A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins

A label-free DNA aptamer-based impedance biosensor for the detection of E. coli outer membrane proteins (OMPs) was developed. Two single stranded DNA sequences were tested as recognition elements and compared. The aptamer capture probes were immobilized, with and without 6-mercapto-1-hexanol (MCH) on a gold electrode. Each step of the modification process was characterized by Faradaic impedance spectroscopy (FIS). A linear relationship between the electron-transfer resistance (Ret) and E. coli OMPs concentration was demonstrated in a dynamic detection range of 1 × 10−7–2 × 10−6 M. Moreover, the aptasensor showed selectivity despite the presence of other possible water contaminates and could be regenerated under low pH condition. The developed biosensor shows great potential to be incorporated in a biochip and used for in situ detection of E. coli OMPs in water samples.

Dynamic epigenetic changes in immune responses to infection in human dendritic cells

La méthylation de l'ADN est une marque épigénétique importante chez les mammifères. Malgré le fait que la méthylation de la cytosine en 5' (5mC) soit reconnue comme une modification épigénétique stable, il devient de plus en plus reconnu qu'elle soit un processus plus dynamique impliquant des voies de méthylation et de déméthylation actives. La dynamique de la méthylation de l'ADN est désormais bien caractérisée dans le développement et dans le fonctionnement cellulaire des mammifères. Très peu est cependant connu concernant les implications régulatrices dans les réponses immunitaires. Pour se faire, nous avons effectué des analyses du niveau de transcription des gènes ainsi que du profilage épigénétique de cellules dendritiques (DCs) humaines. Ceux-ci ont été faits avant et après infection par le pathogène Mycobacterium tuberculosis (MTB). Nos résultats fournissent le premier portrait génomique du remodelage épigénétique survenant dans les DCs en réponse à une infection bactérienne. Nous avons constaté que les changements dans la méthylation de l'ADN sont omniprésents, identifiant 3,926 régions différentiellement méthylées lors des infections par MTB (MTB-RDMs). Les MTB-RDMs montrent un chevauchement frappant avec les régions génomiques marquées par les histones associées avec des régions amplificatrices. De plus, nos analyses ont révélées que les MTB-RDMs sont activement liées par des facteurs de transcription associés à l'immunité avant même d'être infecté par MTB, suggérant ces domaines comme étant des éléments d'activation dans un état de dormance. Nos données suggèrent que les changements actifs dans la méthylation jouent un rôle essentiel pour contrôler la réponse cellulaire des DCs à l'infection bactérienne.

Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer

Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines. Keywords:VLP, Electroporation, Electrotransfer, HIV vaccine, DNA vaccine

Transgenerational, dynamic methylation of stomata genes in response to low relative humidity

Transgenerational inheritance of abiotic stress-induced epigenetic modifications in plants has potential adaptive significance and might condition the offspring to improve the response to the same stress, but this is at least partly dependent on the potency, penetrance and persistence of the transmitted epigenetic marks. We examined transgenerational inheritance of low Relative Humidity-induced DNA methylation for two gene loci in the stomatal developmental pathway in Arabidopsis thaliana and the abundance of associated short-interfering RNAs (siRNAs). Heritability of low humidity-induced methylation was more predictable and penetrative at one locus (SPEECHLESS, entropy ≤ 0.02; χ2 < 0.001) than the other (FAMA, entropy ≤ 0.17; χ2 ns). Methylation at SPEECHLESS correlated positively with the continued presence of local siRNAs (r2 = 0.87; p = 0.013) which, however, could be disrupted globally in the progeny under repeated stress. Transgenerational methylation and a parental low humidity-induced stomatal phenotype were heritable, but this was reversed in the progeny under repeated treatment in a previously unsuspected manner.

Controlled dehydration of a ruthenium complex-DNA crystal induces reversible DNA kinking

Hydration-dependent DNA deformation has been known since Rosalind Franklin recognised that the relative humidity of the sample had to be maintained to observe a single conformation in DNA fibre diffraction. We now report for the first time the crystal structure, at the atomic level, of a dehydrated form of a DNA duplex and demonstrate the reversible interconversion to the hydrated form at room temperature. This system, containing d(TCGGCGCCGA) in the presence of Λ-[Ru(TAP)2(dppz)]2+ (TAP = 1,4,5,8-tetraazaphenanthrene, dppz = dipyridophenazine), undergoes a partial transition from an A/B hybrid to the A-DNA conformation, at 84-79% relative humidity. This is accompanied by an increase in kink at the central step from 22° to 51°, with a large movement of the terminal bases forming the intercalation site. This transition is reversible on rehydration. Seven datasets, collected from one crystal at room temperature, show the consequences of dehydration at near-atomic resolution. This result highlights that crystals, traditionally thought of as static systems, are still dynamic and therefore can be the subject of further experimentation.

Coding behavioural data for cladistic analysis: using dynamic homology without parsimony

Many of the controversies around the concept of homology rest on the subjectivity inherent to primary homology propositions. Dynamic homology partially solves this problem, but there has been up to now scant application of it outside of the molecular domain. This is probably because morphological and behavioural characters are rich in properties, connections and qualities, so that there is less space for conflicting character delimitations. Here we present a new method for the direct optimization of behavioural data, a method that relies on the richness of this database to delimit the characters, and on dynamic procedures to establish character state identity. We use between-species congruence in the data matrix and topological stability to choose the best cladogram. We test the methodology using sequences of predatory behaviour in a group of spiders that evolved the highly modified predatory technique of spitting glue onto prey. The cladogram recovered is fully compatible with previous analyses in the literature, and thus the method seems consistent. Besides the advantage of enhanced objectivity in character proposition, the new procedure allows the use of complex, context-dependent behavioural characters in an evolutionary framework, an important step towards the practical integration of the evolutionary and ecological perspectives on diversity. (C) The Willi Hennig Society 2010.

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5`-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

Interactions between Bacteriophage DNA and Cationic Biomimetic Particles

The interaction between giant bacteriophage DNA and cationic biomimetic particles was characterized from sizing by dynamic light-scattering, zeta-potential analysis, turbidimetry, determination of colloid stability, visualization from atomic force microscopy (AFM), and determination of cytotoxicity against E. coli from colony forming unities counting. First, polystyrene sulfate (PSS) particles with different sizes were covered by a dioctadecyldimethylammonium bromide (DODAB) bilayer yielding the so-called cationic biomimetic particles (PSS/DODAB). These cationic particles are highly organized, present a narrow size distribution and were obtained over a range of particle sizes. Thereafter, upon adding lambda, T5 or T2-DNA to PSS/DODAB particles, supramolecular assemblies PSS/DODAB/DNA were obtained and characterized over a range of DNA concentrations and particle sizes (80-700 nm). Over the low DNA concentration range, PSS/DODAB/DNA assemblies were cationic, colloidally stable with moderate polydispersity and highly cytotoxic against E. coli. From DNA concentration corresponding to charge neutralization, neutral or anionic supramolecular assemblies PSS/DODAB/DNA exhibited low colloid stability, high polydispersity and moderate cytotoxicity. Some nucleosome mimetic assemblies were observed by AFM at charge neutralization (zeta-potential equal to zero).

Genomic organization and evolution of the 5S ribosomal DNA in Tilapiini fishes

5S rDNA sequences present an intense dynamism and have proved to be valuable as genetic markers to distinguish closed related species and also in the understanding of the evolutionary dynamic of repetitive sequences in the genomes. In order to identify patterns of 5S rDNA organization and their evolution in the genome of fish species, such genomic segment was investigated in the tilapias Oreochromis niloticus and Tilapia rendalli, and in the hybrid O. urolepis hornorum x O. mossambicus. A dual 5S rDNA system was identified in the three analyzed tilapia samples. Although each 5S rDNA class was conserved among the three samples, a distinct 5S rDNA genome organization pattern could be evidenced for each sample. The presence of a dual 5S rDNA system seems to be a general trait among non-related teleost fish orders, suggesting that evolutionary events of duplication have occurred before the divergence of the main groups of teleost fishes.

Physicochemical characterization of nanoparticles formed between DNA and phosphorylcholine substituted chitosans

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

Stability of breathers in simple mechanical models for DNA

This work studies through the Floquet theory the stability of breathers generated by the anti-continuous limit. We used the Peyrard-Bishop model for DNA and two kinds of nonlinear potential: the Morse potential and a potential with a hump. The comparison of their stability was done in function of the coupling parameter. We also investigate the dynamic behaviour of the system in stable and unstable regions. Qualitatively, the dynamic of mobile breathers resembles DNA.

Genetic variability analysis among clinical Candida spp. isolates using random amplified polymorphic DNA

The patterns of genetic variation of samples of Candida spp. isolated from patients infected with human immunodeficiency virus in Vitória, state of Espírito Santo, Brazil, were examined. Thirty-seven strains were isolated from different anatomical sites obtained from different infection episodes of 11 patients infected with the human immunodeficiency virus (HIV). These samples were subjected to randomly amplified polymorphic DNA (RAPD) analysis using 9 different primers. Reproducible and complex DNA banding patterns were obtained. The experiments indicated evidence of dynamic process of yeast colonization in HIV-infected patients, and also that certain primers are efficient in the identification of species of the Candida genus. Thus, we conclude that RAPD analysis may be useful in providing genotypic characters for Candida species typing in epidemiological investigations, and also for the rapid identification of pathogenic fungi.

Optimal clone identifier based on dynamic rules and process algebra

The rule creation to clone selection in different projects is a hard task to perform by using traditional implementations to control all the processes of the system. The use of an algebraic language is an alternative approach to manage all of system flow in a flexible way. In order to increase the power of versatility and consistency in defining the rules for optimal clone selection, this paper presents the software OCI 2 in which uses process algebra in the flow behavior of the system. OCI 2, controlled by an algebraic approach was applied in the rules elaboration for clone selection containing unique genes in the partial genome of the bacterium Bradyrhizobium elkanii Semia 587 and in the whole genome of the bacterium Xanthomonas axonopodis pv. citri. Copyright© (2009) by the International Society for Research in Science and Technology.

Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery

Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6-8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6-8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation. © 2013 Dobbs et al.