Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles

One of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24 h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.     

Drosophila intersex orthologue in the silkworm, Bombyx mori and related species

Intersex (ix), a gene required for female sexual development in Drosophila, acts in concert with doublesex (dsx) at the end of the sex determination pathway. In the present study a homologue of ix was identified in Bombyx mori. Expression analysis of this gene by RT–PCR and RNase protection assay revealed a diagnostic alternative splice form present only in testis, whereas the most common splice form was found to express in all other tissues from early embryonic developmental stages. The present study provides evidence for the presence of an alternative splice form of ix in three species of silkmoths examined. Taken together with the results of an earlier study on ix in piralid moth, Maruca vitrata (Cavaliere et al. 2009), the present study suggests that the testis-specific splice form may be a characteristic feature of lepidopterans. Though ix lacks a conserved splicing pattern it appears to have retained its functional conservation in terminal sexual differentiation. We speculate that the presence of an additional splice form, perhaps encoding non-functional protein only in testis, may prevent the feminizing effects exerted by the functional IX protein.     

Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design

Candidatus Liberibacter asiaticus is the causal agent of Huanglongbing (HLB) disease of citrus. Current management practices have not been able to control HLB and stop the spread of HLB. The current study is focused on screening small molecule inhibitors against SecA protein of Ca. L. asiaticus. Homology modeling, structure based virtual screening and molecular docking methods have been used to find the novel inhibitory compounds against SecA activity at ATP binding region. At 20 μm 17 compounds showed >50% inhibition and four compounds had more than 65% inhibition. The most active compound has IC₅₀ value of 2.5 μM. The differences between the activities of the compounds are explained by their inter-molecular interactions at ATP binding site.     

Exposure to Exogenous Enkephalins Disrupts Reproductive Development in the Eastern Lubber Grasshopper,Romalea microptera (Insecta: Orthoptera)

Enkephalins play a major role in reproductive physiology in crustaceans; however their role in reproductive development in insects is largely unknown. We investigated the effect of exposure to exogenous leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), and the opioid antagonist naloxone on gonad development in the Eastern lubber grasshopper, Romalea microptera. Injection of either Leu-Enk or naloxone alone significantly increased the testicular index and testicular follicular diameter in males, and the ovarian index, oocyte length, and oocyte diameter in females. In contrast, injection of Met-Enk inhibited all measures of reproductive development in both sexes. Surprisingly, co-injection of naloxone with either enkephalin enhanced the effect associated with administration of the enkephalin alone. This study clearly demonstrates the ability of enkephalins to disrupt insect sexual development and also suggests the existence of conserved enkephaline-dependent regulatory mechanisms in insects and crustaceans.     

A baculovirus-encoded MicroRNA (miRNA) suppresses its host miRNA biogenesis by regulating the exportin-5 cofactor Ran

MicroRNAs have emerged as key players in the regulation of various biological processes in eukaryotes, including host-pathogen interactions. Recent studies suggest that viruses encode miRNAs to manipulate their host gene expression to ensure their effective proliferation, whereas the host limits virus infection by differentially expressing miRNAs that target essential viral genes. Here, we demonstrate that an insect virus, Bombyx mori nucleopolyhedrosis virus (BmNPV), modulates the small-RNA-mediated defense of its host, B. mori, by encoding an miRNA (bmnpv-miR-1) that downregulates the expression of the host GTP-binding nuclear protein Ran, an essential component of the exportin-5-mediated nucleocytoplasmic transport machinery mainly involved in small-RNA transport from the nucleus to the cytoplasm. We demonstrate the sequence-dependent interaction of bmnpv-miR-1 with Ran mRNA using cell culture and in vivo assays, including RNA interference (RNAi) of Ran. Our results clearly show that bmnpv-miR-1 represses Ran, leading to reduction in the host small-RNA population, and consequently, the BmNPV load increases in the infected larvae. Blocking of bmnpv-miR-1 resulted in higher expression levels of Ran and a decrease in BmNPV proliferation. In contrast, blockage of host miRNA, bmo-miR-8, which targets the immediate-early gene of the virus and whose production was repressed upon bmnpv-miR-1 and Ran dsRNA administration, resulted in a significant increase in the virus load in the infected B. mori larvae. The present study provides an insight into one of the evasion strategies used by the virus to counter the host defense for its effective proliferation and has relevance to the development of insect virus control strategies.     

Binding modes of 6,7 di-substituted 4-anilinoquinoline-3-carbonitriles to EGFR

4-Anilino-3-cyanoquinolines were reported to have irreversible binding to epidermal growth factor receptor kinase (EGFRK) by forming a covalent linkage to C773. Our initial docking studies gave results inconsistent with the in vitro data and showed two different binding modes. To perceive the exact mode of binding of these ligands, two models of the ligand-EGFR complexes were considered: (1) reversible binding mode in which the ligand had hydrogen bond interactions at the binding site and (2) irreversible binding mode wherein the ligand's Michael acceptor side chain has proximity to the sulfhydryl group of C773 of EGFR, thereby enabling a covalent interaction. The irreversible binding mode correlated better than reversible binding mode with respect to in vitro data. However, our results indicate that both modes are being adopted by the ligands and could be utilized to design more potent EGFRK inhibitors.     

MICAS: a fully automated web server for microsatellite extraction and analysis from prokaryote and viral genomic sequences

MICAS is a web server for extracting microsatellite information from completely sequenced prokaryote and viral genomes, or user-submitted sequences. This server provides an integrated platform for MICdb (database of prokaryote and viral microsatellites), W-SSRF (simple sequence repeat finding program) and Autoprimer (primer design software). MICAS, through dynamic HTML page generation, helps in the systematic extraction of microsatellite information from selected genomes hosted on MICdb or from user-submitted sequences. Further, it assists in the design of primers with the help of Autoprimer, for sequences containing selected microsatellite tracts.     

Sources of variability and effect of experimental approach on expression profiling data interpretation

Background  

We provide a systematic study of the sources of variability in expression profiling data using 56 RNAs isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 murine cell culture and tissue RNAs (42 Affymetrix U74Av2 arrays).     

Molecular phylogeny of the nasuta subgroup of Drosophila based on 12S rRNA, 16S rRNA and CoI mitochondrial genes, RAPD and ISSR polymorphisms

The nasuta subgroup is a cluster of morphologically almost similar forms with a wide range of geographic distribution. During the last three decades nature of inter-relationship among the members has been investigated at different levels of organization. The phylogenetic relationships of the members of the nasuta subgroup of the immigrans species group of Drosophila was made by employing Random Amplified Polymorphic DNA (RAPD), Inter Simple Sequence Repeats-PCR (ISSR-PCR) polymorphisms, mitochondrial 12S rRNA, 16S rRNA and Cytochrome C Oxidase subunit I (CoI) gene sequences. The phylogenetic tree generated by RAPD analysis is in nearly complete congruence with the classification based on morphophenotypic characters. The 12S and 16S rRNA genes were highly conserved across the nasuta subgroup and revealed only 3 and 4 variable sites respectively, of which only one site was informative. The CoI gene, on the other hand, revealed 57 variable sites of which 25 sites were informative. All the three species of orbital sheen complex were included in a major cluster in the phylogenetic trees derived from mitochondrial gene sequence data consistent with the morphophenotypic classification. The CoI analysis placed two species of frontal sheen complex, D. n. nasuta and D. n. albomicans in two different clades and this is inconsistent with morphological classification. The molecular clock suggested that divergence between the kohkoa complex and the albomicans complex occurred approximately 2.2 MYA, indicating recent evolution of the nasuta subgroup. The higher transition bias in the mitochondrial genes reported in the present study also suggested recent evolution of the nasuta subgroup.