Progress towards Elimination of HIV Mother-to-Child Transmission in the Dominican Republic from 1999 to 2011

In 1999, prevention of mother-to-child transmission (pMTCT) using antiretrovirals was introduced in the Dominican Republic (DR). Highly active antiretroviral therapy (HAART) was introduced for immunosuppressed persons in 2004 and for pMTCT in 2008. To assess progress towards MTCT elimination, data from requisitions for HIV nucleic acid amplification tests for diagnosis of HIV infection in perinatally exposed infants born in the DR from 1999 to 2011 were analyzed. The MTCT rate was 142/1,274 (11.1%) in 1999–2008 and 12/302 (4.0%) in 2009–2011 (), with a rate of 154/1,576 (9.8%) for both periods combined. This decline was associated with significant increases in the proportions of women who received prenatal HAART (from 12.3% to 67.9%) and infants who received exclusive formula feeding (from 76.3% to 86.1%) and declines in proportions of women who received no prenatal antiretrovirals (from 31.9% to 12.2%) or received only single-dose nevirapine (from 39.5% to 19.5%). In 2007, over 95% of DR pregnant women received prenatal care, HIV testing, and professionally attended delivery. However, only 58% of women in underserved sugarcane plantation communities (2007) and 76% in HIV sentinel surveillance hospitals (2003–2005) received their HIV test results. HIV-MTCT elimination is feasible but persistent lack of access to critical pMTCT measures must be addressed.

Current status of gene therapy for breast cancer: progress and challenges

Breast cancer is characterized by a series of genetic mutations and is therefore ideally placed for gene therapy intervention. The aim of gene therapy is to deliver a nucleic acid-based drug to either correct or destroy the cells harboring the genetic aberration. More recently, cancer gene therapy has evolved to also encompass delivery of RNA interference technologies, as well as cancer DNA vaccines. However, the bottleneck in creating such nucleic acid pharmaceuticals lies in the delivery. Deliverability of DNA is limited as it is prone to circulating nucleases; therefore, numerous strategies have been employed to aid with biological transport. This review will discuss some of the viral and nonviral approaches to breast cancer gene therapy, and present the findings of clinical trials of these therapies in breast cancer patients. Also detailed are some of the most recent developments in nonviral approaches to targeting in breast cancer gene therapy, including transcriptional control, and the development of recombinant, multifunctional bio-inspired systems. Lastly, DNA vaccines for breast cancer are documented, with comment on requirements for successful pharmaceutical product development.

Diagnosis of canine brucellosis: Comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer

A pair of primers directed to 16S-23S rDNA interspacer (ITS) was designed directed to Brucella genetic sequences in order to develop a polymerase chain reaction (PCR) putatively capable of amplifying DNA from any Brucella species. Nucleic acid extracts from whole-blood from naive dogs were spiked with decreasing amounts of Brucella canis RM6/66 DNA and the resulting solutions were tested by PCR. In addition, the ability of PCR to amplify Brucella spp. genetic sequences from naturally infected dogs was evaluated using 210 whole-blood samples of dogs from 19 kennels. The whole-blood samples collected were subjected to blood culture and PCR. Serodiagnosis was performed using the rapid slide agglutination test with and without 2-mercaptoethanol. The DNA from whole blood was extracted using proteinase-K, sodium dodecyl sulphate and cetyl trimethyl ammonium bromide followed by phenol-chloroform purification. The PCR was capable of detecting as little as 3.8 fg of Brucella DNA mixed with 450 ng of host DNA. Theoretically, 3.8 fg of Brucella DNA represents the total genomic mass of fewer than two bacterial cells. The PCR diagnostic sensitivity and specificity were 100%. From the results observed in the present study, we conclude that PCR could be used as confirmatory test for diagnosis of B. canis infection.

Multicentre validation study of nucleic acids extraction from FFPE tissues

In most pathology laboratories worldwide, formalin-fixed paraffin embedded (FFPE) samples are the only tissue specimens available for routine diagnostics. Although commercial kits for diagnostic molecular pathology testing are becoming available, most of the current diagnostic tests are laboratory-based assays. Thus, there is a need for standardized procedures in molecular pathology, starting from the extraction of nucleic acids. To evaluate the current methods for extracting nucleic acids from FFPE tissues, 13 European laboratories, participating to the European FP6 program IMPACTS (www.impactsnetwork.eu), isolated nucleic acids from four diagnostic FFPE tissues using their routine methods, followed by quality assessment. The DNA-extraction protocols ranged from homemade protocols to commercial kits. Except for one homemade protocol, the majority gave comparable results in terms of the quality of the extracted DNA measured by the ability to amplify differently sized control gene fragments by PCR. For array-applications or tests that require an accurately determined DNA-input, we recommend using silica based adsorption columns for DNA recovery. For RNA extractions, the best results were obtained using chromatography column based commercial kits, which resulted in the highest quantity and best assayable RNA. Quality testing using RT-PCR gave successful amplification of 200 bp-250 bp PCR products from most tested tissues. Modifications of the proteinase-K digestion time led to better results, even when commercial kits were applied. The results of the study emphasize the need for quality control of the nucleic acid extracts with standardised methods to prevent false negative results and to allow data comparison among different diagnostic laboratories.

A phase II study of the modulation of 5-fluorouracil and folinic acid with high-dose infusional hydroxyurea in metastatic colorectal carcinoma

Background: Hydroxyurea (HU), an inhibitor of ribonucleotide reductase, may potentiate the activity of 5-fluorouracil (5-FU) and folinic acid (FA) by reducing the deoxyribonucleotide pool available for DNA synthesis and repair. However as HU may inhibit the formation of 5-fluoro-2-deoxyuridine-5- monophosphate (FdUMP), one of the principal active metabolites of 5-FU, the scheduling of HU may be critical. In vitro experiments suggest that administration of HU following 5-FU, maintaining the concentration in the region of I mM for six or more hours, significantly enhances the efficacy of 5-FU. Patients and methods: 5-FU/FA was given as follows: days 1 and 2 - FA 250 mg/m 2 (max. 350 mg) over two hours followed by 5-FU 400 mg/m 2 by intravenous bolus (ivb) over 15 minutes and subsequently 5-FU 400 mg/m 2 infusion (ivi) over 22 hours. HU was administered on day 3 immediately after the 5-FU with 3 g ivb over 15 minutes followed by 12 g ivi over 12 hours. Results: Thirty patients were entered into the study. Median survival was nine months (range 1-51 + months). There were eight partial responses (28%, 95% CI: 13%-47%). The median duration of response was 6.5 (range 4-9 months). Grade 3-4 toxicities included neutropenia (grade 3 in eight patients and grade 4 in five), anaemia (grade 3 in one patient) and diarrhoea (grade 3 in two patients). Neutropenia was associated with pyrexia in two patients. Phlebitis at the infusion site occurred in five patients. The treatment was complicated by pulmonary embolism in one patient and deep venous thrombosis in another. Conclusion: HU administered in this schedule is well tolerated. Based on these results and those of other phase II studies, a randomised phase III study of 5-FU, FA and HU versus 5-FU and FA using the standard de Gramont schedule is recommended.

Applications of a sugar-based surveillance system to track arboviruses in wild mosquito populations

Effective arbovirus surveillance is essential to ensure the implementation of control strategies, such as mosquito suppression, vaccination, or dissemination of public warnings. Traditional strategies employed for arbovirus surveillance, such as detection of virus or virus-specific antibodies in sentinel animals, or detection of virus in hematophagous arthropods, have limitations as an early-warning system. A system was recently developed that involves collecting mosquitoes in CO2-baited traps, where the insects expectorate virus on sugar-baited nucleic acid preservation cards. The cards are then submitted for virus detection using molecular assays. We report the application of this system for detecting flaviviruses and alphaviruses in wild mosquito populations in northern Australia. This study was the first to employ nonpowered passive box traps (PBTs) that were designed to house cards baited with honey as the sugar source. Overall, 20/144 (13.9%) of PBTs from different weeks contained at least one virus-positive card. West Nile virus Kunjin subtype (WNVKUN), Ross River virus (RRV), and Barmah Forest virus (BFV) were detected, being identified in 13/20, 5/20, and 2/20 of positive PBTs, respectively. Importantly, sentinel chickens deployed to detect flavivirus activity did not seroconvert at two Northern Territory sites where four PBTs yielded WNVKUN. Sufficient WNVKUN and RRV RNA was expectorated onto some of the honey-soaked cards to provide a template for gene sequencing, enhancing the utility of the sugar-bait surveillance system for investigating the ecology, emergence, and movement of arboviruses. © 2014, Mary Ann Liebert, Inc.

Specificity in proteinânucleic acid interaction: Solubility study on amino acidânucleoside interaction

The chemical basis of the specificity of proteinnucleic acid interaction, as seen in many biochemical phenomena such as the organization of nucleoprotein complexes (~hro~atin. ribosomes) and gene expression and its regulation, IS not yet understood.A knowledge of such specific interactions is also essential for tracing the chemical evolution of life based an the coupling between protein and nucleic acid and the origin of genetic code [I ,I?].

A gradient PCR-based screen for use in site-directed mutagenesis

Site-directed mutagenesis is widely used to study protein and nucleic acid structure and function. Despite recent advancements in the efficiency of procedures for site-directed mutagenesis, the fraction of site-directed mutants by most procedures rarely exceeds 50% on a routine basis and is never 100%. Hence it is typically necessary to sequence two or three clones each time a site-directed mutant is constructed. We describe a simple and robust gradient-PCR-based screen for distinguishing site-directed mutants from the starting, unmutated plasmid. The procedure can use either purified plasmid DNA or colony PCR, starting from a single colony. The screen utilizes the primer used for mutagenesis and a common outside primer that can be used for all other mutants constructed with the same template. Over 30 site-specific mutants in a variety of templates were successfully screened and all of the mutations detected were subsequently confirmed by DNA sequencing. A single base pair mismatch could be detected in an oligonucleotide of 36 bases. Detection efficiency was relatively independent of starting template concentration and the nature of the outside primer used. (C) 2003 Elsevier Science (USA). All rights reserved.

Multi-dimensional Goodness-of-Fit Tests for Spectrum Sensing Based on Stochastic Distances

In this paper, we study two multi-dimensional Goodness-of-Fit tests for spectrum sensing in cognitive radios. The multi-dimensional scenario refers to multiple CR nodes, each with multiple antennas, that record multiple observations from multiple primary users for spectrum sensing. These tests, viz., the Interpoint Distance (ID) based test and the h, f distance based tests are constructed based on the properties of stochastic distances. The ID test is studied in detail for a single CR node case, and a possible extension to handle multiple nodes is discussed. On the other hand, the h, f test is applicable in a multi-node setup. A robustness feature of the KL distance based test is discussed, which has connections with Middleton's class A model. Through Monte-Carlo simulations, the proposed tests are shown to outperform the existing techniques such as the eigenvalue ratio based test, John's test, and the sphericity test, in several scenarios.

The application of metallointercalators in recognition of and charge transport in nucleic acids

Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety bind to DNA through the major groove. These metallointercalators can recognize DNA sites and perform reactions on DNA as a substrate. The site-specific metallointercalator Λ-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription factor, yAP-1, from an oligonucleotide that contains a common binding site. The demonstration that metal complexes can prevent transcription factor binding to DNA site-specifically is an important step in using metallointercalators as therapeutics.

The distinctive photochemistry of metallointercalators can also be applied to promote long range charge transport in DNA. Experiments using duplexes with regions 4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying order showed that radical migration is more dependent on the sequence of bases, and less dependent on the distance between the guanine doublets. This result suggests that mechanistic proposals of long range charge transport must involve all the bases.

RNA/DNA hybrids show charge migration to guanines from a remote site, thus demonstrating that nucleic acid stacking other than B-form can serve as a radical bridge. Double crossover DNA assemblies also provide a medium for charge transport at distances up to 100 Å from the site of radical introduction by a tethered metal complex. This radical migration was found to be robust to mismatches, and limited to individual, electronically distinct base stacks. In single DNA crossover assemblies, which have considerably greater flexibility, charge migration proceeds to both base stacks due to conformational isomers not present in the rigid and tightly annealed double crossovers.

Finally, a rapid, efficient, gel-based technique was developed to investigate thymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated via the bases of a thymine-thymine interface; reversal of this ligation is easily visualized by gel electrophoresis. This assay was used to show that the repair of thymine dimers from a distance through DNA charge transport can be accomplished with different photooxidants.

Thus, nucleic acids that support long range charge transport have been shown to include A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a method for thymine dimer repair detection using charge transport was developed. These observations underscore and extend the remarkable finding that DNA can serve a medium for charge transport via the heteroaromatic base stack.

Multifunctional label-free electrochemical biosensor based on an integrated aptamer

Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A An electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA).

Flourescent Switch Constructed Based on Hemin-Sensitive Anionic Conjugated Polymer and Its Applications in DNA-Related Sensors

Here, a fluorescent switch is constructed combining hemin, hemin aptamer, and a newly synthesized anionic conjugated polymer (ACP), poly(9,9-bis(6'-phosphate-hexyl) fluorenealt-1,4-phenylene) sodium salt (PFHPNa/PFP). In the "off-state", the fluorescence of PFP is sensitively quenched by hemin, with a high K-sv value of similar to 10(7). While in the "on-state", the formation of the aptamer/hemin complex recovers the fluorescence intensity. The fluorescent switch is sensitive and selective to hemin. To testify the universality and practicality of the fluorescent switch, a series of label-free DNA-related sensing platforms are developed, containing three DNA sensing strategies and one ATP recognition strategy. The fluorescent switch developed is simple, sensitive, and universal, which extends applications of the anionic conjugated polymers.

Colorimetric recognition of the coralyne-poly(dA) interaction using unmodified gold nanoparticle probes, and further detection of coralyne based upon this recognition system

Among the functional nucleic acids studied, adenine-rich nucleic acids have attracted attention due to their critical roles in many biological processes and self-assembly-based nanomaterials, especially deoxyribonucleic acids (abbreviated as poly(dA)). Therefore the ligands binding to poly(dA) might serve as potential therapeutic agents. Coralyne, a kind of planar alkaloid, has been firstly found that it could bind strongly to poly(dA). This work herein reports an approach for visual sensing of the coralyne-poly(dA) interaction. This method was based on the coralyne inducing poly(dA) into the homo-adenine DNA duplex and the difference in electrostatic affinity between single-stranded DNA and double-stranded DNA with gold nanoparticles (GNPs). Furthermore, we applied the recognition process of the interaction between coralyne and poly(dA) into specific coralyne detection with the assistance of certain software (such as Photoshop). A linear response from 0 to 728 nM was obtained for coralyne, and a detection limit of 91 nM was achieved.

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.

New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.

Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson-Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1'-C1' distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1'-C1' distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5'-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.