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naked DNA [keywords]
- A simple cassette as point-of-care diagnostic device for naked-eye colorimetric bacteria detection. [JOURNAL ARTICLE]
- Analyst 2013 Dec 4.
Effective pathogen detection is necessary for treatment of infectious diseases. Point of care (POC) devices have tremendously improved the global human heath. However, design criteria for sample processing POC devices for pathogen detection in limited infrastructure are challenging and can make a significant contribution to global health by providing rapid and sensitive detection of bacteria in food, water, and patient samples. In this paper, we demonstrate a novel portable POC diagnostic device that is simple to assemble for genetic detection of bacterial pathogens by isothermal DNA amplification. The device is fabricated with very low production cost, using simple methods and easy-to-access materials on a flexible ribbon polyethylene substrate. We showed that the device is capable of detection of 30 CFU mL(-1) of E. coli and 200 CFU mL(-1) of S. aureus in less than 1 hour. Through numerical simulations, we estimated that the device can be extended to high-throughput detection simultaneously performing a minimum of 36 analyses. This robust and sensitive detection device can be assembled and operated by non-specialist personnel, particularly for multiple bacterial pathogen detections in low-resource settings.
- MAR-mediated integration of plasmid vectors for in vivo gene transfer and regulation. [JOURNAL ARTICLE]
- BMC Mol Biol 2013 Dec 2; 14(1):26.
The in vivo transfer of naked plasmid DNA into organs such as muscles is commonly used to assess the expression of prophylactic or therapeutic genes in animal disease models.In this study, we devised vectors allowing a tight regulation of transgene expression in mice from such non-viral vectors using a doxycycline-controlled network of activator and repressor proteins. Using these vectors, we demonstrate proper physiological response as consequence of the induced expression of two therapeutically relevant proteins, namely erythropoietin and utrophin. Kinetic studies showed that the induction of transgene expression was only transient, unless epigenetic regulatory elements termed Matrix Attachment Regions, or MAR, were inserted upstream of the regulated promoters. Using episomal plasmid rescue and quantitative PCR assays, we observed that similar amounts of plasmids remained in muscles after electrotransfer with or without MAR elements, but that a significant portion had integrated into the muscle fiber chromosomes. Interestingly, the MAR elements were found to promote plasmid genomic integration but to oppose silencing effects in vivo, thereby mediating long-term expression.This study thus elucidates some of the determinants of transient or sustained expression from the use of non-viral regulated vectors in vivo.
- Mycobacterium avium Complex in Day Care Hot Water Systems, and Persistence of Live Cells and DNA in Hot Water Pipes. [JOURNAL ARTICLE]
- Curr Microbiol 2013 Nov 23.
The Mycobacterium avium complex (MAC) is a group of opportunistic human pathogens that may thrive in engineered water systems. MAC has been shown to occur in drinking water supplies based on surface water, but less is known about the occurrence and persistence of live cells and DNA in public hot water systems based on groundwater. In this study, we examined the occurrence of MAC in hot water systems of public day care centers and determined the persistence of live and dead M. avium cells and naked DNA in model systems with the modern plumbing material cross-linked polyethylene (PEX). The occurrence of MAC and co-occurrence of Legionella spp. and Legionella pneumophila were determined using cultivation and qPCR. Co-occurrences of MAC and Legionella were detected in water and/or biofilms in all hot water systems at temperatures between 40 and 54 °C. Moderate correlations were observed between abundance of culturable MAC and that of MAC genome copies, and between MAC and total eubacterial genome copies. No quantitative relationship was observed between occurrence of Legionella and that of MAC. Persistence in hot water of live and dead M. avium cells and naked DNA was studied using PEX laboratory model systems at 44 °C. Naked DNA and DNA in dead M. avium cells persisted for weeks. Live M. avium increased tenfold in water and biofilms on PEX. The results suggest that water and biofilms in groundwater-based hot water systems can constitute reservoirs of MAC, and that amplifiable naked DNA is relatively short-lived, whereas PEX plumbing material supports persistence and proliferation of M. avium.
- Naked Replication Forks Break apRPArt. [Journal Article]
- Cell 2013 Nov 21; 155(5):979-80.
Stalled replication forks occasionally collapse, leading to potentially catastrophic DNA double-strand breaks. Now, Toledo et al. (2013) reveal that fork breakage occurs when the pool of the single-strand DNA-binding protein RPA becomes exhausted. This study has important implications for the origin and treatment of cancers with high levels of replicative stress.
- Karyotype and cytogenetic mapping of 9 classes of repetitive DNAs in the genome of the naked catfish Mystus bocourti (Siluriformes, Bagridae). [JOURNAL ARTICLE]
- Mol Cytogenet 2013 Nov 22; 6(1):51.
In the present study, conventional and molecular cytogenetic studies were performed in the naked catfish Mystus bocourti (Siluriformes, Bagridae). Besides the conventional Giemsa staining, fluorescence in situ hybridization (FISH) using nine classes of repetitive DNAs namely 5S and 18S rDNAs, U2 snRNA, the microsatellites (CA)15 and (GA)15, telomeric repeats, and the retrotransposable elements Rex1, 3 and 6. was also performed, RESULTS: M. bocourti had a 2n = 56 chromosomes with a karyotype composed by 11 m + 11sm + 6st/a and a fundamental number (NF) equal to 100 in both sexes. Heteromorphic sex chromosome cannot be identified. The U2 snRNA, 5S and 18S rDNA were present in only one pair of chromosomes but none of them in a syntenic position. Microsatellites (CA)15 and (GA)15 showed hybridization signals at subtelomeric regions of all chromosomes with a stronger accumulation into one specific chromosomal pair. FISH with the telomeric probe revealed hybridization signals on each telomere of all chromosomes and interstitial telomeric sites (ITS) were not detected. The retrotransposable elements Rex 1, 3 and 6 were generally spread throughout the genome.In general, the repetitive sequences were not randomly distributed in the genome, suggesting a pattern of compartmentalization on the heterochromatic region of the chromosomes. Little is known about the structure and organization of bagrid genomes and the knowledge of the chromosomal distribution of repetitive DNA sequences in M. bocorti represents the first step for achieving an integrated view of the their genomes.
- Label-free DNA detection using two-dimensional periodic relief grating as a visualized platform for diagnosis of breast cancer recurrence after surgery. [JOURNAL ARTICLE]
- Biosens Bioelectron 2013 Nov 1.:35-41.
In this study we fabricated a nanopillar array of silicon oxide, involving very-large-scale integration (VLSI) and reactive ion etching (RIE), as two-dimensional periodic relief gratings (2DPRGs) on Si surfaces. Thiolated oligonucleotide was successively immobilized on the thiol functionalized surfaces of 2DPRGs by disulfide bond as an optical probe to detect a human genomic DNA (hgDNA584), related to breast cancer recurrence after surgery, from a biological specimen. The oligonucleotide-bound 2DPRG alone produces insignificant structure change, but upon hybridization with hgDNA584 leads to a dramatic change of the pillar scale due to hgDNA584 filling inside the 2DPRG layers. The performance of the sensor was evaluated by capturing hgDNA584 on the oligonucleotide-bound 2DPRGs and measuring the effective refractive index (neff), resulting of color change from pure blue to red, observed by naked eyes along an incident angle of 20-30°. The surface-bound 2DPRG based assay with the chemoresponsive diffraction grating signal transduction scheme results in an experimentally simple DNA detection protocol, displaying attributes of both detection methodologies: the high sensitivity and selectivity afforded by 2DPRG probes and the experimental simplicity, and miniaturization potential provided by the diffraction-based sensing technology.
- Molecular adaptation of telomere associated genes in mammals. [JOURNAL ARTICLE]
- BMC Evol Biol 2013 Nov 15; 13(1):251.
Placental mammals display a huge range of life history traits, including size, longevity, metabolic rate and germ line generation time. Although a number of general trends have been proposed between these traits, there are exceptions that warrant further investigation. Species such as naked mole rat, human and certain bat species all exhibit extreme longevity with respect to body size. It has long been established that telomeres and telomere maintenance have a clear role in ageing but it has not yet been established whether there is evidence for adaptation in telomere maintenance proteins that could account for increased longevity in these species.Here we carry out a molecular investigation of selective pressure variation, specifically focusing on telomere associated genes across placental mammals. In general we observe a large number of instances of positive selection acting on telomere genes. Although these signatures of selection overall are not significantly correlated with either longevity or body size we do identify positive selection in the microbat species Myotis lucifugus in functionally important regions of the telomere maintenance genes DKC1 and TERT, and in naked mole rat in the DNA repair gene BRCA1.These results demonstrate the multifarious selective pressures acting across the mammal phylogeny driving lineage-specific adaptations of telomere associated genes. Our results show that regardless of the longevity of a species, these proteins have evolved under positive selection thereby removing increased longevity as the single selective force driving this rapid rate of evolution. However, evidence of molecular adaptations specific to naked mole rat and Myotis lucifugus highlight functionally significant regions in genes that may alter the way in which telomeres are regulated and maintained in these longer-lived species.
- Naked eye detection of trace cancer biomarkers based on biobarcode and enzyme-assisted DNA recycling hybrid amplifications. [Journal Article]
- Biosens Bioelectron 2014 Mar 15.:494-8.
Naked eye-based detection has received increasing research interest due to the simplicity nature of this type of assay. However, improving the sensitivity of the naked eye detection method for the monitoring of trace amount of target molecules remains a major challenge. Herein, we describe a biobarcode and an enzyme-assisted DNA recycling hybrid amplification strategy for naked eye detection of sub-picomolar carcinoembryonic antigen (CEA), a cancer biomarker. The presence of CEA and the corresponding antibodies results in the formation of immunocomplexes and the capture of the biobarcodes in a microplate. The massive barcode DNAs released from the biobarcodes hybridize with the G-quadruplex inactive hairpin DNA probes and form catalytic nicking sites for N.BstNBI endonuclease, which cleaves the barcode DNA/hairpin partial dsDNA, releases the G-quadruplex active sequences and recycles the barcode DNA. Due to the barcode DNA recycling process, numerous G-quadruplex active sequences are generated and associate with hemin to form peroxidase mimicking enzymes, which convert colorless ABTS(2-) to green color intensified ABTS(•-) to achieve naked eye detection of CEA down to 0.025ngmL(-1) (0.14pM). The naked eye detection strategy reported herein can be applied also to complicated serum sample matrix, making this approach hold great promise for point-of-care diagnostic applications.
- Synthetic DNA Vaccines: Improved Vaccine Potency by Electroporation and Co-Delivered Genetic Adjuvants. [REVIEW]
- Front Immunol 2013.:354.
In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.
- Colorimetric Determination of DNA Methylation Based on the Strength of the Hydrophobic Interactions between DNA and Gold Nanoparticles. [Journal Article]
- ACS Appl Mater Interfaces 2013 Nov 27; 5(22):12045-51.
A simple, novel colorimetric nanosensor for DNA methylation based on the strength of hydrophobic interaction between DNA and gold nanoparticles was proposed. The nanosensing of oligonucleotides with four nitrogen bases was first demonstrated by dividing the bases into two groups (A/T and C/G) using the representative colors that correspond to Watson-Crick base pairing. By treatment of the genomic DNA with sodium bisulfite followed by PCR amplification, the methylation level of nasopharyngeal carcinoma cells treated with 5-aza-2'-deoxycytidine for up to 5 days could be discriminated by naked eye observation. Furthermore, 12 cancer cell lines that demonstrate heterogeneity with respect to DNA methylation could also be distinguished using the nanosensor, even for amplicons as long as 342 bp. These results demonstrate that the proposed colorimetric nanosensor could potentially be useful in epigenetic studies.