Characterization of Penaeus vannamei mitogenome focusing on genetic diversity.

The diversity of the Penaeus vannamei mitochondrial genome has still been poorly characterized, there are no validated mitochondrial markers available for populational studies, and the heteroplasmy has not yet been investigated in this species. In this study, metagenomic reads extracted from the muscle of a single individual were used to assemble the mitochondrial genome (mtDNA). These data associated with mitochondrial genomes previously described allowed to evaluate the inter-individual variability and heteroplasmy. Comparison among 45 mtDNA control regions led to the detection of conserved and variable segments and the characterization of two hypervariable regions. The analysis of diversity revealed mostly low frequency polymorphisms, and heteroplasmy was found in practically all mitochondrial genes, with a high occurrence of indels. These results indicate that the design of mitochondrial markers for P. vannamei must be done with caution. The mapping of conserved and variable regions and the characterization of heteroplasmy presented here will contribute to increasing the efficiency of mitochondrial markers for population or individual studies.

Comparative metagenomics reveals different hydrocarbon degradative abilities from enriched oil-drilling waste.

The oil drilling process generates large volumes of waste with inadequate treatments. Here, oil drilling waste (ODW) microbial communities demonstrate different hydrocarbon degradative abilities when exposed to distinct nutrient enrichments as revealed by comparative metagenomics. The ODW was enriched in Luria Broth (LBE) and Potato Dextrose (PDE) media to examine the structure and functional variations of microbial consortia. Two metagenomes were sequenced on Ion Torrent platform and analyzed using MG-RAST. The STAMP software was used to analyze statistically significant differences amongst different attributes of metagenomes. The microbial diversity presented in the different enrichments was distinct and heterogeneous. The metabolic pathways and enzymes were mainly related to the aerobic hydrocarbons degradation. Moreover, our results showed efficient biodegradation after 15 days of treatment for aliphatic hydrocarbons (C8-C33) and polycyclic aromatic hydrocarbons (PAHs), with a total of about 50.5% and 46.4% for LBE and 44.6% and 37.9% for PDE, respectively. The results obtained suggest the idea that the enzymatic apparatus have the potential to degrade petroleum compounds.

Metagenome enrichment approach used for selection of oil-degrading bacteria consortia for drill cutting residue bioremediation.

Drill cuttings leave behind thousands of tons of residues without adequate treatment, generating a large environmental liability. Therefore knowledge about the microbial community of drilling residue may be useful for developing bioremediation strategies. In this work, samples of drilling residue were enriched in different culture media in the presence of petroleum, aiming to select potentially oil-degrading bacteria and biosurfactant producers. Total DNA was extracted directly from the drill cutting samples and from two enriched consortia and sequenced using the Ion Torrent platform. Taxonomic analysis revealed the predominance of Proteobacteria in the metagenome from the drill cuttings, while Firmicutes was enriched in consortia samples. Functional analysis using the Biosurfactants and Biodegradation Database (BioSurfDB) revealed a similar pattern among the three samples regarding hydrocarbon degradation and biosurfactants production pathways. However, some statistical differences were observed between samples. Namely, the pathways related to the degradation of fatty acids, chloroalkanes, and chloroalkanes were enriched in consortia samples. The degradation colorimetric assay using dichlorophenolindophenol as an indicator was positive for several hydrocarbon substrates. The consortia were also able to produce biosurfactants, with biosynthesis of iturin, lichnysin, and surfactin among the more abundant pathways. A microcosms assay followed by gas chromatography analysis showed the efficacy of the consortia in degrading alkanes, as we observed a reduction of around 66% and 30% for each consortium in total alkanes. These data suggest the potential use of these consortia in the bioremediation of drilling residue based on autochthonous bioaugmentation.

Direct sequencing of the white spot syndrome virus from Brazil: Genome assembly and new insights on phylogeny.

White spot syndrome virus (WSSV) has been the cause of great economic losses in world shrimp farming. In this work the genome of a Brazilian WSSV isolate was determined from direct sequencing of total DNA extracted from an infected whiteleg shrimp, and assembled based on a chimera template approach. Comparisons between WSSV-BR and other isolates revealed that the Brazilian virus has a relatively small genome, and is very similar to isolates from Thailand and Mexico. A phylogenetic relationship using different approaches has demonstrated that these isolates share a common evolutionary history. An analysis of conflicting phylogenetic signals also considering genomes of other isolates revealed that the evolutionary history of WSSV may be related to recombination events. We observed that these events can also be traced at some level by analyzing the homologous regions in the WSSV genome. The existence of recombination events introduces a new point of view that must be considered in the evolutionary history of WSSV.

DNA repair protein APE1 is involved in host response during pneumococcal meningitis and its expression can be modulated by vitamin B6.

BACKGROUND: The production of reactive oxygen species (ROS) during pneumococcal meningitis (PM) leads to severe DNA damage in the neurons and is the major cause of cell death during infection. Hence, the use of antioxidants as adjuvant therapy has been investigated. Previous studies have demonstrated the possible participation of apurinic/apyrimidinic endonuclease (APE1) during PM. The aims of this study were to investigate the APE1 expression in the cortical and hippocampal tissues of infant Wistar rats infected with Streptococcus pneumoniae and its association with cell death and understand the role of vitamin B6 (vitB6) as a protective factor against cell death. METHODS: APE1 expression and oxidative stress markers were analyzed at two-time points, 20 and 24 h post infection (p.i.), in the cortex (CX) and hippocampus (HC) of rats supplemented with vitB6. Statistical analyses were performed by the nonparametric Kruskal-Wallis test using Dunn's post test. RESULTS: Our results showed high protein levels of APE1 in CX and HC of infected rats. In the CX, at 20 h p.i., vitB6 supplementation led to the reduction of expression of APE1 and apoptosis-inducing factor, while no significant changes in the transcript levels of caspase-3 were observed. Furthermore, levels of carbonyl content and glutamate in the CX were reduced by vitB6 supplementation at the same time point of 20 h p.i.. Since our data showed a significant effect of vitB6 on the CX at 20 h p.i. rather than that at 24 h p.i., we evaluated the effect of administering a second dose of vitB6 at 18 h p.i. and sacrifice at 24 h p.i.. Reduction in the oxidative stress and APE1 levels were observed, although the latter was not significant. Although the levels of APE1 was not significantly changed in the HC with vitB6 adjuvant therapy, vitB6 supplementation prevented the formation of the truncated form of APE1 (34 kDa) that is associated with apoptosis. CONCLUSIONS: Our data suggest that PM affects APE1 expression, which can be modulated by vitB6. Additionally, vitB6 contributes to the reduction of glutamate and ROS levels. Besides the potential to reduce cell death and oxidative stress during neuroinflammation, vitB6 showed enhanced effect on the CX than on the HC during PM.

Biogeographical distribution analysis of hydrocarbon degrading and biosurfactant producing genes suggests that near-equatorial biomes have higher abundance of genes with potential for bioremediation.

BACKGROUND: Bacterial and Archaeal communities have a complex, symbiotic role in crude oil bioremediation. Their biosurfactants and degradation enzymes have been in the spotlight, mainly due to the awareness of ecosystem pollution caused by crude oil accidents and their use. Initially, the scientific community studied the role of individual microbial species by characterizing and optimizing their biosurfactant and oil degradation genes, studying their individual distribution. However, with the advances in genomics, in particular with the use of New-Generation-Sequencing and Metagenomics, it is now possible to have a macro view of the complex pathways related to the symbiotic degradation of hydrocarbons and surfactant production. It is now possible, although more challenging, to obtain the DNA information of an entire microbial community before automatically characterizing it. By characterizing and understanding the interconnected role of microorganisms and the role of degradation and biosurfactant genes in an ecosystem, it becomes possible to develop new biotechnological approaches for bioremediation use. This paper analyzes 46 different metagenome samples, spanning 20 biomes from different geographies obtained from different research projects. RESULTS: A metagenomics bioinformatics pipeline, focused on the biodegradation and biosurfactant-production pathways, genes and organisms, was applied. Our main results show that: (1) surfactation and degradation are correlated events, and therefore should be studied together; (2) terrestrial biomes present more degradation genes, especially cyclic compounds, and less surfactation genes, when compared to water biomes; and (3) latitude has a significant influence on the diversity of genes involved in biodegradation and biosurfactant production. This suggests that microbiomes found near the equator are richer in genes that have a role in these processes and thus have a higher biotechnological potential. CONCLUSION: In this work we have focused on the biogeographical distribution of hydrocarbon degrading and biosurfactant producing genes. Our principle results can be seen as an important step forward in the application of bioremediation techniques, by considering the biostimulation, optimization or manipulation of a starting microbial consortia from the areas with higher degradation and biosurfactant producing genetic diversity.

Stearylamine-Containing Cationic Nanoemulsion as a Promising Carrier for Gene Delivery.

The drawbacks related to the use of viral vectors in gene therapy have been stimulated the research in non-viral strategies such as cationic nanoemulsions. The aim of this work was to develop a stearylamine-containing nanoemulsion for gene therapy purpose. The formulation was chosen from a Pseudo-Ternary Phase Diagram and had its long-term stability assessed by Dynamic Light Scattering and Phase Analysis Light Scattering during 180 days at 4 degrees C, 25 degrees C and 40 degrees C. Besides, studies of sterilization and scale up of the product were conducted. It was demonstrated that the proposed system was stable up to 180 days when stored at 4 degrees C and could be sterilized by a 0.22 microm filter pore without changes on its characteristics. The scale up was possible by adjusting the volume to the sonication time. Because the nanoemulsion presented a droplet size smaller than 200 nm and a zeta potential higher than 30 mV, this system was able to correctly complex the plasmid model PIRES2-EGFP, as confirmed by the agarosis gel electrophoresis assay. The nanoemulsion toxicity evaluated over lung fetus human cells (MRC-5) was dose-dependent. However, it does not appear to be a limiting factor for further experiments aiming gene transfection. As a conclusion, stearylamine-containing cationic nanoemulsions can be used for gene therapy, since it presents suitable characteristics, stability and possibility of sterilization.

Cockayne syndrome-derived neurons display reduced synapse density and altered neural network synchrony.

Cockayne syndrome (CS) is a rare genetic disorder in which 80% of cases are caused by mutations in the Excision Repair Cross-Complementation group 6 gene (ERCC6). The encoded ERCC6 protein is more commonly referred to as Cockayne Syndrome B protein (CSB). Classical symptoms of CS patients include failure to thrive and a severe neuropathology characterized by microcephaly, hypomyelination, calcification and neuronal loss. Modeling the neurological aspect of this disease has proven difficult since murine models fail to mirror classical neurological symptoms. Therefore, a robust human in vitro cellular model would advance our fundamental understanding of the disease and reveal potential therapeutic targets. Herein, we successfully derived functional CS neural networks from human CS induced pluripotent stem cells (iPSCs) providing a new tool to facilitate studying this devastating disease. We identified dysregulation of the Growth Hormone/Insulin-like Growth Factor-1 (GH/IGF-1) pathway as well as pathways related to synapse formation, maintenance and neuronal differentiation in CSB neurons using unbiased RNA-seq gene expression analyses. Moreover, when compared to unaffected controls, CSB-deficient neural networks displayed altered electrophysiological activity, including decreased synchrony, and reduced synapse density. Collectively, our work reveals that CSB is required for normal neuronal function and we have established an alternative to previously available models to further study neural-specific aspects of CS.

ExoMeg1: a new exonuclease from metagenomic library.

DNA repair mechanisms are responsible for maintaining the integrity of DNA and are essential to life. However, our knowledge of DNA repair mechanisms is based on model organisms such as Escherichia coli, and little is known about free living and uncultured microorganisms. In this study, a functional screening was applied in a metagenomic library with the goal of discovering new genes involved in the maintenance of genomic integrity. One clone was identified and the sequence analysis showed an open reading frame homolog to a hypothetical protein annotated as a member of the Exo_Endo_Phos superfamily. This novel enzyme shows 3'-5' exonuclease activity on single and double strand DNA substrates and it is divalent metal-dependent, EDTA-sensitive and salt resistant. The clone carrying the hypothetical ORF was able to complement strains deficient in recombination or base excision repair, suggesting that the new enzyme may be acting on the repair of single strand breaks with 3' blockers, which are substrates for these repair pathways. Because this is the first report of an enzyme obtained from a metagenomic approach showing exonuclease activity, it was named ExoMeg1. The metagenomic approach has proved to be a useful tool for identifying new genes of uncultured microorganisms.

BioSurfDB: knowledge and algorithms to support biosurfactants and biodegradation studies.

Crude oil extraction, transportation and use provoke the contamination of countless ecosystems. Therefore, bioremediation through surfactants mobilization or biodegradation is an important subject, both economically and environmentally. Bioremediation research had a great boost with the recent advances in Metagenomics, as it enabled the sequencing of uncultured microorganisms providing new insights on surfactant-producing and/or oil-degrading bacteria. Many research studies are making available genomic data from unknown organisms obtained from metagenomics analysis of oil-contaminated environmental samples. These new datasets are presently demanding the development of new tools and data repositories tailored for the biological analysis in a context of bioremediation data analysis. This work presents BioSurfDB, www.biosurfdb.org, a curated relational information system integrating data from: (i) metagenomes; (ii) organisms; (iii) biodegradation relevant genes; proteins and their metabolic pathways; (iv) bioremediation experiments results, with specific pollutants treatment efficiencies by surfactant producing organisms; and (v) a biosurfactant-curated list, grouped by producing organism, surfactant name, class and reference. The main goal of this repository is to gather information on the characterization of biological compounds and mechanisms involved in biosurfactant production and/or biodegradation and make it available in a curated way and associated with a number of computational tools to support studies of genomic and metagenomic data.

The kynurenine pathway is involved in bacterial meningitis.

BACKGROUND: Bacterial meningitis (BM) is characterized by an intense host inflammatory reaction, which contributes to the development of brain damage and neuronal sequelae. Activation of the kynurenine (KYN) pathway (KP) has been reported in various neurological diseases as a consequence of inflammation. Previously, the KP was shown to be activated in animal models of BM, and the association of the SNP AADAT + 401C/T (kynurenine aminotransferase II - KAT II) with the host immune response to BM has been described. The aim of this study was to investigate the involvement of the KP during BM in humans by assessing the concentrations of KYN metabolites in the cerebrospinal fluid (CSF) of BM patients and their relationship with the inflammatory response compared to aseptic meningitis (AM) and non-meningitis (NM) groups. METHODS: The concentrations of tryptophan (TRP), KYN, kynurenic acid (KYNA) and anthranilic acid (AA) were assessed by HPLC from CSF samples of patients hospitalized in the Giselda Trigueiro Hospital in Natal (Rio Grande do Norte, Brazil). The KYN/TRP ratio was used as an index of indoleamine 2,3-dioxygenase (IDO) activity, and cytokines were measured using a multiplex cytokine assay. The KYNA level was also analyzed in relation to AADAT + 401C/T genotypes. RESULTS: In CSF from patients with BM, elevated levels of KYN, KYNA, AA, IDO activity and cytokines were observed. The cytokines INF-γ and IL-1Ra showed a positive correlation with IDO activity, and TNF-α and IL-10 were positively correlated with KYN and KYNA, respectively. Furthermore, the highest levels of KYNA were associated with the AADAT + 401 C/T variant allele. CONCLUSION: This study suggests a downward modulatory effect of the KP on CSF inflammation during BM.

Taxonomic and functional profiles of soil samples from Atlantic forest and Caatinga biomes in northeastern Brazil.

Although microorganisms play crucial roles in ecosystems, metagenomic analyses of soil samples are quite scarce, especially in the Southern Hemisphere. In this work, the microbial diversity of soil samples from an Atlantic Forest and Caatinga was analyzed using a metagenomic approach. Proteobacteria and Actinobacteria were the dominant phyla in both samples. Among which, a significant proportion of stress-resistant bacteria associated to organic matter degradation was found. Sequences related to metabolism of amino acids, nitrogen, and DNA and stress resistance were more frequent in Caatinga soil, while the forest sample showed the highest occurrence of hits annotated in phosphorous metabolism, defense mechanisms, and aromatic compound degradation subsystems. The principal component analysis (PCA) showed that our samples are close to the desert metagenomes in relation to taxonomy, but are more similar to rhizosphere microbiota in relation to the functional profiles. The data indicate that soil characteristics affect the taxonomic and functional distribution; these characteristics include low nutrient content, high drainage (both are sandy soils), vegetation, and exposure to stress. In both samples, a rapid turnover of organic matter with low greenhouse gas emission was suggested by the functional profiles obtained, reinforcing the importance of preserving natural areas.

Cerebrospinal-fluid cytokine and chemokine profile in patients with pneumococcal and meningococcal meningitis.

BACKGROUND: Bacterial meningitis is characterized by an intense inflammatory reaction contributing to neuronal damage. The aim of this study was to obtain a comparative analysis of cytokines and chemokines in patients with pneumococcal (PM) and meningococcal meningitis (MM) considering that a clear difference between the immune response induced by these pathogens remains unclear. METHODS: The cyto/chemokines, IL-1ß, IL-2, IL-6, TNF-α, IFN-γ, IL-10, IL-1Ra, CXCL8/IL-8, CCL2/MCP-1, CLL3/MIP-1α, CCL4/MIP-1γ and G-CSF, were measured in cerebrospinal fluid (CSF) samples from patients with PM and MM. Additionally, a literature review about the expression of cytokines in CSF samples of patients with MB was made. RESULTS: Concerning cytokines levels, only IFN-γ was significantly higher in patients with Streptococcus pneumoniae compared to those with Neisseria meningitidis, regardless of the time when the lumbar puncture (LP) was made. Furthermore, when samples were compared considering the timing of the LP, higher levels of TNF-α (P <0.05) were observed in MM patients whose LP was made within 48 h from the initial symptoms of disease. We also observed that the index of release of cyto/chemokines per cell was significantly higher in PM. From the literature review, it was observed that TNF-α, IL-1ß and IL-6 are the best studied cytokines, while reports describing the concentration of the cytokine IL-2, IL-1Ra, G-CSF and CCL4/MIP-1ß in CSF samples of patients with bacterial meningitis were not found. CONCLUSION: The data obtained in this study and the previously published data show a similar profile of cytokine expression during PM and MM. Nevertheless, the high levels of IFN-γ and the ability to release high levels of cytokines with a low number of cells are important factors to be considered in the pathogenesis of PM and thereby should be further investigated. Moreover, differences in the early response induced by the pathogens were observed. However, the differences observed are not sufficient to trigger changes in the current therapy of corticosteroids adopted in both the PM and MM.

Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey.

BACKGROUND: The scorpion Tityus stigmurus is widely distributed in Northeastern Brazil and known to cause severe human envenoming, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the gene expression profile from the non-stimulated venom gland of Tityus stigmurus scorpion. RESULTS: A cDNA library was constructed and 540 clones were sequenced and grouped into 153 clusters, with one or more ESTs (expressed sequence tags). Forty-one percent of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMP-like) being the most abundant, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. CONCLUSIONS: This investigation provides the first global view of gene expression of the venom gland from Tityus stigmurus under resting conditions. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or non yet described types of venom peptides and proteins from the Buthidae family.

DNA damage by singlet oxygen and cellular protective mechanisms.

Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.

A characterization of a MutM/Fpg ortholog in sugarcane--A monocot plant.

Plant genomic projects, such as Arabidopsis thaliana, rice, and maize, have provided excellent tools for comparative genome analysis on Base Excision DNA Repair (BER). A data-mining study associated with the SUCEST Genome project identified two EST clusters that shared homology to the bacteria MutM/Fpg protein. Comparative analyses presented here show a duplication of the MutM/Fpg gene in sugarcane, wheat and rice. The complementation assays show that both cDNAs from sugarcane are able to complement the Fpg and MutY-glycosylase deficiency in a double mutant Escherichia coli strain (CC104mutMmutY), reducing the spontaneous mutation frequency by 10-fold. The expression analyses by semi-quantitative RT-PCR show that these two mRNAs have different expression levels.

Base excision repair in sugarcane

Danos no DNA podem ser induzidos por um grande número de agentes físicos e químicos presentes no ambiente, como também por compostos produzidos pelo próprio metabolismo celular. Estes danos podem interferir com processos celulares como replicaçäo e transcriçäo, levando a morte celular e/ou mutações. Os baixos níveis de mutaçäo nas células säo devidos à presença de vias enzimáticas, que reparam os danos no DNA. Diversos genes de reparo de DNA têm sido clonados e seus produtos caracterizados, principalmente em bactérias, leveduras e mamíferos. O interesse no estudo de mecanismos de reparo de DNA advém de seu envolvimento com a proteçäo da integridade da informaçäo genética. A alta conservaçäo observada para a maioria dos genes relacionados ao reparo de DNA, especialmente em eucariotos, aponta para sua importância para a manutençäo da vida na terra. Em plantas, o conhecimento sobre os mecanismos de reparo de DNA é ainda reduzido. Os primeiros genes de reparo foram recentemente clonados e o mecanismo de açäo de seus produtos está por ser caracterizado. Nosso objetivo neste trabalho de data mining foi identificar, no banco de dados gerados pelo projeto Genoma da Cana de Açúcar (Sugarcane Expressed Tag Project-SUCEST), genes relacionados ao reparo por excisäo de bases (BER). Esta busca foi feita através do programa tblastn. Em cana de açúcar, foram identificados clusters homólogos para a maioria das proteínas BER analisadas e um alto grau de conservaçäo foi observado. Os melhores resultados foram obtidos com proteínas BER de Arabidopsis thaliana. Para alguns homólogos BER de cana de açúcar, a presença de mais de uma forma de mRNA é possível, como definido pela ocorrência de mais de um cluster homólogo.