Targeted correction of a thalassemia-associated β-globin mutation induced by pseudo-complementary peptide nucleic acids

β-Thalassemia is a genetic disorder caused by mutations in the β-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA–pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction

Report of a TREAT-NMD/World Duchenne Organisation Meeting on Dystrophin Quantification Methodology

Representatives of academia, patient organisations, industry and the United States Food and Drug Administration attended a workshop on dystrophin quantification methodology. The aims of the workshop were to provide an overview of methods used to quantify dystrophin levels in human skeletal muscle and their applicability to clinical trial samples, outline the gaps with regards to validating the methods for robust clinical applications prior to regulatory agency review, and to align future efforts towards further optimizing these methods. The workshop facilitated a constructive but also critical discussion on the potential and limitations of techniques currently used in the field of translational research (western blot and immunofluorescence analysis) and emerging techniques (mass spectrometry and capillary western immunoassay). Notably, all participants reported variation in dystrophin levels between muscle biopsies from different healthy individuals and agreed on the need for a common reference sample

Disinfection of Ocular Cells and Tissues by Atmospheric-Pressure Cold Plasma

Background: Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage. Methodology and Principal Findings: We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2',7'-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2) was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis. Conclusions: A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses

Constrained flexibility in PNA: DNA binding studies with bridged aminopropylglycyl PNA

Introduction of methylene bridges in aegPNA and apgPNA molecules give rise to cyclic five and six membered ring structures. Synthesis of a new six membered cyclic PNA monomer, aminopipecolyl PNA (pipPNA) is reported. Incorporation of pipPNA into PNA oligomers and comparative binding with target DNA sequences is studied

Assessment of Ground Water Quality in Gajanan Colony, Ahmednagar. by Water Quality Index (WQI)

A water quality analysis was carried out in Ahmednagar city. Different parameters were analyzed that are pH, alkalinity, turbidity, TDS, total hardness and chlorides. The sampling sites showed that the physicocmc al parameters were within the water quality standards and the quality of water was found to be fit for drinking.Water is an essential natural resource for sustaining life and environment but over the last few decades the water quality is deteriorating due to it's over exploitation. Water quality is essential parameter to be studied when the overall focus is sustainable development keeping mankind at focal point. Groundwater is the major source of drinking water in rural as well as in urban areas and over 94% of the drinking water demand is met by groundwater.Assessment ground water quality is necessary as it controls it's as usability for drinking purpose .The study was carried out by the physic-chemical and biological analysis of ground water quality

Environmental status of some beaches in Daman and South Gujarat, India

641-649Beaches are the prime choice of amusement, hence determining its quality is significant to public health. The existing status of water and sediment quality of four beaches along the north-west coast of India is evaluated at high and low tide (for three weeks) during the pre-monsoon season. Water samples were measured for hydrography, nutrients, Petroleum Hydrocarbons (PHc) and Fecal Coliform (FC). Sediments were analyzed for heavy metals, PHc and FC. Results indicated higher concentrations of ammonia (28.7 μmol/l) and phosphate (57.3 μmol/l) in the waters of Tadgam beach owing to the proximity to industrial discharge. Metals such as Fe, Al, Mn, Cr, Zn, Cu, Ni, Co, and Hg were found to be above Lowest Effective Range (LER) as compared to Cr and Fe, those exceed Severe Effective Range (SER). Elevated levels of FC in both water and sediment in all the studied beaches indicated microbial contamination relative to anthropogenic activities