Identification of novel angiogenesis inhibitors

Vascular endothelial growth factor (VEGF) is a key stimulant of angiogenesis, which is the process of generating new capillary blood vessels. Inhibition of the vascular endothelial growth factor receptor (VEGFR) kinase is known to result in blockage of angiogenesis. A pharmacophore was developed based on the binding of ATP to the hinge region of the kinase domain of VEGFR and a database search of 18,000 compounds was conducted. Selected hits were assessed for their ability to limit the induction of web-like network of capillary tubes by the human umbilical vascular endothelial cells. Two compounds (1 and 4) showed good inhibitory ability to prevent sprouting and closed polygon formation of the tubular networks, promising them to be lead compounds. Compound 4 showed 60% inhibition at 0.05 microM.     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.     

Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA

Molecular phylogeny of some of the economically important silkmoths was derived using three mitochondrial genes, 12S rRNA, 16S rRNA, and COI, and the control region (CR). Maximum likelihood (ML) analyses showed two distinct clades, one consisting of moths from Bombycidae family and the other from Saturniidae family. The mitochondrial CR showed length polymorphisms with indels. The ML analyses for complete mitochondrial genome sequences of Bombyx mori (strains Aojuku, C108, Backokjam, and Xiafang), Japanese and Chinese strains of B. mandarina (Japanese mandarina and Chinese mandarina) and, Antheraea pernyi revealed two distinct clades, one comprising of B. mori strains and the other with B. mandarina, and A. pernyi forming an outgroup. Pairwise distances revealed that all of the strains of B. mori studied are closer to Chinese than to Japanese mandarina. Phylogenetic analyses based on whole mitochondrial genome sequences, the finding of a tandem triplication of a 126bp repeat element only in Japanese mandarina, and chromosome number variation in B. mandarina suggest that B. mori must have shared its recent common ancestor with Chinese mandarina. Another wild species of the Bombycidae family, Theophila religiosa, whose phylogenetic status was not clear, clustered together with the other bombycid moths in the study. Analysis of the interspecific hybrid, A. proylei gave evidence for paternal inheritance of mitochondrial DNA.     

GREG cells, a dysferlin-deficient myogenic mouse cell line

The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.     

Glucocorticoid-treated mice are an inappropriate positive control for long-term preclinical studies in the mdx mouse

Background

Dmd^mdx (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.

Methods and Findings

We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.

Conclusion

Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials.     

Genetic Diversity and Population Structure of Indian Golden Silkmoth (Antheraea assama)

Background

The Indian golden saturniid silkmoth (Antheraea assama), popularly known as muga silkmoth, is a semi-domesticated silk producing insect confined to a narrow habitat range of the northeastern region of India. Owing to the prevailing socio-political problems, the muga silkworm habitats in the northeastern region have not been accessible hampering the phylogeography studies of this rare silkmoth. Recently, we have been successful in our attempt to collect muga cocoon samples, although to a limited extent, from their natural habitats. Out of 87 microsatellite markers developed previously for A. assama, 13 informative markers were employed to genotype 97 individuals from six populations and analyzed their population structure and genetic variation.

Methodology/Principal Findings

We observed highly significant genetic diversity in one of the populations (WWS-1, a population derived from West Garo Hills region of Meghalaya state). Further analysis with and without WWS-1 population revealed that dramatic genetic differentiation (global F_ST = 0.301) was due to high genetic diversity contributed by WWS-1 population. Analysis of the remaining five populations (excluding WWS-1) showed a marked reduction in the number of alleles at all the employed loci. Structure analysis showed the presence of only two clusters: one formed by WWS-1 population and the other included the remaining five populations, inferring that there is no significant genetic diversity within and between these five populations, and suggesting that these five populations are probably derived from a single population. Patterns of recent population bottlenecks were not evident in any of the six populations studied.

Conclusions/Significance

A. assama inhabiting the WWS-1 region revealed very high genetic diversity, and was genetically divergent from the five populations studied. The efforts should be continued to identify and study such populations from this region as well as other muga silkworm habitats. The information generated will be very useful in conservation of dwindling muga culture in Northeast India.