Isothermal titration calorimetry and surface plasmon resonance analysis using the dynamic approach

Biophysical techniques such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) are routinely used to ascertain the global binding mechanisms of protein-protein or protein-ligand interaction. Recently, Dumas etal, have explicitly modelled the instrument response of the ligand dilution and analysed the ITC thermogram to obtain kinetic rate constants. Adopting a similar approach, we have integrated the dynamic instrument response with the binding mechanism to simulate the ITC profiles of equivalent and independent binding sites, equivalent and sequential binding sites and aggregating systems. The results were benchmarked against the standard commercial software Origin-ITC. Further, the experimental ITC chromatograms of 2′-CMP + RNASE and BH3I-1 + hBCL_XL interactions were analysed and shown to be comparable with that of the conventional analysis. Dynamic approach was applied to simulate the SPR profiles of a two-state model, and could reproduce the experimental profile accurately.     

Biogenic approach to synthesize rod shaped Gd2O3 nanoparticles and its optimization using response surface methodology-Box–Behnken design model

The rare earth metal oxide nanoparticles such as gadolinium oxide nanoparticles (Gd₂O₃ NPs) have been synthesized by green synthesis process using methanolic extract of Moringa oleifera (M oleifera) peel. In this process, the Gd₂O₃ NPs formation was observed at 280–300 nm in UV–Vis spectroscopy. The XRD pattern of the synthesized Gd₂O₃ NPs was exactly matched with JCPDS No 3-065-3181which confirms the crystalline nature of Gd₂O₃ NPs. In addition, Energy-dispersive X-ray spectroscopy (EDX) analysis was stated that Gd and O elements were present as 70.31 and 29.69%, respectively in Gd₂O₃ NPs. The SEM and TEM analysis were said Gd₂O₃ NPs are in rod shape and 26 ± 2 nm in size. Further the synthesized Gd₂O₃ NPs were confirmed by X-ray photoemission spectroscopy (XPS). The synthesized Gd₂O₃ NPs were further examined for anti-fungal activity against Alternaria saloni (A saloni) and Sclerrotium rolfsii (S rolfsii) and it showed moderate activity. Also, Gd₂O₃ NPs evaluated as good antibacterial agent against different Gram +ve and Gram −ve bacteria. Moreover, the toxicity of the Gd₂O₃ NPs on red blood cells (RBCs) of the human blood was determined using hemolytic assay, the obtained results were stated the synthesized Gd₂O₃ NPs are nontoxic to the human erythrocytes. The photocatalytic activity against malachite green (MG) dye was tested and confirmed as 92% of dye was degraded within 2 hr by Gd₂O₃ NPs. The results were stated the green synthesized Gd₂O₃ NPs are good anti-fungal agents, nontoxic and we can use as a photocatalyst. Copyright © 2019 John Wiley & Sons, Ltd.     

RAPD, SCAR and conserved 18S rDNA markers for a red-listed and endemic medicinal plant species, Knema andamanica (Myristicaceae)

Knema andamanica is a red-listed endemic medicinal species of Myristicaceae restricted to Andaman and Nicobar (A&N) Islands, India. This species is used in tribal medicines and has immense bioprospective potential. With a view to generate suitable genomic markers for classification and identification, we have generated RAPD, SCAR and conserved 18S rDNA markers from K. andamanica. A unique 585 bp fragment, that distinguished it from seven other related species of Myristicaceae was first amplified using the random primer OPE 06 and converted to SCAR marker (GenBank accession # JN228256). The conserved sequences of 18S rDNA loci from K. andamanica were also amplified and sequenced (GenBank accession #JN228265). The sequence revealed deviations including 18 variable regions and 15 indels that were unique to K. andamanica. These markers can help in definite identification of K. andamanica even at the juvenile stages.     

Cloning of hok gene into anhydrotetracycline inducible pASK75 vector reveals potent antimicrobial effect of 19 amino acid long N-terminal fragment of hok peptide

An important toxin-antitoxin (TA) system hok/sok, encoded by R1 plasmid of Escherichia coli, is involved in the post segregation killing of cells that have lost the plasmid. The lethal properties of hok protein have been utilized for the environmental containment of microbes and the development of potential vaccine candidates. This study aimed to demonstrate the potent anti-microbial property of a 19 amino acid (AA) long N-terminal fragment of hok peptide. This was accomplished by designing a conditional suicide system based on hok gene expression cloned in an anhydrotetracycline (aTc) inducible vector – pASK75. Heat shock and electroporation were utilized for the transformation of Escherichia coli and Vibrio cholerae cells, respectively. The minimal induction concentration (MI_dC) of aTc, determined by analyzing the expression of green fluorescent protein cloned separately into pASK75 vector, was 30 ng/mL. As hok gene was synthesized de novo (using recombinant polymerase chain reaction) in our study, various random sized hok fragments were generated (as a result of the error-prone nature of Taq polymerase). The smallest hok fragment able to bring about effective antimicrobial killing was a 19 AA long N-terminal fragment of hok having the wild type sequence, except for the carboxy terminus AA residue. The MI_dC of aTc in our experiments was 6-fold lower than previously reported, making our bacterial clones suitable for use in mammalian systems as potential vaccine candidates. Based on our experiments, we hypothesize the 19 AA long N-terminal fragment of hok peptide to be the smallest possible hok fragment sufficient to bring about effective antimicrobial killing.     

Scoring function to predict solubility mutagenesis

Background  

Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT) protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention.     

Monosaccharides bound to hemoglobins in normal and diabetic individuals. Evidence for glucose, mannose and galactose as sugars released by methanolysis of the different hemoglobin components

Direct evidence is given for the presence of glucose, mannose and galactose as the products of hydrolysis of hemoglobins A1a1, A1a2, A1b, A1c and A0. The presence of galactose cannot be explained by the earlier hypothesis of Amadori rearrangement and suggests the existence of further complex rearrangements. Monosaccharide content of the different hemoglobin components varies from 0.2-2.0 mol/mol of alpha beta dimer with an increase of 1.5-2.0-times in diabetic components. This increase is not accompanied by net charge differences, suggesting that additionally bound sugars are not responsible for the pI modification of these hemoglobins. The pattern of glucose, mannose and galactose ratio in normal individuals divides these hemoglobins into two classes, hemoglobins A1b, A1c and A0 (ratio 0.60:0.25:0.15) on one hand and hemoglobins A1a1 and A1a2 (ratio 0.40:0.40: 0.20) on the other. These findings suggest that diverse mechanisms for sugar binding might exist between these two classes of glycosylated hemoglobins. This difference disappears in diabetic components suggesting that the non-NH2-terminal sites are glycosylated in all components by a common mechanism. Increase in glucose at the expense of mannose and galactose, as observed in diabetics, could be an indicator of recent glycosylation.     

Recovery of two exotic parasites,Trichogramma brasiliensis [Hym.: Trichogrammatidae] andEucelatoria bryani [Dip.: Tachinidae] fromHeliothis armigera [Lep.: Noctuidae] in tomato fields

Heliothis armigera (Hübner) is a major pest of several crops especially tomato and pulses in India. Indigenous natural enemies likeCarcelia illota Curr.,Campoletis chlorideae Uchida,Trichogramma chilonis Ishii andHexamermis sp are unable to exert adequate control ofH. armigera in tomato. Field trials were conducted with 2 exotic parasites,Trichogramma brasiliensis (Ashmead) andEucelatoria bryani Sabrosky in tomato fields around Bangalore. Both the parasites were recovered fromH. armigera in tomato fields. Field parasitism inH. armigera byT. brasiliensis ranged from 34.6 to 51.3%. WithE. bryani, the parasitism ranged from 0.0 to 8.0%.     

Effect of aminophylline preperfusion on digoxin-induced cardiac arrest in isolated frog heart.

Digoxin (DGN) and aminophylline (theophylline ethylenediamine, APH) being frequently prescribed cardioactive drugs, the present study investigated the effect of APH (10(-4) M) preperfusion on DGN-cardiotoxicity employing the isolated frog heart preparation. The mean DGN perfusion time (sec) and mean DGN exposure (microgram/10 mg heart wt.) for cardiac arrest were the parameters studied. APH preperfusion caused a significant elevation in both the parameters, signifying that it afforded protection against DGN-cardiotoxicity. This protective effect was not observed with the preperfusion of ethylenediamine (EDA) instead of APH, which led to the inference that the protective effect of APH was solely due to its theophylline component. The present finding that APH-pretreatment might modulate DGN-cardiotoxicity, of considerable pharmaco-toxicological interest.