Directed evolution of a recombinase for improved genomic integration at a native human sequence

We previously established that a unidirectional site-specific recombinase, the phage C31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.     

Anti-inflammatory potential of an ethyl acetate fraction isolated from Justicia gendarussa roots through inhibition of iNOS and COX-2 expression via NF-κB pathway

Justicia gendarussa Burm.f. (J. gendarussa) is a plant used as traditional medicine in different parts of India and China to treat inflammatory disorders like rheumatoid arthritis. But its mechanism of anti-inflammatory action is still unclear. Hence in this context, the objective of our study is to reveal the mechanism of anti-inflammatory activity of J. gendarussa which would form an additional proof to the traditional knowledge of this plant. The anti-inflammatory function and mechanism(s) of action was studied in an ethyl acetate fraction isolated from methanolic extract of J. gendarussa roots (EJG). Anti-inflammatory studies were conducted on rats using partitioned fractions isolated from methanolic extract of J. gendarussa roots. In carrageenan-induced rat paw edema, ethyl acetate fraction brought about 80% and 93% edema inhibition at 3rd and 5th hour at a dose of 50 mg/kg, when compared to other extracts and Voveran. We investigated whether EJG inhibits the release of cycloxygenase (COX), 5-lipoxygenase (5-LOX), interleukin-6 (IL-6) and nuclear factor kappa B (NF-κB) in LPS stimulated human peripheral blood mononuclear cells (hPBMCs). Results shows that EJG dose dependently inhibited LPS-activated COX, 5-LOX, IL-6, and NF-κB in hPBMCs. EJG also reduced LPS induced levels of iNOS and COX-2 mRNA expression in hPBMCs. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory activity of EJG and therefore, we report the first confirmation of the anti-inflammatory potential of this traditionally employed herbal medicine in vitro.     

DNA template-assisted modulation of horseradish peroxidase activity

The precise alignment of DNA molecules by Watson-Crick base-pairing combined with its polymeric characteristics have allowed DNA to be used as a template or scaffold for assembling materials. In this work, we investigate the role of calf-thymus DNA as a template for enhancing the horseradish peroxidase (HRP)-mediated oxidation of phenol and phenolic derivatives. The HRP-catalyzed oxidation of phenol into polyphenolic products and in presence of 4-aminoantipyrine into quinoneimine dye complexes is studied. Visible spectroscopy reveals an increased yield of both products of the enzymatic reaction in presence of calf-thymus DNA and is attributed to the prearrangement of the corresponding substrates on the DNA. The concentrations of calf-thymus DNA and the substrates are found to affect the nature of prearrangement and subsequent formation of polymeric or co-oxidation products. Also, phenolic derivatives with different aromatic substitutions display divergent propensities towards product formation in presence of the DNA template. Our results demonstrate the ability of calf-thymus DNA to modulate the activity of HRP and exercise control on the nature of products formed. This work highlights the potential of using DNA as a template for influencing enzymatic reactions involving aromatic substrates.     

Combined Effect of Enterocin and Lipase From Enterococcus faecium NCIM5363 Against Food Borne Pathogens: Mode of Action Studies

Food borne diseases have a major impact on public health whose epidemiology is rapidly changing. The whole cells of pathogens involved or their toxins/metabolites affect the human health apart from spoiling sensory properties of the food products finally affecting the food industry as well as consumer health. With pathogens developing mechanisms of antibiotic resistance, there has been an increased need to replace antibiotics as well as chemical additives with naturally occurring bacteriocins. Bacteriocins are known to act mainly against Gram-positive pathogens and with little or no effect towards Gram-negative enteric bacteria. In the present study, combination effect of lipase and bacteriocin produced by Enterococcus faecium NCIM5363, a highly lipolytic lactic acid bacterium against various food pathogens was assessed. The lipase in combination with enterocin exhibited a lethal effect against Gram-negative pathogens. Scanning electron microscopy studies carried out to ascertain the constitutive mode of action of lipase and enterocin revealed that the lipase degrades the cell wall of Gram-negative bacteria and creates a pore through which enterocin enters thereby resulting in cell death. The novelty of this work is the fact that this is the first report revealing the synergistic effect of lipase with enterocin against Gram-negative bacteria.     

Molecular modeling,docking and ADMET studies towards development of novel Disopyramide analogs for potential inhibition of human voltage gated sodium channel proteins

The sodium “channelopathies” are the first among the ion channel diseases identified and have attracted widespread clinical and scientific interests. Human voltage gated sodium channels are sites of action of several antiarrhythmic drugs, local anesthetics and related antiepileptic drugs. The present study aims to optimize the activity of Disopyramide, by modification in its structures which may improve the drug action by reducing its side effects. Herein, we have selected Human voltage-gated sodium channel protein type 5 as a potent molecular target. Nearly eighty analogs of Disopyramide are designed and optimized. Thirty are selected for energy minimization using Discovery studio and the LigPrep 2.5. Prior to docking, the active sites of all the proteins are identified. The processing, optimization and minimization of all the proteins is done in Protein preparation wizard. The docking study is performed using the GLIDE. Finally top five ranked lead molecules with better dock scores are identified as having strong binding affinity to 2KAV protein than Disopyramide based on XP G scores. These five leads are further docked with other similar voltage gated sodium channel proteins (PDB IDs: 2KBI, 4DCK, 2L53 and 4DJC) and the best scoring analog with each protein is identified. Drug likeliness and comparative bioactivity analysis for all the analogs is done using QikProp 3.4. Results have shown that the top five lead molecules would have the potential to act as better drugs as compared to Disopyramide and would be of interest as promising starting point for designing compounds against various Sodium channelopathies.     

Cytoskeletal elements and intracellular transport

Recent advances in the understanding of the functions of various components of the cytoskeleton indicate that, besides serving a structural role, the cytoskeletal elements may regulate the transport of several proteins in the cell. Studies reveal that there are co-operative interactions between the actin and microtubule cytoskeletons including functional overlap in the transport influenced by different motor families. Multiple motors are probably involved in the control of the dynamics of many proteins and intriguing hints about how these motors are co-ordinated are appearing. It has been shown that some of the intermediate elements also participate in selected intracellular transport mechanisms. In view of the author's preoccupation with the steroid receptor systems, special attention has been given to the role of the cytoskeletal elements, particularly actin, in the intracellular transport of steroid receptors and receptor-related proteins.     

A Computationally Efficient Correlational Neural Network for Automated Prediction of Chronic Kidney Disease

ObjectivesIn this paper, we propose a computationally efficient Correlational Neural Network (CorrNN) learning model and an automated diagnosis system for detecting Chronic Kidney Disease (CKD). A Support Vector Machine (SVM) classifier is integrated with the CorrNN model for improving the prediction accuracy.Material and methodsThe proposed hybrid model is trained and tested with a novel sensing module. We have monitored the concentration of urea in the saliva sample to detect the disease. Experiments are carried out to test the model with real-time samples and to compare its performance with conventional Convolutional Neural Network (CNN) and other traditional data classification methods.ResultsThe proposed method outperforms the conventional methods in terms of computational speed and prediction accuracy. The CorrNN-SVM combined network achieved a prediction accuracy of 98.67%. The experimental evaluations show a reduction in overall computation time of about 9.85% compared to the conventional CNN algorithm.ConclusionThe use of the SVM classifier has improved the capability of the network to make predictions more accurately. The proposed framework substantially advances the current methodology, and it provides more precise results compared to other data classification methods.