Molecular detection and characterization of phytoplasma associated with China aster (Callistephus chinensis) phyllody in India

China aster (Callistephus chinensis L.) is one of the most popular annual flowering plant grown through-out the world. Phyllody disease of China aster is a phytoplasma associated disease that induces severe economic losses. Phytoplasmal disease in China aster was assessed for phytoplasma by direct polymerase chain reaction primed by using phytoplasma universal primer pairs PI/P7. A 1.8 Kb DNA fragments encoding the portion of phyto-plasma 16SrDNA amplified by PCR was cloned and sequenced. Sequencing of the PCR product and BLAST analy-sis indicated that China aster phyllody phytoplasma strain shared maximum sequence identity (99%) with strains of Peanut Witches’ broom (16SrII) phytoplasma group. Phylogenetic relationship of 16SrDNA sequence of China aster phyllody phytoplasma strain in the present study confirmed association of Peanut Witches’ broom (16SrII) group of phytoplasmas with China aster phyllody disease in India

Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage

Molecular detection and characterization of phytoplasma associated with China aster (Callistephus chinensis) phyllody in India

China aster (Callistephus chinensis L.) is one of the most popular annual flowering plant grown through-out the world. Phyllody disease of China aster is a phytoplasma associated disease that induces severe economic losses. Phytoplasmal disease in China aster was assessed for phytoplasma by direct polymerase chain reaction primed by using phytoplasma universal primer pairs PI/P7. A 1.8 Kb DNA fragments encoding the portion of phyto-plasma 16SrDNA amplified by PCR was cloned and sequenced. Sequencing of the PCR product and BLAST analy-sis indicated that China aster phyllody phytoplasma strain shared maximum sequence identity (99%) with strains of Peanut Witches’ broom (16SrII) phytoplasma group. Phylogenetic relationship of 16SrDNA sequence of China aster phyllody phytoplasma strain in the present study confirmed association of Peanut Witches’ broom (16SrII) group of phytoplasmas with China aster phyllody disease in India

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties