A simple boiling-based DNA extraction for RAPD profiling of landfarm soil to provide representative metagenomic content

Landfarm soils are employed in industrial and petrochemical residue bioremediation. This process induces selective pressure directed towards microorganisms capable of degrading toxic compounds. Detailed description of taxa in these environments is difficult due to a lack of knowledge of culture conditions required for unknown microorganisms. A metagenomic approach permits identification of organisms without the need for culture. However, a DNA extraction step is first required, which can bias taxonomic representativeness and interfere with cloning steps by extracting interference substances. We developed a simplified DNA extraction procedure coupled with metagenomic DNA amplification in an effort to overcome these limitations. The amplified sequences were used to generate a metagenomic data set and the taxonomic and functional representativeness were evaluated in comparison with a data set built with DNA extracted by conventional methods. The simplified and optimized method of RAPD to access metagenomic information provides better representativeness of the taxonomical and metabolic aspects of the environmental samples.

New insights into Trimezieae (Iridaceae) phylogeny: what do molecular data tell us?

The Neotropical tribe Trimezieae are taxonomically difficult. They are generally characterized by the absence of the features used to delimit their sister group Tigridieae. Delimiting the four genera that make up Trimezieae is also problematic. Previous family-level phylogenetic analyses have not examined the monophyly of the tribe or relationships within it. Reconstructing the phylogeny of Trimezieae will allow us to evaluate the status of the tribe and genera and to examine the suitability of characters traditionally used in their taxonomy. Maximum parsimony and Bayesian phylogenetic analyses are presented for 37 species representing all four genera of Trimezieae. Analyses were based on nrITS sequences and a combined plastid dataset. Ancestral character state reconstructions were used to investigate the evolution of ten morphological characters previously considered taxonomically useful. Analyses of nrITS and plastid datasets strongly support the monophyly of Trimezieae and recover four principal clades with varying levels of support; these clades do not correspond to the currently recognized genera. Relationships within the four clades are not consistently resolved, although the conflicting resolutions are not strongly supported in individual analyses. Ancestral character state reconstructions suggest considerable homoplasy, especially in the floral characters used to delimit Pseudotrimezia. The results strongly support recognition of Trimezieae as a tribe but suggest that both generic- and species-level taxonomy need revision. Further molecular analyses, with increased sampling of taxa and markers, are needed to support any revision. Such analyses will help determine the causes of discordance between the plastid and nuclear data and provide a framework for identifying potential morphological synapomorphies for infra-tribal groups. The results also suggest Trimezieae provide a promising model for evolutionary research.

Prediction of tolerance in children with IgE mediated cow's milk allergy by microarray profiling and chemometric approach

The sera of a retrospective cohort (n = 41) composed of children with well characterized cow's milk allergy collected from multiple visits were analyzed using a protein microarray system measuring four classes of immunoglobulins. The frequency of the visits, age and gender distribution reflected real situation faced by the clinicians at a pediatric reference center for food allergy in 530 Paulo, Brazil. The profiling array results have shown that total IgG and IgA share similar specificity whilst IgM and in particular IgE are distantly related. The correlation of specificity of IgE and IgA is variable amongst the patients and this relationship cannot be used to predict atopy or the onset of tolerance to milk. The array profiling technique has corroborated the clinical selection criteria for this cohort albeit it clearly suggested that 4 out of the 41 patients might have allergies other than milk origin. There was also a good correlation between the array data and ImmunoCAP results, casein in particular. By using qualitative and quantitative multivariate analysis routines it was possible to produce validated statistical models to predict with reasonable accuracy the onset of tolerance to milk proteins. If expanded to larger study groups, the array profiling in combination with the multivariate techniques show potential to improve the prognostic of milk allergic patients. (C) 2012 Elsevier B.V. All rights reserved.

Cloning and characterisation of a novel chromosome end repeat enriched with homopolymeric (dA)/(dT) DNA in Rhynchosciara americana (Diptera: Sciaridae)

Short tandem DNA repeats and telomerase compose the telomere structure in the vast majority of eukaryotic organisms. However, such a conserved organisation has not been found in dipterans. While telomeric DNA in Drosophila is composed of specific retrotransposons, complex terminal tandem repeats are present in chromosomes of Anopheles and chironomid species. In the sciarid Rhynchosciara americana, short repeats (16 and 22 bp long) tandemly arrayed seem to reach chromosome ends. Moreover, in situ hybridisation data using homopolymeric RNA probes suggested in this species the existence of a third putative chromosome end repeat enriched with (dA).(dT) homopolymers. In this work, chromosome micro-dissection and PCR primed by homopolymeric primers were employed to clone these repeats. Named T-14 and 93 % AT-rich, the repetitive unit is 14 bp long and appears organised in tandem arrays. It is localised in five non-centromeric ends and in four interstitial bands of R. americana chromosomes. To date, T-14 is the shortest repeat that has been characterised in chromosome ends of dipterans. As observed for short tandem repeats identified previously in chromosome ends of R. americana, the T-14 probe hybridised to bridges connecting non-homologous polytene chromosome ends, indicative of close association of T-14 repeats with the very end of the chromosomes. The results of this work suggest that R. americana represents an additional example of organism provided with more than one DNA sequence that is able to reach chromosome termini.

Therapeutic DNA Vaccine Encoding Peptide P10 against Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides brasiliensis, is the most prevalent invasive fungal disease in South America. Systemic mycoses are the 10th most common cause of death among infectious diseases in Brazil and PCM is responsible for more than 50% of deaths due to fungal infections. PCM is typically treated with sulfonamides, amphotericin B or azoles, although complete eradication of the fungus may not occur and relapsing disease is frequently reported. A 15-mer peptide from the major diagnostic antigen gp43, named P10, can induce a strong T-CD4+ helper-1 immune response in mice. The TEPITOPE algorithm and experimental data have confirmed that most HLA-DR molecules can present P10, which suggests that P10 is a candidate antigen for a PCM vaccine. In the current work, the therapeutic efficacy of plasmid immunization with P10 and/or IL-12 inserts was tested in murine models of PCM. When given prior to or after infection with P. brasiliensis virulent Pb 18 isolate, plasmid-vaccination with P10 and/or IL-12 inserts successfully reduced the fungal burden in lungs of infected mice. In fact, intramuscular administration of a combination of plasmids expressing P10 and IL-12 given weekly for one month, followed by single injections every month for 3 months restored normal lung architecture and eradicated the fungus in mice that were infected one month prior to treatment. The data indicate that immunization with these plasmids is a powerful procedure for prevention and treatment of experimental PCM, with the perspective of being also effective in human patients.

Anisotropic Brownian motion in ordered phases of DNA fragments

Using Fluorescence Recovery After Photobleaching, we investigate the Brownian motion of DNA rod-like fragments in two distinct anisotropic phases with a local nematic symmetry. The height of the measurement volume ensures the averaging of the anisotropy of the in-plane diffusive motion parallel or perpendicular to the local nematic director in aligned domains. Still, as shown in using a model specifically designed to handle such a situation and predicting a non-Gaussian shape for the bleached spot as fluorescence recovery proceeds, the two distinct diffusion coefficients of the DNA particles can be retrieved from data analysis. In the first system investigated (a ternary DNA-lipid lamellar complex), the magnitude and anisotropy of the diffusion coefficient of the DNA fragments confined by the lipid bilayers are obtained for the first time. In the second, binary DNA-solvent system, the magnitude of the diffusion coefficient is found to decrease markedly as DNA concentration is increased from isotropic to cholesteric phase. In addition, the diffusion coefficient anisotropy measured within cholesteric domains in the phase coexistence region increases with concentration, and eventually reaches a high value in the cholesteric phase.

Counter-ion effect on surfactant-DNA gel particles as controlled DNA delivery systems

The ability to entrap drugs within vehicles and subsequently release them has led to new treatments for a number of diseases. Based on an associative phase separation and interfacial diffusion approach, we developed a way to prepare DNA gel particles without adding any kind of cross-linker or organic solvent. Among the various agents studied, cationic surfactants offered particularly efficient control for encapsulation and DNA release from these DNA gel particles. The driving force for this strong association is the electrostatic interaction between the two components, as induced by the entropic increase due to the release of the respective counter-ions. However, little is known about the influence of the respective counter-ions on this surfactant-DNA interaction. Here we examined the effect of different counter-ions on the formation and properties of the DNA gel particles by mixing DNA (either single-(ssDNA) or double-stranded (dsDNA)) with the single chain surfactant dodecyltrimethylammonium (DTA). In particular, we used as counter-ions of this surfactant the hydrogen sulfate and trifluoromethane sulfonate anions and the two halides, chloride and bromide. Effects on the morphology of the particles obtained, the encapsulation of DNA and its release, as well as the haemocompatibility of these particles are presented, using counter-ion structure and DNA conformation as controlling parameters. Analysis of the data indicates that the degree of counter-ion dissociation from the surfactant micelles and the polar/hydrophobic character of the counter-ion are important parameters in the final properties of the particles. The stronger interaction with amphiphiles for ssDNA than for dsDNA suggests the important role of hydrophobic interactions in DNA.

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma. Modern Pathology (2012) 25, 1345-1353; doi: 10.1038/modpathol.2012.96; published online 8 June 2012

Chk1 phosphorylation of Metnase enhances DNA repair but inhibits replication fork restart

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. Oncogene (2012) 31, 4245-4254; doi:10.1038/onc.2011.586; published online 9 January 2012

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen (102), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with 102, they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT. (c) 2012 Elsevier Inc. All rights reserved.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase 2 (ALDH2) and glutathione Stransferase pi 1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatin A increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G0/G1 and S in HEK293 cells, whereas HEK293/SET showed G2/M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

A feature selection approach for identification of signature genes from SAGE data

Abstract Background One goal of gene expression profiling is to identify signature genes that robustly distinguish different types or grades of tumors. Several tumor classifiers based on expression profiling have been proposed using microarray technique. Due to important differences in the probabilistic models of microarray and SAGE technologies, it is important to develop suitable techniques to select specific genes from SAGE measurements. Results A new framework to select specific genes that distinguish different biological states based on the analysis of SAGE data is proposed. The new framework applies the bolstered error for the identification of strong genes that separate the biological states in a feature space defined by the gene expression of a training set. Credibility intervals defined from a probabilistic model of SAGE measurements are used to identify the genes that distinguish the different states with more reliability among all gene groups selected by the strong genes method. A score taking into account the credibility and the bolstered error values in order to rank the groups of considered genes is proposed. Results obtained using SAGE data from gliomas are presented, thus corroborating the introduced methodology. Conclusion The model representing counting data, such as SAGE, provides additional statistical information that allows a more robust analysis. The additional statistical information provided by the probabilistic model is incorporated in the methodology described in the paper. The introduced method is suitable to identify signature genes that lead to a good separation of the biological states using SAGE and may be adapted for other counting methods such as Massive Parallel Signature Sequencing (MPSS) or the recent Sequencing-By-Synthesis (SBS) technique. Some of such genes identified by the proposed method may be useful to generate classifiers.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Tissue distribution of DNA-Hsp65/TDM-loaded PLGA microspheres and uptake by phagocytic cells

This study aimed to demonstrate that microspheres, used as delivery vehicle of DNA-Hsp65/TDM [plasmid DNA encoding heat shock protein 65 (Hsp65) coencapsulated with trehalose dimycolate (TDM) into PLGA microspheres], are widely spread among several organs after intramuscular administration in BALB/c mice. In general, we showed that these particles were phagocytosed by antigen presenting cells, such as macrophages and dendritic cells. Besides, it was demonstrated herein that draining lymph node cells presented a significant increase in the number of cells expressing costimulatory molecules (CD80 and CD86) and MHC class II, and also that the administration of the DNA-Hsp65/TDM and vector/TDM formulations resulted in the up-regulation of CD80, CD86 and MHC class II expression when compared to control formulations (vector/TDM and empty). Regarding the intracellular trafficking we observed that following phagocytosis, the microspheres were not found in the late endosomes and/or lysosomes, until 15 days after internalization, and we suggest that these constructions were hydrolysed in early compartments. Overall, these data expand our knowledge on PLGA [poly (lactic-co- glycolic acid)] microspheres as gene carriers in vaccination strategies, as well as open perspectives for their potential use in clinical practice.