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naked DNA [keywords]
- Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes. [JOURNAL ARTICLE]
- Anal Chem 2014 Nov 21.
We have developed a self-reporting isothermal system for visual bacterial pathogen detection with single base resolution. The new DNA diagnostic is based on combination of Peptide Nucleic Acid (PNA) technology, rolling circle amplification (RCA) and DNAzymes. PNAs are used as exceedingly selective chemical tools that bind genomic DNA at a predetermined sequence under non-denaturing conditions. After assembly of the PNA-DNA construct a padlock probe is circularized on the free strand. The probe incorporates a G-quadruplex structure flanked by nicking enzyme recognition sites. The assembled circle serves as a template for a novel hybrid RCA strategy that allows for exponential amplification and production of short single-stranded DNA pieces. These DNA fragments fold into G-quadruplex structures and when complexed with hemin become functional DNAzymes. The catalytic activity of each DNAzyme unit leads to colorimetric detection and provides the second amplification step. The combination of PNA, RCA, and DNAzymes allows for sequence-specific and highly sensitive detection of bacteria with a colorimetric output observed with the naked eye. Herein, we apply this method for the discrimination of Escherichia coli, Salmonella typhimurium, and Clostridium difficile genomes and achieve a limit of detection of ranging between 1 and 100fM depending on the target species.
- Next-Generation Environmental Diversity Surveys of Foraminifera: Preparing the Future. [REVIEW]
- Biol Bull 2014 Oct; 227(2):93-106.
Foraminifera are commonly defined as marine testate protists, and their diversity is mainly assessed on the basis of the morphology of their agglutinated or mineralized tests. Diversity surveys based on environmental DNA (eDNA) have dramatically changed this view by revealing an unexpected diversity of naked and organic-walled lineages as well as detecting foraminiferal lineages in soil and freshwater environments. Moreover, single-cell analyses have allowed discrimination among genetically distinctive types within almost every described morphospecies. In view of these studies, the foraminiferal diversity appeared to be largely underestimated, but its accurate estimation was impeded by the low speed and coverage of a cloning-based eDNA approach. With the advent of high-throughput sequencing (HTS) technologies, these limitations disappeared in favor of exhaustive descriptions of foraminiferal diversity in numerous samples. Yet, the biases and errors identified in early HTS studies raised some questions about the accuracy of HTS data and their biological interpretation. Among the most controversial issues affecting the reliability of HTS diversity estimates are (1) the impact of technical and biological biases, (2) the sensitivity and specificity of taxonomic sequence assignment, (3) the ability to distinguish rare species, and (4) the quantitative interpretation of HTS data. Here, we document the lessons learned from previous HTS surveys and present the current advances and applications focusing on foraminiferal eDNA. We discuss the problems associated with HTS approaches and predict the future trends and avenues that hold promises for surveying foraminiferal diversity accurately and efficiently.
- The first report of luminescent liver tissue in fishes: Evolution and structure of bioluminescent organs in the deep-sea naked barracudinas (Aulopiformes: Lestidiidae). [JOURNAL ARTICLE]
- J Morphol 2014 Nov 18.
Bioluminescent organs that provide ventral camouflage are common among fishes in the meso-bathypelagic zones of the deep sea. However, the anatomical structures that have been modified to produce light vary substantially among different groups of fishes. Although the anatomical structure and evolutionary derivation of some of these organs have been well studied, the light organs of the naked barracudinas have received little scientific attention. This study describes the anatomy and evolution of bioluminescent organs in the Lestidiidae (naked barracudinas) in the context of a new phylogeny of barracudinas and closely related alepisauroid fishes. Gross and histological examination of bioluminescent organs or homologous structures from preserved museum specimens indicate that the ventral light organ is derived from hepatopancreatic tissue and that the antorbital spot in Lestrolepis is, in fact, a second dermal light organ. In the context of the phylogeny generated from DNA-sequence data from eight gene fragments (7 nuclear and 1 mitochondrial), a complex liver with a narrow ventral strand running along the ventral midline evolves first in the Lestidiidae. The ventral hepatopancreatic tissue later evolves into a ventral bioluminescent organ in the ancestor of Lestidium and Lestrolepis with the lineage leading to the genus Lestrolepis evolving a dermal antorbital bioluminescent organ, likely for light-intensity matching. This is the first described hepatopancreatic bioluminescent organ in fishes. J. Morphol., 2014. © 2014 Wiley Periodicals, Inc.
- Concatenated Logic Circuits Based on a Three-Way DNA Junction: A Keypad-Lock Security System with Visible Readout and an Automatic Reset Function. [JOURNAL ARTICLE]
- Angew Chem Int Ed Engl 2014 Nov 17.
Concatenated logic circuits operating as a biocomputing keypad-lock security system with an automatic reset function have been successfully constructed on the basis of toehold-mediated strand displacement and three-way-DNA-junction architecture. In comparison with previously reported keypad locks, the distinctive advantage of the proposed security system is that it can be reset and cycled spontaneously a large number of times without an external stimulus, thus making practical applications possible. By the use of a split-G-quadruplex DNAzyme as the signal reporter, the output of the keypad lock can be recognized readily by the naked eye. The "lock" is opened only when the inputs are introduced in an exact order. This requirement provides defense against illegal invasion to protect information at the molecular scale.
- Development of the MITO-Porter, a Nano Device for Mitochondrial Drug Delivery via Membrane Fusion. [Journal Article]
- Yakugaku Zasshi 2014; 134(11):1143-55.
Many human diseases have been reported to be associated with mitochondrial dysfunction. Therefore, mitochondrial therapy would be expected to be useful and productive in the treatment of various diseases. To achieve such an innovative therapy, it will be necessary to deliver therapeutic agents into mitochondria. However, only a limited number of methods are available for accomplishing this. We previously developed the MITO-Porter, a liposome-based carrier that permits macromolecular cargos to be transported into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule confirmed the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, when we attempted the mitochondrial delivery of bongkrekic acid (BKA), an antiapoptosis agent, the MITO-Porter enhanced the antiapoptosis effect compared with naked BKA. To construct a device with enhanced performance, the MITO-Porter was coated with cell membrane-fusogenic outer envelopes to produce the dual function (DF)-MITO-Porter. Intracellular observations indicated that the DF-MITO-Porter was more effective in delivering exogenous macromolecules into mitochondria than the conventional MITO-Porter. Furthermore, when biomacromolecules were delivered using the DF-MITO-Porter to estimate the mitochondrial gene targeting of the carrier, the results confirmed that the MITO-Porter system has the potential for use in therapies aimed at mitochondrial DNA. This paper sumarizes our findings on mitochondrial drug delivery systems that are directed toward mitochondrial medicine development and mitochondrial gene therapy. It is expected that the MITO-Porter system will open new research areas in mitochondrial drug delivery systems and have a significant impact on the medical and life sciences.
- A new species of Harpacticella Sars, 1908 (Copepoda, Harpacticoida), from a tidal pool on Jeju Island, Korea. [Journal Article]
- Zookeys 2014; (445):13-30.
A new species of the genus Harpacticella Sars, 1908 is described from a tidal pool on Jeju Island, Korea. Harpacticellajejuensis sp. n. is closely related to Harpacticellaitoi Chang & Kim, 1991, with regard to the structure of P1 exp-1 and enp-1, the length of P1 exp-1 and exp-2, and the setal number of the P5 exopod in males. However, the new species is clearly distinguishable from Harpacticellaitoi by the combined following characters: six setae on the P5 exopod in females, one naked seta on the inner margin of P1 exp-2, the short endopod of P1 compared to the exopod, and a naked long seta on the proximal inner margin of the P5 exopod of males. The mtCOI partial sequence is provided as a DNA barcode for the new species.
- Rapid Detection of Staphylococcus Aureus by Loop-Mediated Isothermal Amplification. [JOURNAL ARTICLE]
- Appl Biochem Biotechnol 2014 Oct 28.
Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), is a major bacterial pathogen associated with nosocomial and community-acquired S. aureus infections all over the world. A rapid detection assay for staphylococcal gene of nuc and mecA is needed. In this study, a rapid identification assay based on the loop-mediated isothermal amplification (LAMP) method was established. PCR and LAMP assays were used to detect Staphylococcus aureus and other related species for nuc and mecA. With optimization of the primers and reaction temperature, the LAMP successfully amplified the genes under isothermal conditions at 62 °C within 60 min, of which the results were identical with those of the conventional PCR methods. The detection limits of the LAMP for nuc and mecA were 1.47 and 14.7 pg/μl DNA per tube, respectively, by naked eye inspections, while the detection limits of the PCR for nuc and mecA were 14.7 pg/μl and 147 pg/μl DNA, respectively. Finally, The LAMP method was then applied to clinical blood plaque samples. The LAMP and PCR demonstrated identical results for the plaque samples with the culture assay. Together, the LAMP offers an alternative detection assay for nuc and mecA with a great advantage of the sensitivity and rapidity.
- Non canonical cell-to-cell DNA transfer in Thermus spp. is insensitive to Argonaute-mediated interference. [JOURNAL ARTICLE]
- J Bacteriol 2014 Oct 20.
Horizontal gene transfer drives the rapid evolution of bacterial populations. Classical processes that promote lateral flow of genetic information are conserved throughout the prokaryotic world. However, some species hold non-conserved transfer mechanisms which are poorly known. This is the case for the ancient extreme thermophile Thermus thermophilus. In this work, we show that T. thermophilus strains are capable of exchanging large DNA fragments by a novel mechanism which requires cell-to-cell contacts and employs components of the natural transformation machinery. This process facilitates bidirectional transfer of virtually any DNA locus, but favors by tend folds those found in the megaplasmid compared to those in the chromosome. In contrast to naked DNA acquisition by transformation, the system does not activate the recently described DNA-DNA interference mechanism mediated by the prokaryotic Argonaute protein, thus allowing the organism to discriminate between DNA transferred from a mate and exogenous DNA acquired from unknown hosts. This Argonaute-mediated discrimination may be tentatively viewed as a strategy to safely share potentially "useful" traits by the components of a given population of Thermus spp without increasing the genome size of its individuals.
- Distribution of human-specific Bacteroidales and fecal indicators in an urban watershed impacted by sewage pollution using RNA and DNA based quantitative PCR assays. [JOURNAL ARTICLE]
- Appl Environ Microbiol 2014 Oct 17.
The identification of fecal pollution sources is commonly carried out using DNA-based methods. However, there is evidence that DNA can be associated with dead cells or present as "naked DNA" in the environment. Furthermore, it has been shown that rRNA-targeted RT-qPCR assays can be more sensitive than rDNA-based qPCR assays since metabolically active cells usually contain higher numbers of ribosomes than quiescent cells. To this end, we compared the detection frequency of host specific markers and fecal bacteria using RNA-based RT-qPCR and DNA-based qPCR methods for water samples collected in sites impacted by combined sewer overflows. As a group, fecal bacteria were more frequently detected in most sites using RNA-based methods. Specifically, 8%, 87%, and 85% of the samples positive for general enterococci, Enterococcus faecalis, and E. faecium markers respectively, were detected using RT-qPCR, but not with the qPCR assay counterpart. On average, two human-specific Bacteroidales markers were not detected when using DNA in 12% of the samples while they were positive for all samples when using RNA (cDNA) as the template. Moreover, signal intensity was up to three orders of magnitude higher in RT-qPCR assays than in qPCR assays. The human-specific Bacteroidales markers exhibited moderate correlation with conventional fecal indicators using RT-qPCR results, suggesting the persistence of non-human sources of fecal pollution or the presence of false positive signals. In general, the results from this study suggest that RNA-based assays can increase the detection sensitivity of fecal bacteria in urban watersheds impacted with human fecal sources.
- Single molecule fluorescence methodologies for investigating transcription factor binding kinetics to nucleosomes and DNA. [JOURNAL ARTICLE]
- Methods 2014 Oct 7.
Site specific DNA binding complexes must bind their DNA target sites and then reside there for a sufficient amount of time for proper regulation of DNA processing including transcription, replication and DNA repair. In eukaryotes, the occupancy of DNA binding complexes at their target sites is regulated by chromatin structure and dynamics. Methodologies that probe both the binding and dissociation kinetics of DNA binding proteins with naked and nucleosomal DNA are essential for understanding the mechanisms by which these complexes function. Here, we describe single-molecule fluorescence methodologies for quantifying the binding and dissociation kinetics of transcription factors at a target site within DNA, nucleosomes and nucleosome arrays. This approach allowed for the unexpected observation that nucleosomes impact not only binding but also dissociation kinetics of transcription factors and is well-suited for the investigation of numerous DNA processing complexes that directly interact with DNA organized into chromatin.