CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.     

Green synthesis and stabilization of gold nanoparticles in chemically modified chitosan matrices

Chitosan-N-2-methylhydroxypyridine-6-methylcorboxylate (Ch-PDC) and chitosan-N-2-methylhydroxypyridine-6-methylhydroxy thiocarbohydrazide (Ch-PDC-Th) were synthesized for the first time using chitosan as precursor. Chitosan, Ch-PDC, Ch-PDC-Th were used in the synthesis of gold nanoparticles (AuNP) in aqueous medium. Chitosan and Ch-PDC-Th possess reducing properties which enabled the 'green' synthesis of AuNPs. The stabilization of the AuNPs was as a result of the thiocarbide (SC) and amine (NH(2)) groups in the chitosan matrix. The modified chitosan, its derivatives and the resulting AuNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Ultraviolet-visible (UV-vis) spectroscopy, Raman scattering measurements, powder X-ray diffraction (PXRD) and thermo gravimetric analysis (TGA). Particle size, morphology, segregation and individuality of the AuNPs were examined by transmission electron microscope (TEM) and energy dispersion spectroscopy (EDS). An average AuNPs size of 20 nm was observed for chitosan and Ch-PDC-Th while Ch-PDC was 50 nm. In comparison, AuNPs resulting from Ch-PDC-Th precursor has the most enhanced Raman and fluorescent intensities and was stable for over 2 months.     

Measuring Social Networks for Medical Research in Lower-Income Settings

Social networks are believed to affect health-related behaviors and health. Data to examine the links between social relationships and health in low- and middle-income country settings are limited. We provide guidance for introducing an instrument to collect social network data as part of epidemiological surveys, drawing on experience in urban India. We describe development and fielding of an instrument to collect social network information relevant to health behaviors among adults participating in a large, population-based study of non-communicable diseases in Delhi, India. We discuss basic characteristics of social networks relevant to health including network size, health behaviors of network partners (i.e., network exposures), network homogeneity, network diversity, strength of ties, and multiplexity. Data on these characteristics can be collected using a short instrument of 11 items asked about up to 5 network members and 3 items about the network generally, administered in approximately 20 minutes. We found high willingness to respond to questions about social networks (97% response). Respondents identified an average of 3.8 network members, most often relatives (80% of network ties), particularly blood relationships. Ninety-one percent of respondents reported that their primary contacts for discussing health concerns were relatives. Among all listed ties, 91% of most frequent snack partners and 64% of exercise partners in the last two weeks were relatives. These results demonstrate that family relationships are the crux of social networks in some settings, including among adults in urban India. Collecting basic information about social networks can be feasibly and effectively done within ongoing epidemiological studies.     

Proteome analysis reveals a large merozoite surface protein-1 associated complex on the Plasmodium falciparum merozoite surface

Plasmodium merozoite surface protein-1 (MSP-1) is an essential antigen for the merozoite invasion of erythrocytes. A key challenge to the development of an effective malaria vaccine that can block the erythrocyte invasion is to establish the molecular interaction(s) among the parasite surface proteins as well as with the host cell encoded receptors. In the present study, we applied molecular interactions and proteome approaches to identify PfMSP-1 associated complex on the merozoite surface. Proteomic analysis identified a major malaria surface protein, PfRhopH3 interacting with PfMSP-1(42). Pull-down experiments with merozoite lysate using anti-PfMSP-1 or anti-PfRhopH3 antibodies showed 16 bands that when identified by tandem mass spectrometry corresponded to11 parasite proteins: PfMSP-3, PfMSP-6, PfMSP-7, PfMSP-9, PfRhopH3, PfRhopH1, PfRAP-1, PfRAP-2, and two RAP domain containing proteins. This MSP-1 associated complex was specifically seen at schizont/merozoite stages but not the next ring stage. We could also identify many of these proteins in culture supernatant, suggesting the shedding of the complex. Interestingly, the PfRhopH3 protein also showed binding to the human erythrocyte and anti-PfRhopH3 antibodies blocked the erythrocyte invasion of the merozoites. These results have potential implications in the development of PfMSP-1 based blood stage malaria vaccine.     

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein

Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efficient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10?mM of oxalic acid whereas only 15 isolates were grow at 50?mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23?%) after 60?min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents.     

Identification of Novel Targets for miR-29a Using miRNA Proteomics

MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3' Untranslated Regions (3' UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.