Improved methods for total and chloroplast protein extraction from Cajanus species for two-dimensional gel electrophoresis and mass spectrometry.

The recent advances in pigeon pea genomics, including high-quality whole genome and chloroplast genome sequence information helped develop improved varieties. However, a comprehensive Cajanus proteome, including the organelle proteome, is yet to be fully mapped. The spatial delineation of pigeon pea proteins at sub-cellular levels and inter-organelle communication could offer valuable insights into its defense mechanism against various stresses. However, the major bottleneck in the proteomic study is the lack of a suitable method of protein extraction and sample preparation compatible with two-dimensional gel electrophoresis (2D-PAGE), liquid chromatography-mass spectrometry (LCMS), or matrix-assisted laser desorption ionization-time of flight (MALDi-ToF). Our study introduces two efficient methods, one for isolating total proteins and another for organelle (chloroplast) proteins from various Cajanus spp. For total protein extraction, we have optimized a protocol using phenol in combination with a reducing agent (DTT) and protease inhibitor cocktail, also washing (6-7 times) with ice-cold acetone after overnight protein precipitation of total proteins. Our modified extraction method using phenol for total leaf protein yielded approximately 2-fold more proteins than the previously reported protocols from C. cajan (3.18 ± 0.11 mg/gm) and C. scarabaeoides (2.06 ± 0.08 mg/gm). We have also optimized a protocol for plastid protein extraction, which yielded 1.33 ± 0.25 mg/10 gm plastid proteins from C. cajan and 0.88 ± 0.19 mg/10 gm plastid proteins from C. scarabaeoides. The 2D-PAGE analysis revealed 678 ± 08 reproducible total protein spots from C. cajan and 597 ± 22 protein spots from C. scarabaeoides. Similarly, we found 566 ± 10 and 486 ± 14 reproducible chloroplast protein spots in C. cajan and C. scarabaeoides, respectively. We confirmed the plastid protein fractions through immunoblot analysis using antibodies against LHCb1/LHCⅡ type Ⅰ protein. We found both methods suitable for 2D-PAGE and mass spectrometry (MS). This is the first report on developing protocols for total and chloroplastic protein extraction of Cajanus spp. suitable for advanced proteomics research.

Synthesis of ZnO/Fe3O4/rGO nanocomposites and evaluation of antibacterial activities towards E. coli and S. aureus.

Antibacterial activity of nanoparticles (NPs) and nanocomposites (NCs) has received wide spread attention in biomedical applications. In this direction, the authors prepared zinc oxide (ZnO), iron oxide (Fe3O4), and their composite including reduced graphene oxide (rGO) by hydrothermal method. The structural and microstructural properties of the synthesised NPs and NCs were investigated by XRD, FT-IR, UV-Vis, TGA, and TEM analysis. PEG-coated ZnO and Fe3O4 form in hexagonal wurtzite and inverse spinel structures, respectively. ZnO forms in rod-shaped (aspect ratio of ∼3) morphology, whereas well-dispersed spherical-shaped morphology of ∼10 nm is observed in Fe3O4 NPs. The ZnO/Fe3O4 composite possesses a homogeneous distribution of above two phases and shows a very good colloidal stability in aqueous solvent. These synthesised particles exhibited varying antibacterial activity against gram-positive strain Staphylococcus aureus (S. aureus) and gram-negative strain Escherichia coli (E. coli). The nanocomposite exhibits a better cidal effect on E. coli when compared to S. aureus when treated with 1 mg/ml concentration. Further, the addition of rGO has intensified the anti-bacterial effect to a much higher extent due to synergistic influence of individual components.

Exploration and evaluation of proton source-assisted photocatalyst for hydrogen generation.

The present paper reports about the superior performance of some amine-based proton sources in enhancing the photocatalytic efficiency of Fe2O3-TiO2 composite during a water-splitting reaction. The band gap of the Fe2O3-TiO2 catalyst is tuned to 2.3 eV by varying the Fe content. The heterojunctions generated in the photocatalyst facilitate effective charge carrier migration suppressing the electron-hole recombination rate. The enhanced photocatalytic activity of the catalyst is studied using an experimental setup comprising a solar simulator (AAA) and a hydrogen gas chromatograph. The effect of proton sources viz, aniline hydrogen chloride (AH), diethylamine hydrogen chloride (DAH) and triethylamine hydrogen chloride (TAH), on the photocatalytic performance of the catalyst is explored and studied in detail. These proton sources serve as electron donors that stimulate photogenerated electron-hole separation that results in high quantum efficiency of the Fe2O3-TiO2 photocatalyst. A very high hydrogen generation rate of 880 µmol h-1 is achieved with the DAH-assisted Fe2O3-TiO2, whereas it is just 323 µmol h-1 with the Fe2O3-TiO2 alone. The enhancement in the hydrogen generation rate is attributed to the high basic nature, distinct hole scavenging action, low electron-hole recombination rate and the swift interfacial charge - transfer process. The effect of other proton source-assisted catalysts are also discussed in detail.

Parkinson's Disease Associated α-Synuclein Familial Mutants Promote Dopaminergic Neuronal Death in Drosophila melanogaster.

α-Synuclein (α-Syn) aggregation and amyloid formation are associated with loss of dopaminergic neurons in Parkinson's disease (PD). In addition, familial mutations in α-Syn are shown to be one of the definite causes of PD. Here we have extensively studied familial PD associated α-Syn G51D, H50Q, and E46K mutations using Drosophila model system. Our data showed that flies expressing α-Syn familial mutants have a shorter lifespan and exhibit more climbing defects compared to wild-type (WT) flies in an age-dependent manner. The immunofluorescence studies of the brain from the old flies showed more dopaminergic neuronal cell death in all mutants compared to WT. This adverse effect of α-Syn familial mutations is highly correlated with the sustained population of oligomer production and retention in mutant flies. Furthermore, this was supported by our in vitro studies, where significantly higher amount of oligomer was observed in mutants compared to WT. The data suggest that the sustained population of oligomer formation and retention could be a major cause of cell death in α-Syn familial mutants.