Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout (KO) mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the eGFP gene in the tibialis anterior muscle of the Dmd KO mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out-of-frame to in-frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9 has great potential for the treatment of DMD and other neuromuscular diseases

The Antimicrobial Effect of Silver Ion Impregnation into Endodontic Sealer against Streptococcus mutans.

Pulpal and periradicular diseases are primarily caused by bacterial invasion of the root canal system as a result of caries progression. The presence of residual bacteria at the time of root canal completion (obturation) is associated with significantly higher rate of treatment failure. Re-infection of obturated root canals can be potentially prevented by enhancing the antibacterial activities of root canal obturation materials. We evaluated, in an in vitro model, the antimicrobial efficacy of silver ions added to a common endodontic sealer. For that purpose we performed growth inhibition studies and bacterial viability tests. We measured the zone of inhibition, optical density and performed confocal laser scanning microscopy. Our results show that the silver ions enhance the antimicrobial activity of the root canal sealer against Streptococcus mutans. This study approach may hold promise for studying other biologically based therapies and therefore increasing the success rate of routine orthograde root canal treatment

Application of finite element code to characterize mechanical properties of complex microstructured materials

A technique to solve the periodic homogenization problem is described systematically in this work. The method is to solve the cell problems by imposing eigenstrains in terms of a thermal or a piezoelectric strain to the representative volume element (RVE). Homogenized coefficients are then calculated from stress solutions of those cell problems. As a dual approach, an imposed stress field can also be applied to solve the cell problems. Numerical examples of characterization mechanical properties of complicated microstructure materials are examined. The obtained results show good agreements with the published data. Comparisons show that the technique in this study can be effectively used to characterize the mechanical properties of complex microstructured materials

QUANTITATIVE DETERMINATION AND PREPARATIVE ISOLATION OF TWO MAJOR ALKALOIDS FROM THE VIETNAMESE MEDICINAL HERB EVODIAE FRUCTUS

Objective: To develop a simple and accurate HPLC-DAD method for simultaneous determination, the content of major components: limonin, evodiamine, and rutaecarpine in Evodiae fructus and evaluation the quality of Evodiae fructus sold in markets. Methods: Open column chromatography was used to separate and purify rutaecarpine and evodiamine, the two major alkaloids from Evodiae fructus extract as a laboratory standard. Chromatographic separation was achieved using a Germini C18 column (150 mm × 4.6 mm I.D., 5 µm), detected at 210 nm. The mobile phase consisted of acetonitrile (A), methanol (B), and water (C). The validated method simultaneously determined alkaloid content in 40 batches of samples collected from markets in different regions of Vietnam. Results: In one-step purification, our method yielded 326 mg of rutaecarpine and 128 mg of evodiamine from 3.2 g of crude extract, with purities of 98.9 and 98.5%, respectively. The structures of these compounds were identified using 1H NMR and 13C NMR. There was a significant correlation between alkaloid content and fruit size, with a Spearman correlation coefficient of>0.5 (p<0.001), and there was a large difference in alkaloid contents between three maturity degrees of the fruit. Open-mouth fruits and fruits with average sizes of 4 to 6 mm had the highest alkaloid contents, whereas closed-mouth fruits had the lowest. Conclusion: This study provided information on the standardization and quality control of evodiamine and rutaecarpine in Evodiae fructus, as well as a foundation for further pharmacological and toxicological studies

Innovative in silico approaches to address avian flu using grid technology

The recent years have seen the emergence of diseases which have spread very quickly all around the world either through human travels like SARS or animal migration like avian flu. Among the biggest challenges raised by infectious emerging diseases, one is related to the constant mutation of the viruses which turns them into continuously moving targets for drug and vaccine discovery. Another challenge is related to the early detection and surveillance of the diseases as new cases can appear just anywhere due to the globalization of exchanges and the circulation of people and animals around the earth, as recently demonstrated by the avian flu epidemics. For 3 years now, a collaboration of teams in Europe and Asia has been exploring some innovative in silico approaches to better tackle avian flu taking advantage of the very large computing resources available on international grid infrastructures. Grids were used to study the impact of mutations on the effectiveness of existing drugs against H5N1 and to find potentially new leads active on mutated strains. Grids allow also the integration of distributed data in a completely secured way. The paper presents how we are currently exploring how to integrate the existing data sources towards a global surveillance network for molecular epidemiology.Comment: 7 pages, submitted to Infectious Disorders - Drug Target

Mapping of Two High Mobility Group Protein Genes for Growth and Composition traits in Pig

Using information from the human genome two new candidate genes for growth and composition traits were studied. The porcine high mobility group isoforms protein [HMGI(Y) and HMGIC] genes were chosen based on their presumed role in fat cell growth and differentiation. The HMGI(Y) gene was assigned to pig chromosome 7 by both linkage and physical mapping methods. This assignment agrees with other comparative mapping studies as the human HMGI(Y) gene maps to human chromosome 6p21, which is known to share a homology with pig chromosome 7. Interestingly, the pig HMGIC gene was assigned to the pig chromosome 1 by both methods. The localization of these candidate genes in the pig genome could improve the power of analyses for quantitative traits associated with growth and meat quality traits

Structure-Guided Mutagenesis Reveals the Catalytic Residue that Controls the Regiospecificity of C6-Indole Prenyltransferases

Indole is a significant structural moiety and functionalization of the C−H bond in indole-containing molecules expands their chemical space, and modifies their properties and/or activities. Indole prenyltransferases (IPTs) catalyze the direct regiospecific installation of prenyl moieties on indole-derived compounds. IPTs have shown relaxed substrate flexibility enabling them to be used as tools for indole functionalization. However, the mechanism by which certain IPTs target a specific carbon position is not fully understood. Herein, we use structure-guided site-directed mutagenesis, in vitro enzymatic reactions, kinetics and structural-elucidation of analogs to verify the key catalytic residues that control the regiospecificity of all characterized regiospecific C6 IPTs. The presented results also demonstrate that substitution of PriB_His312 to Tyr leads to the synthesis of analogs prenylated at different positions than C6. This work contributes to understanding of how certain IPTs can access a challenging position in indole-derived compounds

Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency.

Type 1 diabetes affects 20 million patients worldwide. Insulin is the primary and commonly the sole therapy for type 1 diabetes. However, only a minority of patients attain the targeted glucose control and reduced adverse events. We tested urocortin 2 gene transfer as single-agent therapy for insulin deficiency using two mouse models. Urocortin 2 gene transfer reduced blood glucose for months after a single intravenous injection, through increased skeletal muscle insulin sensitivity, increased insulin release in response to glucose stimulation, and increased plasma insulin levels before and during euglycemic clamp. The combined increases in both insulin availability and sensitivity resulted in improved glycemic indices-events that were not anticipated in these insulin-deficient models. In addition, urocortin 2 gene transfer reduced ocular manifestations of long-standing insulin deficiency such as vascular leak and improved retinal function. Finally, mortality was reduced by urocortin 2 gene transfer. The mechanisms for these beneficial effects included increased activities of AMP-activated protein kinase and Akt (protein kinase B) in skeletal muscle, increased skeletal muscle glucose uptake, and increased insulin release. These data suggest that urocortin 2 gene transfer may be a viable therapy for new onset type 1 diabetes and might reduce insulin needs in later stage disease