Development of a pilot-scale production process and characterization of a recombinant Japanese encephalitis virus envelope domain III protein expressed in Escherichia coli

Japanese encephalitis virus (JEV) is the most important cause of encephalitis in most Asian regions. JEV envelope domain III (JEV EDIII) protein is involved in binding to host receptors, and it contains specific epitopes that elicit virus-neutralizing antibodies. A highly immunogenic, recombinant JEV EDIII protein was expressed in Escherichia coli. In order to take this vaccine candidate for further studies, recombinant JEV EDIII protein was produced employing a pilot-scale fermentation process. Recombinant JEV EDIII protein expressed as inclusion bodies (IBs) was solubilized in 8?M urea and renatured by on-column refolding protocol in the presence of glycerol. A three-step purification process comprising of affinity chromatography, ion-exchange chromatography (IEX) based on salt, and IEX based on pH was developed. About ~124?mg of highly purified and biologically active EDIII protein was obtained from 100?g of biomass. Biological function of the purified EDIII protein was confirmed by their ability to generate EDIII-specific antibodies in mice that could neutralize the virus. These findings suggest that recombinant JEV EDIII protein in combination with compatible adjuvant is highly immunogenic and elicit high-titer neutralizing antibodies. Thus, recombinant JEV EDIII protein produced at large scale can be a potential vaccine candidate.     

Biodegradation of oil in oily sludges from steel mills

Lab-scale batch studies were conducted to determine the biodegradability of oil associated with oily sludge from a steel mill using two microbial cultures enriched in the laboratory. After 60 days of biodegradation the residual oil content in mill sludge was reduced from 4.5-5% to 2.7-3.0%, corresponding to 40-45% loss with respect to initial. The rate of degradation was different for the two enrichment cultures studied. Significant loss of oil was observed in the un-inoculated controls while loss in the azide killed controls was negligible. Bioavailability limitations and the presence of structurally complex high molecular weight hydrocarbons in lubricating oil are responsible for the slow rate of degradation. Significant loss of oil in un-inoculated controls indicated the presence of indigenous microorganisms in oily mill sludge. The association of biomass with sludge solids and presence of a high level of residual oil may adversely affect the recyclability of iron-fines associated with the sludge.     

Comparative Study of Propranolol hydrochloride Release from Matrix Tablets with Kollidon®SR or Hydroxy Propyl Methyl Cellulose

The release of propranolol hydrochloride from matrix tablets with hydroxy propyl methyl cellulose (HPMC K15M) or KollidonSR at different concentrations was investigated with a view to developing twice daily sustained release dosage form. A hydrophilic matrix-based tablet using different concentrations of HPMC K15M or KollidonSR was developed using direct compression technique to contain 80 mg of propranolol hydrochloride. The resulting matrix tablets prepared with HPMC K15M or KollidonSR fulfilled all the official requirements of tablet dosage forms. Formulations were evaluated for the release of propranolol hydrochloride over a period of 12 h in pH 6.8 phosphate buffer using USP type II dissolution apparatus. Propranolol hydrochloride and pure KollidonSR or HPMC K15M compatibility interactions was investigated by using Fourier-transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). FTIR spectroscopic and DSC studies revealed that there was no well defined chemical interaction between propranolol hydrochloride with KollidonSR or HPMC K15M. Tablets were exposed to 40 degrees C/75% of RH in open disc for stability. The in vitro drug release study revealed that HPMC K15 at a concentration of 40% of the dosage form weight was able to control the release of propranolol hydrochloride for 12 h, exhibit non-Fickian diffusion with first-order release kinetics where as at 40% KollidonSR same dosage forms show zero-order release kinetics. In conclusion, the in vitro release profile and the mathematical models indicate that release of propranolol hydrochloride can be effectively controlled from a single tablet using HPMC K15M or KollidonSR matrix system.     

Biotic stress induced demolition of thylakoid structure and loss in photoelectron transport of chloroplasts in papaya leaves.

Papaya mosaic virus (PMV) causes severe mosaic symptoms in the papaya (Carica papaya L.) leaves. The PMV-induced alterations in photosystem II (PS II) structure and photochemical functions were probed. An increase in chlorophyll a (Chl a) fluorescence polarization suggests pathogen-induced transformation of thylakoid membrane to a gel phase. This transformation in physical state of thylakoid membrane may result in alteration in topology of pigments on pigment-binding proteins as reflected in pathogen-induced loss in the efficiency of energy transfer from carotenoids to chlorophylls. The fast Chl a fluorescence induction kinetics of healthy and PMV-infected plants by F(O)-F(J)-F(I)-F(P) transients revealed pathogen-induced perturbation on PS II acceptor side electron transfer equilibrium between Q(A) and Q(B) and in the pool size of electron transport acceptors. Pathogen-induced loss in photosynthetic pigments, changes in thylakoid structure and decrease in the ratio of F(V)/F(M) (photochemical potential of PS II) further correlate with the loss in photoelectron transport of PS II as probed by 2,6-dichlorophenol indophenol (DCPIP)-Hill reaction. Restoration of the loss by 1,5-diphenyl carbazide (DPC), an exogenous electron donor, that donates electron directly to reaction centre II bypassing the oxygen evolving system (OES), leads towards the conclusion that OES is one of the major targets of biotic stress. Further, the data suggest that chlorophyll fluorescence could be used as a non-invasive handy tool to assess the loss in photosynthetic efficiency and symptom severity in infected green tissues vis-a-vis the healthy ones.     

Interactions of a few azole derivatives with a transport protein: role of heteroatoms

The interaction of a few azole derivatives, 2‐(4′‐N,N‐dimethylaminophenyl)benzimidazole, 2‐(4′‐N,N‐dimethylaminophenyl)benzoxazole, 2‐(4′‐N,N‐dimethylaminophenyl)oxazolo[4,5‐b]pyridine with bovine serum albumin (BSA) were examined by absorption and fluorescence spectroscopy. The results were compared with the previously studied imidazopyridine derivative 2‐(4′‐N,N‐dimethylaminophenyl)imidazo[4,5‐b]pyridine. Displacement studies were carried out with site selective probes to locate the binding site of these ligands. The spectral shifts and the binding constant vary depending on the nature of the ligand. The fluorescence intensity of both oxazole derivatives 2‐(4′‐N,N‐dimethylaminophenyl)benzoxazole and 2‐(4′‐N,N‐dimethylaminophenyl) oxazolo[4,5‐b]pyridine increases substantially in the presence of BSA, whereas the intensity of 2‐(4′‐N,N‐dimethylaminophenyl)benzimidazole decreases. However, hypsochromic shift is observed in presence of BSA. The results obtained from the docking studies are also in good agreement with the experimental results. The location and orientation of binding depend upon the nature of the ligand. The studies revealed that apart from hydrophobic interaction, hydrogen bonding also plays a vital role in the molecular binding. Oxazoles have higher binding affinity than imidazoles and substitution of extra nitrogen further increases the binding affinity. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of a triazole scaffold compound as an inhibitor of Mycobacterium tuberculosis imidazoleglycerol-phosphate dehydratase

Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), employs ten enzymes including imidazoleglycerol-phosphate dehydratase (IGPD) for de novo biosynthesis of histidine. The absence of histidine-biosynthesis in humans combined with its essentiality for Mtb makes the enzymes of this pathway major anti-TB drug targets. We explored the inhibitory potential of a small molecule β-(1,2,4-Triazole-3-yl)-DL-alanine (DLA) against Mtb IGPD. DLA exhibits an in vitro inhibitory efficacy in the lower micromolar range. Higher-resolution crystal structures of native and substrate-bound Mtb IGPD provided additional structural features of this important drug target. Crystal structure of IGPD-DLA complex at a resolution of 1.75 Å, confirmed that DLA locks down the function of the enzyme by binding in the active site pocket of the IGPD mimicking the substrate-binding mode to a high degree. In our biochemical study, DLA showed an efficient inhibition of Mtb IGPD. Furthermore, DLA also showed bactericidal activity against Mtb and Mycobacterium smegmatis and inhibited their growth in respective culture medium. Importantly, owing to the favorable ADME and physicochemical properties, it serves as an important lead molecule for further derivatizations.     

Cadmium Free Cu2ZnSnS4 Solar Cells with 9.7% Efficiency

Cu₂ZnSnS₄(CZTS) thin‐film solar cell absorbers with different bandgaps can be produced by parameter variation during thermal treatments. Here, the effects of varied annealing time in a sulfur atmosphere and an ordering treatment of the absorber are compared. Chemical changes in the surface due to ordering are examined, and a downshift of the valence band edge is observed. With the goal to obtain different band alignments, these CZTS absorbers are combined with Zn_1?xSn_xO_y (ZTO) or CdS buffer layers to produce complete devices. A high open circuit voltage of 809 mV is obtained for an ordered CZTS absorber with CdS buffer layer, while a 9.7% device is obtained utilizing a Cd free ZTO buffer layer. The best performing devices are produced with a very rapid 1 min sulfurization, resulting in very small grains.