Towards a therapeutic inhibition of dystrophin exon 23 splicing in mdx mouse muscle induced by antisense oligoribonucleotides (splicomers): target sequence optimisation using oligonucleotide arrays

BACKGROUND: The activity of synthetic antisense oligonucleotides (splicomers) designed to block pre-mRNA splicing at specific exons has been demonstrated in a number of model systems, including constitutively spliced exons in mouse dystrophin RNA. Splicomer reagents directed to Duchenne muscular dystrophy (DMD) RNAs might thus circumvent nonsense or frame-shifting mutations, leading to therapeutic expression of partially functional dystrophin, as occurs in the milder, allelic (Becker) form of the disease (BMD). METHODS: Functional and hybridisation array screens have been used to select optimised splicomers directed to exon 23 of dystrophin mRNA which carries a nonsense mutation in the mdx mouse. Splicomers were transfected into cultured primary muscle cells, and dystrophin mRNA assessed for exon exclusion. Splicomers were also administered to the muscles of mdx mice. RESULTS: Oligonucleotide array analyses with dystrophin pre-mRNA probes revealed strong and highly specific hybridisation patterns spanning the exon 23/intron 23 boundary, indicating an open secondary structure conformation in this region of the RNA. Functional screening of splicomer arrays by direct analysis of exon 23 RNA splicing in mdx muscle cultures identified a subset of biologically active reagents which target sequence elements associated with the 5' splice site region of dystrophin intron 23; splicomer-mediated exclusion of exon 23 was specific and dose-responsive up to a level exceeding 50% of dystrophin mRNA, and Western blotting demonstrated de novo expression of dystrophin protein at 2-5% of wild-type levels. Direct intramuscular administration of optimised splicomer reagents in vivo resulted in the reappearance of sarcolemmal dystrophin immunoreactivity in > 30% of muscle fibres in the mdx mouse CONCLUSIONS: These results suggest that correctly designed splicomers may have direct therapeutic value in vivo, not only for DMD, but also for a range of other genetic disorders.     

Molecular phylogeny of elasmobranchs inferred from mitochondrial and nuclear markers

The elasmobranchs (sharks, rays and skates) being the extant survivors of one of the earliest offshoots of the vertebrate evolutionary tree are good model organisms to study the primitive vertebrate conditions. They play a significant role in maintaining the ecological balance and have high economic value. Due to over-exploitation and illegal fishing worldwide, the elasmobranch stocks are being decimated at an alarming rate. Appropriate management measures are necessary for restoring depleted elasmobranch stocks. One approach for restoring stocks is implementation of conservation measures and these measures can be formulated effectively by knowing the evolutionary relationship among the elasmobranchs. In this study, a total of 30 species were chosen for molecular phylogeny studies using mitochondrial cytochrome c oxidase subunit I, 12S ribosomal RNA gene and nuclear Internal Transcribed Spacer 2. Among different genes, the combined dataset of COI and 12S rRNA resulted in a well resolved tree topology with significant bootstrap/posterior probabilities values. The results supported the reciprocal monophyly of sharks and batoids. Within Galeomorphii, Heterodontiformes (bullhead sharks) formed as a sister group to Lamniformes (mackerel sharks): Orectolobiformes (carpet sharks) and to Carcharhiniformes (ground sharks). Within batoids, the Myliobatiformes formed a monophyly group while Pristiformes (sawfishes) and Rhinobatiformes (guitar fishes) formed a sister group to all other batoids.     

Prioritizing Tiger Conservation through Landscape Genetics and Habitat Linkages

Even with global support for tiger (Panthera tigris) conservation their survival is threatened by poaching, habitat loss and isolation. Currently about 3,000 wild tigers persist in small fragmented populations within seven percent of their historic range. Identifying and securing habitat linkages that connect source populations for maintaining landscape-level gene flow is an important long-term conservation strategy for endangered carnivores. However, habitat corridors that link regional tiger populations are often lost to development projects due to lack of objective evidence on their importance. Here, we use individual based genetic analysis in combination with landscape permeability models to identify and prioritize movement corridors across seven tiger populations within the Central Indian Landscape. By using a panel of 11 microsatellites we identified 169 individual tigers from 587 scat and 17 tissue samples. We detected four genetic clusters within Central India with limited gene flow among three of them. Bayesian and likelihood analyses identified 17 tigers as having recent immigrant ancestry. Spatially explicit tiger occupancy obtained from extensive landscape-scale surveys across 76,913 km² of forest habitat was found to be only 21,290 km². After accounting for detection bias, the covariates that best explained tiger occupancy were large, remote, dense forest patches; large ungulate abundance, and low human footprint. We used tiger occupancy probability to parameterize habitat permeability for modeling habitat linkages using least-cost and circuit theory pathway analyses. Pairwise genetic differences (F_ST) between populations were better explained by modeled linkage costs (r>0.5, p<0.05) compared to Euclidean distances, which was in consonance with observed habitat fragmentation. The results of our study highlight that many corridors may still be functional as there is evidence of contemporary migration. Conservation efforts should provide legal status to corridors, use smart green infrastructure to mitigate development impacts, and restore habitats where connectivity has been lost.     

MicroRNA profiles in Sorghum exposed to individual drought or heat or their combination

Sorghum is largely grown for food, fodder and for biofuel production in semi-arid regions where the drought or high temperature or their combination co-occur. Plant microRNAs (miRNAs) are integral to the gene regulatory networks that control almost all biological processes including adaptation to stress conditions. Thus far, plant miRNA profiles under separate drought or heat stresses have been reported but not under combined drought and heat. In this study, we report miRNA profiles in leaves of sorghum exposed to individual drought or heat or their combination. Approximately 29 conserved miRNA families represented by 80 individual miRNAs, 26 families represented by 47 members of less conserved or sorghum-specific miRNA families as well as 8 novel miRNA families have been identified. Of these, 25 miRNAs were found to be differentially regulated in response to stress treatments. The comparative profiling revealed that the miRNA regulation was stronger under heat or combination of heat and drought compared to the drought alone. Furthermore, using degradome sequencing, 48 genes were confirmed as targets for the miRNAs in sorghum. Overall, this study provides a framework for understanding of the miRNA-guided gene regulations under combined stresses.

     

Primula munroi的产自东喜马拉雅的一个新亚种——P. munroi ssp. schizocalyx

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Surfactant Protein D Inhibits HIV-1 Infection of Target Cells via Interference with gp120-CD4 Interaction and Modulates Pro-Inflammatory Cytokine Production

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.     

Nitrogen and harvest management of Conservation Reserve Program (CRP) grassland for sustainable biomass feedstock production

The Biomass Regional Feedstock Partnership has identified grasslands planted under the Conservation Reserve Program (CRP) as a potential source for herbaceous bioenergy feedstock. The goal of this project is to assess the yield potential of CRP grasslands across diverse regions. Consistent with that goal, the objective of this project was to establish yield potential and quality parameters for several different CRP grasslands, representative of different growing environments. Standard field scale agricultural practices were used as management guidelines at each location. The test locations were identified and established based on known regions containing concentrated tracts of CRP grassland and represented variable climatic parameters and production histories. Biomass production potential for CRP land dominated by either warm‐ or cool‐season grass mixtures in each location was evaluated over the course of three growing seasons (2008, 2009, and 2010). Specifically, a mixture of warm‐season perennial grasses was evaluated in North Dakota, Kansas, and Oklahoma, while a cool‐season mixture was evaluated in Montana, Georgia, and Missouri. Maximum biomass yields for the three warm‐season CRP sites ranged from 4.0 to 7.2 Mg ha^?1 and for the three cool‐season CRP sites 3.4–6.0 Mg ha^?1. Our results demonstrate that CRP grassland has potential as a bioenergy feedstock resource if the appropriate management practices are followed.     

Production of thermostable β-amylase and pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation: Screening of nutrients using Plackett-Burman design

Screening of fifteen nutrients belonging to four categories, viz., carbon, nitrogen, salt and complex organic sources was carried out using Plackett-Burman design for the production of thermostable #-amylase and pullulanase by Clostridium thermosulfurogenes SV2 in solid-state fermentation (SSF). This design involves screening of up to `nу' variables in just `n' number of experiments. Regression co-efficients and t-values were calculated by subjecting the experimental data to statistical analysis. Lactose, diammonium hydrogen phosphate, calcium chloride and casein hydrolysate showed higher regression co-efficients in the biomass formation. Among the fifteen nutrients screened, based on their performance in terms of product promoting ability, availability and cost, magnesium chloride, potato starch, ferrous sulphate, pearl millet flour and corn steep liquor were identified as most effective and, therefore, selected for inclusion in further optimization studies.     

Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis. Amplicons of 265 bp and 447 bp of htrA and yueF, respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with TaqI produced DNA bands of 90 bp and 175 bp with ssp. lactis, and 66 bp and 199 bp with ssp. cremoris. Similarly, restriction of PCR product of 447 bp size with AluI produced digested fragments of 125 bp and 322 bp with ssp. lactis, and 71 bp and 376 bp with ssp. cremoris. The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with TaqI and AluI, respectively.