit of NF-κB in F-HABP07 cells, an important signature of ROS induced signalling cascade providing us an opportunity to use it as a screening system for ROS scavengers. Using known antioxidants on our designer cell line, we have demonstrated a dose dependant reduction in ROS generation and observed inhibition of p65 subunit of NF-κB nuclear translocation, increase in glutathione content and down-regulation of apoptotic marker Bax establishing its antioxidant biosensing capacity. With the help of this cell line, we for the first time demonstrated serpentine, one of the active components from the roots of Rauwolfia serpentina (a traditional medicinal plant), to be a novel non-cytotoxic antioxidant. The authenticity of this cell line screening system based discovery was validated using standard chemical assays thus, opening up new therapeutic avenues for this herbal compound and the use of this designer cell line.     

Piperine Attenuates Cardiovascular, Liver and Metabolic Changes in High Carbohydrate, High Fat-Fed Rats

Black pepper is used worldwide to enhance food flavor. We investigated dietary supplementation with piperine, the active principle of black pepper, to high carbohydrate, high fat (HCHF) diet-fed rats as a model of human metabolic syndrome. Rats were fed with either HCHF diet (carbohydrate, 52%; fat, 24%; 25% fructose in drinking water) or cornstarch (CS) diet for a total of 16 weeks. Diets of the treatment groups (CS + piperine and HCHF + piperine) were supplemented with piperine for the last 8 weeks of this protocol. After 16 weeks, rats fed with HCHF diet developed hypertension, elevated oxidative stress and inflammation-induced cardiac changes (infiltration of inflammatory cells in heart, increase in count and degranulation of mast cells in heart, cardiac fibrosis and increase in ventricular stiffness), reduced responsiveness of aortic rings, impaired glucose tolerance, abdominal obesity together with liver fibrosis, fat deposition and increased plasma liver enzymes. Supplementation with piperine (375 mg/kg food; approximately 30 mg/kg/day) in HCHF-fed rats normalized blood pressure, improved glucose tolerance and reactivity of aortic rings, reduced plasma parameters of oxidative stress and inflammation, attenuated cardiac and hepatic inflammatory cell infiltration and fibrosis and improved liver function. These changes clearly suggest that piperine reduces symptoms of human metabolic syndrome in HCHF-fed rats by reducing inflammation and oxidative stress.     

Bystander-type effects mediated by long-lived inflammatory signaling in irradiated bone marrow

Radiation-induced bystander and abscopal effects, in which DNA damage is produced in nonirradiated cells as a consequence of communication with irradiated cells, indicate mechanisms of inducing damage and cell death additional to the conventional model of deposition of energy in the cell nucleus at the time of irradiation. In this study we show that signals generated in vivo in the bone marrow of mice irradiated with 4 Gy γ rays 18 h to 15 months previously are able to induce DNA damage and apoptosis in nonirradiated bone marrow cells but that comparable signals are not detected at earlier times postirradiation or at doses below 100 mGy. Bone marrow cells of both CBA/Ca and C57BL/6 genotypes exhibit responses to signals produced by either irradiated CBA/Ca or C57BL/6 mice, and the responses are mediated by the cytokines FasL and TNF-α converging on a COX-2-dependent pathway. The findings are consistent with indirect inflammatory signaling induced as a response to the initial radiation damage rather than to direct signaling between irradiated and nonirradiated cells. The findings also demonstrate the importance of studying tissue responses when considering the mechanisms underlying the consequences of radiation exposures.     

Suppression of DNA damage in human peripheral blood lymphocytes by a juice concentrate: a randomized, double-blind, placebo-controlled trial

Chronic inflammation contributes to many prevalent diseases worldwide, and it is widely accepted that inflammatory molecules contribute to DNA damage. In this ancillary study, we investigated the influence of an encapsulated fruit and vegetable juice powder concentrate on peripheral blood lymphocytes (PBL) DNA damage. Using a double-blind, placebo-controlled approach, subjects were randomly assigned capsules containing placebo, or one of two formulations of the juice powder. Blood was drawn at baseline and after 60 days of capsule consumption. We found DNA damage in isolated PBL is suppressed after consumption of the encapsulated juice powder, and damage was correlated with the level of systemic inflammation. These data suggest a potential health benefit by consuming the juice concentrate capsules through their ability to suppress DNA damage as measured in surrogate tissues (PBL).     

In silico prediction of drug resistance due to S247R mutation of Influenza H1N1 neuraminidase protein

We present here in silico studies on antiviral drug resistance due to a novel mutation of influenza A/H1N1 neuraminidase (NA) protein. Influenza A/H1N1 virus was responsible for a recent pandemic and is currently circulating among the seasonal influenza strains. M2 and NA are the two major viral proteins related to pathogenesis in humans and have been targeted for drug designing. Among them, NA is preferred because the ligand-binding site of NA is highly conserved between different strains of influenza virus. Different mutations of the NA active site residues leading to drug resistance or susceptibility of the virus were studied earlier. We report here a novel mutation (S247R) in the NA protein that was sequenced earlier from the nasopharyngeal swab from Sri Lanka and Thailand in the year 2009 and 2011, respectively. Another mutation (S247N) was already known to confer resistance to oseltamivir. We did a comparative study of these two mutations vis-a-vis the drug-sensitive wild type NA to understand the mechanism of drug resistance of S247N and to predict the probability of the novel S247R mutation to become resistant to the currently available drugs, oseltamivir and zanamivir. We performed molecular docking- and molecular dynamics-based analysis of both the mutant proteins and showed that mutation of S247R affects drug binding to the protein by positional displacement due to altered active site cavity architecture, which in turn reduces the affinity of the drug molecules to the NA active site. Our analysis shows that S247R may have high probability of being resistant.