Production of scleroglucan from Sclerotium rolfsii MTCC 2156

Scleroglucan, a neutral homopolysaccaride consisting of a linear chain of beta-D-(1-3)-glucopyranosyl and beta-D-(1-6)-glucopyranosyl groups, was produced by pure culture fermentation from Sclerotium rolfsii MTCC 2156 by submerged culture. Fermentation process was optimized in two steps. In the first step, one-factor-at-a-time method was used to investigate the effects of medium constituents such as carbon and nitrogen sources. In the second step, concentration of medium components was optimized using an L16-orthogonal array method. In all, 10 different carbon sources and eight different nitrogen sources were evaluated. Maximum yield of 16.58 g/l was obtained in a medium containing sucrose as a carbon source and sodium nitrate and yeast extract as nitrogen source.     

Uncialamycin as a novel payload for antibody drug conjugate (ADC) based targeted cancer therapy

Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50?=?0.88?nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.     

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis–Menten kinetic parameters (k_m and V_max) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.     

Antimicrobial activity of <Emphasis Type="Italic">Alcaligenes sp</Emphasis>. HPC 1271 against multidrug resistant bacteria

Alcaligenes sp. HPC 1271 demonstrated antibacterial activity against multidrug resistant bacteria, Enterobacter sp., resistant to sulfamethoxazole, ampicillin, azithromycin, and tetracycline, as well as against Serratia sp. GMX1, resistant to the same antibiotics with the addition of netilmicin. The cell-free culture supernatant was analyzed for possible antibacterials by HPLC, and the active fraction was further identified by LC-MS. Results suggest the production of tunicamycin, a nucleoside antibiotic. The draft genome of this bacterial isolate was analyzed, and the 4.2 Mb sequence data revealed six secondary metabolite-producing clusters, identified using antiSMASH platform as ectoine, butyrolactone, phosphonate, terpene, polyketides, and nonribosomal peptide synthase (NRPS). Additionally, the draft genome demonstrated homology to the tunicamycin-producing gene cluster and also defined 30 ORFs linked to protein secretion that could also play a role in the antibacterial activity observed. Gene expression analysis demonstrated that both NRPS and dTDP-glucose 4,6-dehydratase gene clusters are functional and could be involved in antibacterial biosynthesis.     

Protein engineering and de novo designing of a biocatalyst

Proteins as a biomolecule have been recognized as a “molecule with manifold biological functions”. The functions not only include the structural, regulatory and transportation processes inside the body but also its capacity as an extremely specific catalyst for various biochemical reactions. Nature has been quite admirably using proteins as biocatalysts which are known as enzymes. Properties like higher reaction rate, good specificity, faster kinetics, production of lesser by‐products and their non‐hazardous nature make enzymes the most suitable targets for a process chemist to exploit. At the same time, limitations like a narrow range of substrates, requirement of coenzymes, lesser stability, smaller shelf‐life, along with difficulties in procuring these enzymes, make this biocatalysis field quite challenging. For exploiting a broad range of applications related to therapeutics, biosensors, biotechnology, nanotechnology etc., de novo designing of proteins is of utmost importance. Enzymes with altered, specific and modified properties might be designed by utilizing the prior knowledge of structure and function of a protein with the help of computational modeling. Various protein engineering techniques like directed evolution, rational designing and immobilization strategies etc. have already been extensively used to address some of the issues. This review aims to update the repertoire of the advancements in the field of protein engineering, which can help in laying some guiding principles about designing, modifying and altering their usage for commercial industrial purposes. This possibility of effective and novel designing of peptides and proteins might further facilitate our understanding about the structure, function and folding patterns along with their inter‐relationships. Copyright © 2016 John Wiley & Sons, Ltd.     

Evaluation of Curcumin Capped Copper Nanoparticles as Possible Inhibitors of Human Breast Cancer Cells and Angiogenesis: a Comparative Study with Native Curcumin

Synthesis of metal nanoparticles for improving therapeutic index and drug delivery is coming up as an attractive strategy in the mainstream of cancer therapeutic research. In the present study, curcumin-capped copper nanoparticles (CU-NPs) were evaluated as possible inhibitors of in vivo angiogenesis, pro-angiogenic cytokines involved in promoting tumor angiogenesis along with inhibition of cell proliferation and migration of breast cancer cell line MDA-MB-231. The antiangiogenic potential was assessed using in vivo chorioallantoic membrane (CAM) model. 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based cytotoxicity assay was used to assess the effect of CU-NPs against proliferation of breast cancer cell line. The wound healing migration assay was used to evaluate the effects of CU-NPs on the migration ability of breast cancer cell line. Native curcumin (CU) was used as a reference compound for comparison purpose. The result of the present investigation indicates that CU-NPs could not demonstrate impressive antiangiogenic or anticancer activities significantly as compared to native CU. The possible mechanisms of experimental outcomes are discussed in the light of the methods of nanoparticle synthesis in concert with the current state of the art literature.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

Microfluidic modeling of cell-cell interactions in malaria pathogenesis

The clinical outcomes of human infections by Plasmodium falciparum remain highly unpredictable. A complete understanding of the complex interactions between host cells and the parasite will require in vitro experimental models that simultaneously capture diverse host-parasite interactions relevant to pathogenesis. Here we show that advanced microfluidic devices concurrently model (a) adhesion of infected red blood cells to host cell ligands, (b) rheological responses to changing dimensions of capillaries with shapes and sizes similar to small blood vessels, and (c) phagocytosis of infected erythrocytes by macrophages. All of this is accomplished under physiologically relevant flow conditions for up to 20 h. Using select examples, we demonstrate how this enabling technology can be applied in novel, integrated ways to dissect interactions between host cell ligands and parasitized erythrocytes in synthetic capillaries. The devices are cheap and portable and require small sample volumes; thus, they have the potential to be widely used in research laboratories and at field sites with access to fresh patient samples.     

Estimation of carvedilol in human plasma by using HPLC-fluorescence detector and its application to pharmacokinetic study

A simple, precise and sensitive high performance liquid chromatography procedure has been developed for determination of carvedilol in human plasma. The method was developed on Lichrosphere R CN column using a mobile phase of acetonitrile/20 mM ammonium acetate buffer with 0.1% triethylamine (pH adjusted to 4.5) (40/60, v/v). The peaks were detected by using fluorescence detector (excitation wavelength 282 nm and emission wavelength 340 nm). Carvedilol and domperidone (internal standard) were extracted by liquid-liquid extraction procedure using dichloromethane. This method was specific and had a linearity range of 1-128 ng/ml with intra- and inter-day precision (%C.V.) less than 15%. The accuracy ranges from 87.3 to 100.88% and the recovery of carvedilol was 69.90%. The stability studies showed that carvedilol in human plasma was stable during short-term period for sample preparation and analysis. This method was used to assay the carvedilol in human plasma samples obtained from subjects who had been given an oral tablet of 12.5 mg carvedilol.