Effect of ionic strength solution on the stability of chitosan-DNA nanoparticles

Chitosan-DNA nanoparticles employed in gene therapy protocols consist of a neutralised, stoichiometric core and a shell of the excess of chitosan which stabilises the particles against further coagulation. At low ionic strength, these nanoparticles possess a high stability; however, as the ionic strength increases, it weakens the electrostatic repulsion which can play a decisive part in the formation of highly aggregated particles. In this study, new results about the effect of ionic strength on the colloidal stability of chitosan-DNA nanoparticles were obtained by studying the interaction between chitosans of increasing molecular weights (5, 10, 16, 29, 57 and 150 kDa) and calf thymus DNA. The physicochemical properties of polyplexes were investigated by means of dynamic light scattering, static fluorescence spectroscopy, optic microscopy, transmission electronic microscopy and gel electrophoresis. After subsequent addition of salt to the nanoparticles solution, secondary aggregation increased the size of the polyplexes. The nanoparticles stability decreased drastically at the ionic strengths 150 and 500 mM, which caused the corresponding decrease in the thickness of the stabilising shell. The morphologies of chitosan/DNA nanoparticles at those ionic strengths were a mixture of large spherical aggregates, toroids and rods. The results indicated that to obtain stable chitosan-DNA nanoparticles, besides molecular weight and N/P ratio, it is quite important to control the ionic strength of the solution. © 2013 Copyright Taylor and Francis Group, LLC.

Tracking the evolution of sex chromosome systems in Melanoplinae grasshoppers through chromosomal mapping of repetitive DNA sequences

Background: The accumulation of repetitive DNA during sex chromosome differentiation is a common feature of many eukaryotes and becomes more evident after recombination has been restricted or abolished. The accumulated repetitive sequences include multigene families, microsatellites, satellite DNAs and mobile elements, all of which are important for the structural remodeling of heterochromatin. In grasshoppers, derived sex chromosome systems, such as neo-XY♂/XX♀ and neo-X1X2Y♂/X 1X1X2X2♀, are frequently observed in the Melanoplinae subfamily. However, no studies concerning the evolution of sex chromosomes in Melanoplinae have addressed the role of the repetitive DNA sequences. To further investigate the evolution of sex chromosomes in grasshoppers, we used classical cytogenetic and FISH analyses to examine the repetitive DNA sequences in six phylogenetically related Melanoplinae species with X0♂/XX♀, neo-XY♂/XX♀ and neo-X1X2Y♂/X1X1X 2X2♀ sex chromosome systems. Results: Our data indicate a non-spreading of heterochromatic blocks and pool of repetitive DNAs (C 0 t-1 DNA) in the sex chromosomes; however, the spreading of multigene families among the neo-sex chromosomes of Eurotettix and Dichromatos was remarkable, particularly for 5S rDNA. In autosomes, FISH mapping of multigene families revealed distinct patterns of chromosomal organization at the intra- and intergenomic levels. Conclusions: These results suggest a common origin and subsequent differential accumulation of repetitive DNAs in the sex chromosomes of Dichromatos and an independent origin of the sex chromosomes of the neo-XY and neo-X1X2Y systems. Our data indicate a possible role for repetitive DNAs in the diversification of sex chromosome systems in grasshoppers. © 2013Palacios-Gimenez et al.; licensee BioMed Central Ltd.

Chromosomal organization of repetitive DNA sequences in Astyanax bockmanni (Teleostei, Characiformes): dispersive location, association and co-localization in the genome

Repetitive DNA sequences constitute a great portion of the genome of eukaryotes and are considered key components to comprehend evolutionary mechanisms and karyotypic differentiation. Aiming to contribute to the knowledge of chromosome structure and organization of some repetitive DNA classes in the fish genome, chromosomes of two allopatric populations of Astyanax bockmanni were analyzed using classic cytogenetics techniques and fluorescent in situ hybridization, with probes for ribosomal DNA sequences, histone DNA and transposable elements. These Astyanax populations showed the same diploid number (2n = 50), however with differences in chromosome morphology, distribution of constitutive heterochromatin, and location of 18S rDNA and retroelement Rex3 sites. In contrast, sites for 5S rDNA and H1, H3 and H4 histones showed to be co-located and highly conserved. Our results indicate that dispersion and variability of 18S rDNA and heterochromatin sites are not associated with macro rearrangements in the chromosome structure of these populations. Similarly, distinct evolutionary mechanisms would act upon histone genes and 5S rDNA, contributing to chromosomal association and co-location of these sequences. Data obtained indicate that distinct mechanisms drive the spreading of repetitive DNAs in the genome of A. bockmanni. Also, mobile elements may account for the polymorphism of the major rDNA sites and heterochromatin in this genus. © 2013 Springer Science+Business Media Dordrecht.

Amplificação de DNA circulante para o diagnóstico da cisticercose bovina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Karyotypic and morphological divergence between two cryptic species of Eigenmannia in the Amazon basin with a new occurrence of XX/XY sex chromosomes (Gymnotiformes: Sternopygidae)

Espécies de Eigenmannia estão amplamente distribuídas na região Neotropical, com oito espécies válidas atualmente reconhecidas. Populações de Eigenmannia de três localidades do leste da Amazônia foram investigadas usando técnicas citogenéticas e morfológicas, revelando dois táxons designados aqui comoEigenmannia sp. "A" e Eigenmannia sp. "B". As espécies diferem em três caracteres morfométricos, dois merísticos e um osteológico. Eigenmannia sp. "A" apresenta 2n = 34 (22 m/sm+12st/a) e Eigenmannia sp. "B" apresenta 2n = 38 (14 m/sm+24st/a) e cromossomos sexuais de diferenciação simples, do tipo XX/XY. Em ambas espécies a Heterocromatina Constitutiva (HC) rica em bases A-T está distribuída na região centromérica de todos os cromossomos. Eigenmannia sp. "B" também apresenta blocos de HC na região intersticial dos pares cromossômicos 8, 9 e X que coraram positivamente para CMA₃, indicando regiões ricas em G-C. A NOR está localizada no braço curto do par 17 em Eigenmannia sp. "A" e no braço curto do par 14 em Eigenmannia sp. "B". FISH com sondas de rDNA hibridizaram em regiões de tamanhos diferentes entre os homólogos, sugerindo heteromorfismo. A diferenciação do cromossomo X em Eigenmannia sp. "B" pode ser o resultado de amplificação de sequências repetitivas de DNA.

Chromosomal Mapping of Repetitive DNA and Cytochrome C Oxidase I Sequence Analysis Reveal Differentiation among Sympatric Samples of Astyanax fasciatus (Characiformes, Characidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Amplification of neuromuscular transmission by methylprednisolone involves activation of presynaptic facilitatory adenosine A(2A) receptors and redistribution of synaptic vesicles

The mechanisms underlying improvement of neuromuscular transmission deficits by glucocorticoids are still a matter of debate despite these compounds have been used for decades in the treatment of autoimmune myasthenic syndromes. Besides their immunosuppressive action, corticosteroids may directly facilitate transmitter release during high-frequency motor nerve activity. This effect coincides with the predominant adenosine A(2A) receptor tonus, which coordinates the interplay with other receptors (e.g. muscarinic) on motor nerve endings to sustain acetylcholine (ACh) release that is required to overcome tetanic neuromuscular depression in myasthenics. Using myographic recordings, measurements of evoked [H-3]ACh release and real-time video microscopy with the FM4-64 fluorescent dye, results show that tonic activation of facilitatory A(2A) receptors by endogenous adenosine accumulated during 50 Hz bursts delivered to the rat phrenic nerve is essential for methylprednisolone (03 mM)-induced transmitter release facilitation, because its effect was prevented by the A(2A) receptor antagonist, ZM 241385 (10 nM). Concurrent activation of the positive feedback loop operated by pirenzepine-sensitive muscarinic M-1 autoreceptors may also play a role, whereas the corticosteroid action is restrained by the activation of co-expressed inhibitory M-2 and Al receptors blocked by methoctramine (0.1 mu M) and DPCPX (2.5 nM), respectively. Inhibition of FM4-64 loading (endocytosis) by methylprednisolone following a brief tetanic stimulus (50 Hz for 5 s) suggests that it may negatively modulate synaptic vesicle turnover, thus increasing the release probability of newly recycled vesicles. Interestingly, bulk endocytosis was rehabilitated when methylprednisolone was co-applied with ZM241385. Data suggest that amplification of neuromuscular transmission by methylprednisolone may involve activation of presynaptic facilitatory adenosine A(2A) receptors by endogenous adenosine leading to synaptic vesicle redistribution. (C) 2014 Elsevier Ltd. All rights reserved.

Genotypic characterization of ten microsatellite loci in two Brazilian free range (Caipira) chicken lines

This study aimed to investigate the genetic variability of two Brazilian free range (Caipira) chickens lines using microsatellites analysis of ten loci. It was collected a total of 99 blood samples, which 49 were from Paraiso Pedres (PP) and 50 were from Rubro Negra (RN) lines. The amplification of the DNA fragments was performed by polymerase chain reaction (PCR) and the genotyping was conduct using ABI 3130 sequencer. The allele number variation was among 3 (LEI0254) to 32 (LEI0212) in the PP line, and 4 (LEI0254) to 31 (LEI0212) in the RN line. The allelic average per locus was 13.3 and 13.1 in the PP and RN lines, respectively. The average observed and the expected heterozygosity were 0.650 and 0.820 in the PP line, and 0.671 and 0.804 in the RN line. All of the analyzed loci were informative (PIC>0.5). These results indicate that these free-range animals have a high genetic variability, at least for the majority of the analyzed loci, and this genetic variation is higher than the commercial chickens and similar for the no-commercial birds.

Gallus gallus domesticus are resistant to infection with Neospora caninum tachyzoites of the NC-1 strain

The aim of this study was to experimentally evaluate infection in Gallus gallus domesticus with Neospora caninum tachyzoites of the NC-1 strain. Experimental infection was conducted in 90-day-old chickens, embryonated eggs and bioassays in dogs. In the first experiment, poults were randomly divided into four groups. Groups I and II were provided feed with coccidiostat, whereas groups III and IV received feed without coccidiostat. When the poults from groups I and III reached 90 days of age, they received a subcutaneous inoculation of N. caninum. Once the hens entered their egg-laying period, during the following 30 days, the eggs were collected, identified, weighed and placed in an incubator. On the 70th day after inoculation, all animals, including the chicks, were euthanized. Tissue samples from the adult poultry and chicks were collected for histopathology, immunohistochemistry (IHC) and PCR. Brain tissue and pectoral muscle samples from infected birds were fed to two dogs. Notably, the average weight of the group III eggs was lower than that of the group IV eggs (p <0.05). No changes consistent with infection in adult poultry or chicks were detected by histopathology or IHC; moreover, no amplified parasite DNA was detected in the birds'tissues or dogs'feces. No dog eliminated oocysts. In the second experiment, the embryonated chicken eggs were inoculated with 1 x 10(2) N. caninum tachyzoites, on the 10th day of incubation, and chicks born from these eggs were housed in boxes suitable for the species and received commercial feed and distilled water ad libitum. On the 30th day after infection (DAI), the poultry were euthanized, and their organs were processed as described in experiment I. The amplification of parasite DNA was observed in the spleen and pectoral muscles of one of the birds. The ingestion of bird tissues by dogs did not result in oocyst elimination. These results indicate that the parasite may have been eliminated by the host and that the use of tachyzoites to induce chronic disease might be a poor source for hens. (C) 2014 Elsevier B.V. All rights reserved.

Comparação entre dois métodos de detecção de DNA de papilomavírus humano em carcinoma epidermoide de lábio

Introdução: Recentemente o papilomavírus humano (HPV) tem sido associado à carcinogênese oral. A metodologia empregada na detecção do vírus é uma das maiores causas observadas da grande variabilidade nas taxas de detecção do HPV. Objetivo: Este estudo comparou a sensibilidade de detecção do DNA do HPV em casos de carcinoma epidermoide de lábio utilizando a amplificação do DNA viral por reação em cadeia da polimerase (PCR) ou nPCR. Material e método: Foram utilizadas 33 amostras provenientes de casos de carcinoma epidermoide de lábio. Para as extrações do DNA utilizou-se o sistema QIAamp DNA Mini Kit. Como controle interno utilizou-se o gene da b-globina. Das 33 amostras iniciais, 30 foram positivas para o gene b-globina, sendo utilizadas para detectar o DNA viral. Comparou-se a amplificação do DNA viral pelos métodos da PCR com os oligonucleotídeos MY09/MY11 e nPCR, empregando-se os pares de oligonucleotídeos iniciadores MY09/MY11 e, na segunda etapa, o par GP5+/GP6+. O controle positivo para a presença do DNA do HPV utilizado foi a linhagem de células HeLa e, como controle negativo, a mistura de amplificação sem DNA. A análise dos produtos de PCR e nPCR para HPV foi realizada por eletroforese em gel de poliacrilamida a 8%. Resultados: Utilizando-se o método da PCR, a amplificação do DNA do HPV foi constatada em dois casos. Com a nPCR foi verificada presença de DNA viral em 13 das 30 amostras. Conclusão: Com a utilização da nPCR, a detecção do HPV nos casos estudados aumentou mais de seis vezes.

New species of Moenkhausia Eigenmann (Characiformes: Characidae) from Rio Xingu and Rio Tapajos basins, Brazil, with comments on a putative case of polymorphic Batesian mimicry

A new species of Moenkhausia is described from Rio Xingu and Rio Tapajos basins, Brazil. The new species is distinguished from its congeners, except from Moenkhausia moisae, by having more scales in the lateral series, 43-47 (v. 23-41 in the remaining congeners). The new species is distinguished from M. moisae by its colour pattern, which consists of a dark midlateral stripe, and an asymmetrical caudal blotch (inconspicuous or faded in specimens from the Rio Arinos) continuous with the midlateral stripe (v. narrow dark midlateral line and conspicuous, regularly rounded and symmetrical blotch not continuous with the midlateral line). The new species is putatively assumed to be mimetic to Jupiaba apenima, in the Rio Xingu and Rio Teles Pires drainages, and to Jupiaba yarina in the Rio Arinos. The two species of Jupiaba are sympatric and remarkably similar in size, general external morphology and colouration to the new species. A small difference occurs in the colouration between the two species of Jupiaba and is also observed in the two respectively sympatric morphotypes of the new species of Moenkhausia. The occurrence of polymorphic Batesian mimicry is therefore discussed for neotropical freshwater fishes. (C) 2009 The Authors Journal compilation (C) 2009 The Fisheries Society of the British Isles

COMPARISON OF DIFFERENT PROTOCOLS FOR THE EXTRACTION OF MICROBIAL DNA FROM REEF CORALS

This study aimed to test different protocols for the extraction of microbial DNA from the coral Mussismilia harttii. Four different commercial kits were tested, three of them based on methods for DNA extraction from soil (FastDNA SPIN Kit for soil, MP Bio, PowerSoil DNA Isolation Kit, MoBio, and ZR Soil Microbe DNA Kit, Zymo Research) and one kit for DNA extraction from plants (UltraClean Plant DNA Isolation Kit, MoBio). Five polyps of the same colony of M. harttii were macerated and aliquots were submitted to DNA extraction by the different kits. After extraction, the DNA was quantified and PCR-DGGE was used to study the molecular fingerprint of Bacteria and Eukarya. Among the four kits tested, the ZR Soil Microbe DNA Kit was the most efficient with respect to the amount of DNA extracted, yielding about three times more DNA than the other kits. Also, we observed a higher number and intensities of DGGE bands for both Bacteria and Eukarya with the same kit. Considering these results, we suggested that the ZR Soil Microbe DNA Kit is the best adapted for the study of the microbial communities of corals.

Characterization of flexible DNA films

Polymer electrolytes (PEs) are currently the focus of much attention as potential electrolytes in electrochemical devices such as batteries, display devices and sensors. Deoxyribonucleic acid (DNA) as an important biological macromolecule has electric conducting electrochemical properties and unique three dimensional structures. With the goal of developing a new family of environmentally friendly multifunctional biohybrid materials displaying simultaneously high ionic conductivity we have produced in the present work, flexible films based on DNA, incorporating ionic liquids (ILs). Over the last decade ILs have been employed as a new media in electrochemistry and electroanalysis. The materials studied here have been characterized by means of Differential Scanning Calorimetry, Complex Impedance Spectroscopy and Cyclic Voltammetry. (C) 2012 Elsevier B.V. All rights reserved.