Strand Analysis, a free online program for the computational identification of the best RNA interference (RNAi) targets based on Gibbs free energy

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

Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns

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

Evidence of epigenetic regulation of the tumor suppressor gene cluster flanking RASSF1 in breast cancer cell lines

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

Horizontal transfers of Mariner transposons between mammals and insects

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

LHX6 is a sensitive methylation marker in head and neck carcinomas

Head and neck cancer remains a morbid and often fatal disease and at the present time few effective molecular markers have been identified. The purpose of the present work was to identify new molecular markers for head and neck squamous cell carcinoma (HNSCC). We applied methylation-sensitive arbitrarily primed PCR (MS/APPCR) to isolate sequences differentially methylated in HNSCC. The most frequently hypermethylated fragment we found maps close to a cytosine guanine dinucleotide (CpG) island on chromosome 9q33.2, and hypermethylation of this CpG island was associated with transcriptional silencing of an alternative transcript of the LHX6 gene. Using combined bisulfite restriction analysis (COBRA), hypermethylation of this fragment was detected in 13 of 14 (92.8%) HNSCC cell lines studied and 21 of 32 (65.6%) primary tumors, whereas little or no methylation was seen in 10 normal oral mucosa samples. We extended this investigation to other cancer cell lines and methylation was found in those derived from colon, breast, leukemia and lung, and methylation was also found in 12/14 primary colon tumors. These findings suggest that differentially methylated (DIME)-6 hypermethylation is a good cancer marker in HNSCC as well as in other kinds of neoplasias and confirm the importance of searching for markers of epigenetic dysregulation in cancer.

Identification and Analyses of Candidate Genes for Rpp4-Mediated Resistance to Asian Soybean Rust in Soybean

Asian soybean rust is a formidable threat to soybean (Glycine max) production in many areas of the world, including the United States. Only five sources of resistance have been identified (Resistance to Phakopsora pachyrhizi1 [Rpp1], Rpp2, Rpp3, Rpp4, and Rpp5). Rpp4 was previously identified in the resistant genotype PI459025B and mapped within 2 centimorgans of Satt288 on soybean chromosome 18 (linkage group G). Using simple sequence repeat markers, we developed a bacterial artificial chromosome contig for the Rpp4 locus in the susceptible cv Williams82 (Wm82). Sequencing within this region identified three Rpp4 candidate disease resistance genes (Rpp4C1-Rpp4C3 [Wm82]) with greatest similarity to the lettuce (Lactuca sativa) RGC2 family of coiled coil-nucleotide binding site-leucine rich repeat disease resistance genes. Constructs containing regions of the Wm82 Rpp4 candidate genes were used for virus-induced gene silencing experiments to silence resistance in PI459025B, confirming that orthologous genes confer resistance. Using primers developed from conserved sequences in the Wm82 Rpp4 candidate genes, we identified five Rpp4 candidate genes (Rpp4C1-Rpp4C5 [PI459025B]) from the resistant genotype. Additional markers developed from the Wm82 Rpp4 bacterial artificial chromosome contig further defined the region containing Rpp4 and eliminated Rpp4C1 (PI459025B) and Rpp4C3 (PI459025B) as candidate genes. Sequencing of reverse transcription-polymerase chain reaction products revealed that Rpp4C4 (PI459025B) was highly expressed in the resistant genotype, while expression of the other candidate genes was nearly undetectable. These data support Rpp4C4 (PI459025B) as the single candidate gene for Rpp4-mediated resistance to Asian soybean rust.

Transcription factors expressed in soybean roots under drought stress

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identification and frequency of transposable elements in Eucalyptus

Transposable elements (TE) are major components of eukaryotic genomes and involved in cell regulation and organism evolution. We have analyzed 123,889 expressed sequence tags of the Eucalyptus Genome Project database and found 124 sequences representing 76 TE in 9 groups, of which copia, MuDR and FAR1 groups were the most abundant. The low amount of sequences of TE may reflect the high efficiency of repression of these elements, a process that is called TE silencing. Frequency of groups of TE in Eucalyptus libraries which were prepared with different tissues or physiologic conditions from seedlings or adult plants indicated that developing plants experience the expression of a much wider spectrum of TE groups than that seen in adult plants. These are preliminary results that identify the most relevant TE groups involved with Eucalyptus development, which is important for industrial wood production. Copyright by the Brazilian Society of Genetics.

Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: An evolutionary history

Background: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. Methodology/Principal Findings: Hz formation activity of an α-glucosidase was investigated. Hz formation was inhibited by specific α-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect α-glucosidase was able to inhibit Hz formation. The α-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that α-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of α-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both α-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of α-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of α-glucosidase shows a high similarity to the insect α-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. Conclusions/Significance: Herein the Hz formation is shown to be associated to an a-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that α-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance. © 2009 Mury et al.

Papel da proteína Mx1 na resposta a interferons e no processo neoplásico

The Mx1 protein is encoded by an interferon- induced gene and shares domain organization, homooligomerization capacity and membrane association with the large dynamin-like GTPases. The Mx1 protein is involved in the response to a large number of RNA viruses, such as the bunyavirus family and the influenza virus. Interestingly, it has also been found as a methylation-silenced gene in several types of neoplasm, including head and neck squamous cell carcinoma. In this scenario, MX1 gene silencing is associated with immortalization in several neoplastic cell lines. Thus, Mx1 stands out as one of the key proteins involved in interferon-induced immune response and also plays an important role in cell cycle control. Here we discuss some of the functions of the Mx1 protein, including its antiviral activity, protein folding and involvement in neoplasia, as well as those revealed by investigating its cellular partners.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development.We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the samewas detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos. © 2013 Society for Reproduction and Fertility.

Um balanço critico da década da alfabetizaçâo no brasil

In relation to United Nations Literacy Decade (2003- 2012), are being developed in Brazil many public policies on education and literacy. The results of these public policies indicate both noisy conquest of some advances as the worsening of many historical problems. Among these, we highlight the result of silencing of discussion around the concept of restricted and rudimentary literacy, on which, in line with neoliberal political model, are based educational policies and corresponding systems assessment of skills and competencies of reading and writing, which it is hoped that students learn and which are defi ning the role of the teacher as a mere provider of strategies for such learning. Based on this hypothesis, we present, in a tone of critical balance, refl ections on the Decade of Literacy in order to contribute to the discussion of problems and perspectives for teaching reading and writing in Brazil.

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

A novel function for CUGBP2 in controlling the pro-inflammatory stimulus in H9c2 cells: Subcellular trafficking of messenger molecules

Accumulating evidence demonstrates that chronic inflammation plays an important role in heart hypertrophy and cardiac diseases. However, the fine-tuning of cellular and molecular mechanisms that connect inflammatory process and cardiac diseases is still under investigation. Many reports have demonstrated that the overexpression of the cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids, is correlated with inflammatory processes. Increased level of prostaglandin E2 was also found in animal model of left ventricle of hypertrophy. Based on previous observations that demonstrated a regulatory loop between COX-2 and the RNA-binding protein CUGBP2, we studied cellular and molecular mechanisms of a pro-inflammatory stimulus in a cardiac cell to verify if the above two molecules could be correlated with the inflammatory process in the heart. A cellular model of investigation was established and H9c2 was used.We also demonstrated a regulatory connection between COX-2 and CUGBP2 in the cardiac cells. Based on a set of different assays including gene silencing and fluorescence microscopy, we describe a novel function for the RNA-binding protein CUGBP2 in controlling the pro-inflammatory stimulus: subcellular trafficking of messenger molecules to specific cytoplasmic stress granules to maintain homeostasis. © 2013 International Federation for Cell Biology.

MyD88 or TRAM knockdown regulates interleukin (IL)-6, IL-8, and CXCL12 mRNA expression in human gingival and periodontal ligament fibroblasts

Background: In a previous report, it was shown that Toll-like receptor (TLR) 2 knockdown modulates interleukin (IL)-6 and IL-8 but not the chemokine CXCL12, an important mediator with inflammatory and proangiogenic effects, in human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). This study investigates whether knocking down two important TLR adaptor molecules, such as myeloid differentiation protein 88 (MyD88) and TRIF-related adaptor molecule (TRAM), could affect mRNA expression of IL-6, IL-8, and CXCL12 in HGF and HPDLF. Methods: After small interfering (si) RNA-mediated silencing of MyD88 or TRAM, HGF and HPDLF were stimulated with Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) or two synthetic ligands of TLR2 (Pam2CSK4 and Pam3CSK4) for 6 hours. IL-6, IL-8, and CXCL12 mRNAs were evaluated by quantitative polymerase chain reaction. Results: Knockdown of MyD88 or TRAM partially impaired the IL-8 mRNA upregulation in both fibroblast subpopulations. Similarly, IL-6 upregulation was partially prevented by siMyD88 or siTRAM in HGF stimulated with Pg LPS, as well as in both fibroblast subtypes challenged with Pam2CSK4. Conversely, constitutive CXCL12 mRNA levels were upregulated by MyD88 or TRAM knockdown in non-stimulated cells. Conclusions: These results suggest that TLR adaptor molecules knockdown, such as MyD88 or TRAM, can decrease IL-6 and IL-8 mRNA and increase CXCL12 mRNA expression in HGF and HPDLF. This can be an important step for better understanding the mechanisms that control the inflammatory cytokine and chemokine expression, which in turn contributes to periodontal pathogenesis.