Identification of novel genes and proteoforms in Angiostrongylus costaricensis through a proteogenomic approach

RNA sequencing (RNA-Seq) and mass-spectrometry-based proteomics data are often integrated in proteogenomic studies to assist in the prediction of eukaryote genome features, such as genes, splicing, single-nucleotide (SNVs), and single-amino-acid variants (SAAVs). Most genomes of parasite nematodes are draft versions that lack transcript- and protein-level information and whose gene annotations rely only on computational predictions

APLICAÇÕES DE ÓLEOS ESSENCIAIS NA ODONTOLOGIA: REVISÃO INTEGRATIVA DE LITERATURA

Os óleos essenciais são uma mistura de constituintes voláteis produzidos por plantas aromáticas como metabólitos secundários com propriedades terapêuticas e possibilidade de aplicações na prática odontológica. O presente estudo objetiva realizar uma análise, através de uma revisão integrativa de literatura, das possíveis aplicações dos óleos essenciais na odontologia. Para desenvolvimento do estudo, inicialmente foi realizado o processo de busca nas bases de dados PubMed, SciELO e LILACS. Os critérios de inclusão foram artigos publicados sem intervalo temporal, com conteúdo online inteiramente disponível e publicados em português e inglês. Foram incluídos estudos de coorte, transversal, caso controle, pesquisas aplicadas e estudos piloto. Foram excluídas revisões integrativas, sistemáticas e simples de literatura, livros, capítulos de livro, cartas ao autor, resumos de anais e artigos de opinião. Durante o processo de seleção, foram obtidos 21 artigos, sendo PubMed (n=16); SciELO (n=1); LILACS (n=4).  Destacou-se as possibilidades terapêuticas de aplicações dos óleos essenciais na prática clínica odontológica pelos efeitos antimicrobianos e antibiofilme (redução da placa bacteriana), anti-inflamatória e antinociceptiva (redução da periodontite, gengivite, sangramento em geral e alívio da dor) e de uso diário para saúde bucal. Os principais óleos essenciais são obtidos dos extratos de Casearia sylvestris, Cymbopogon citratus, Cymbopogon flexuosus, Eleutherine plicata, Psidium guajava, Syzygium aromaticum ou Eugenia caryophyllata, Baccharis dracunculifolia, Anacardium occidentale, Melaleuca alternifólia, Thymus zygis, Rosmarinus officinalis, Nigella sativa, Carica papaya e Lippia sidoides. O avanço nas pesquisas e nas aplicações dos óleos essenciais merece atenção pelos profissionais da odontologia, sendo uma atual e importante alternativa terapêutica

SDS‑induced hexameric oligomerization of myotoxin‑II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 μM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

BARYONIC DARK MATTER ?

Snakebite envenomation is a neglected condition that constitutes a public health problem in tropical and subtropical countries, including Brazil. Interestingly, some animals are resistant to snake envenomation due to the presence of inhibitory glycoproteins in their serum that target toxic venom components. DM64 is an acidic glycoprotein isolated from Didelphis aurita (opossum) serum that has been characterized as an inhibitor of the myotoxicity induced by bothropic toxins bearing phospholipase A2 (PLA2) structures. This antitoxic protein can serve as an excellent starting template for the design of novel therapeutics against snakebite envenomation, particularly venom-induced local tissue damage. Therefore, the aim of this work was to produce a recombinant DM64 (rDM64) in the methylotrophic yeast Pichia pastoris and to compare its biological properties with those of native DM64. Yeast fermentation in the presence of Pefabloc, a serine protease inhibitor, stimulated cell growth (~1.5-fold), increased the rDM64 production yield approximately 10-fold and significantly reduced the susceptibility of rDM64 to proteolytic degradation. P. pastoris fermentation products were identified by mass spectrometry and Western blotting. The heterologous protein was efficiently purified from the culture medium by affinity chromatography (with immobilized PLA2 myotoxin) and/or an ion exchange column. Although both native and recombinant DM64 exhibit different glycosylation patterns, they show very similar electrophoretic mobilities after PNGase F treatment. rDM64 formed a noncovalent complex with myotoxin II (Lys49-PLA2) from Bothrops asper and displayed biological activity that was similar to that of native DM64, inhibiting the cytotoxicity of myotoxin II by 92% at a 1:1 molar ratio

Revisiting the Therapeutic Potential of Bothrops jararaca Venom: Screening for Novel Activities Using Connectivity Mapping

Submitted by Sandra Infurna ([email protected]) on 2018-10-05T13:49:56Z No. of bitstreams: 1 carolinaAlves_nicolau_etal_IOC_2081.pdf: 945357 bytes, checksum: bef37e6da5b6ecb11075862afbd0bbee (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-10-05T18:34:49Z (GMT) No. of bitstreams: 1 carolinaAlves_nicolau_etal_IOC_2081.pdf: 945357 bytes, checksum: bef37e6da5b6ecb11075862afbd0bbee (MD5)Made available in DSpace on 2018-10-05T18:34:49Z (GMT). No. of bitstreams: 1 carolinaAlves_nicolau_etal_IOC_2081.pdf: 945357 bytes, checksum: bef37e6da5b6ecb11075862afbd0bbee (MD5) Previous issue date: 2018Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil / University of Virginia. Department of Microbiology, Immunology and Cancer Biology. Charlottesville, VA, USA.University of Virginia. Department of Microbiology, Immunology and Cancer Biology. Charlottesville, VA, USA.University of Virginia. Department of Microbiology, Immunology and Cancer Biology. Charlottesville, VA, USA.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Toxinologia. Rio de Janeiro, RJ, Brasil / Instituto Nacional de Ciência e Tecnologia em Toxinas. Brasília, DF, Brasil.University of Virginia. Department of Microbiology, Immunology and Cancer Biology. Charlottesville, VA, USA.Snake venoms are sources of molecules with proven and potential therapeutic applications. However, most activities assayed in venoms (or their components) are of hemorrhagic, hypotensive, edematogenic, neurotoxic or myotoxic natures. Thus, other relevant activities might remain unknown. Using functional genomics coupled to the connectivity map (C-map) approach, we undertook a wide range indirect search for biological activities within the venom of the South American pit viper Bothrops jararaca. For that effect, venom was incubated with human breast adenocarcinoma cell line (MCF7) followed by RNA extraction and gene expression analysis. A list of 90 differentially expressed genes was submitted to biosimilar drug discovery based on pattern recognition. Among the 100 highest-ranked positively correlated drugs, only the antihypertensive, antimicrobial (both antibiotic and antiparasitic), and antitumor classes had been previously reported for B. jararaca venom. The majority of drug classes identified were related to (1) antimicrobial activity; (2) treatment of neuropsychiatric illnesses (Parkinson's disease, schizophrenia, depression, and epilepsy); (3) treatment of cardiovascular diseases, and (4) anti-inflammatory action. The C-map results also indicated that B. jararaca venom may have components that target G-protein-coupled receptors (muscarinic, serotonergic, histaminergic, dopaminergic, GABA, and adrenergic) and ion channels. Although validation experiments are still necessary, the C-map correlation to drugs with activities previously linked to snake venoms supports the efficacy of this strategy as a broad-spectrum approach for biological activity screening, and rekindles the snake venom-based search for new therapeutic agents

Time-course profiles of rDM64 secretion in <i>Pichia pastoris</i> culture during fermentation.

<p><i>P</i>. <i>pastoris</i> was grown at 30°C and 250 rpm for 288 hours. 0–24 hours: glycerol batch phase. 24–288 hours: methanol induction (fed-batch phase); 1% methanol was added every 24 hours, for 264 hours. Dry cell weight/biomass (circle) and rDM64 concentration (square) were determined in the absence <b>(A)</b> or presence <b>(B)</b> of 0.2 mM Pefabloc. The concentration of rDM64 was measured by immunoassay in the culture medium and was performed in triplicate. Error bars represent the standard deviation of the mean.</p

Characterization of rDM64 protein purified from yeast medium.

<p>(A) Purification of rDM64 from the culture medium supernatant by affinity chromatography with immobilized myotoxin II. (B) After elution, the bound fraction (indicated by an asterisk) was further purified by anion-exchange chromatography using a Mono Q column. The chromatographic fractions were analyzed by 12% SDS-PAGE under reducing conditions and were stained with Coomassie blue (C) and periodic acid-Schiff reagent (D) or by immunoblotting with polyclonal antibodies raised against DM64 (E). Lane 1, native DM64 (5 μg). A typical degradation product ~ 45 kDa in size, generated by sample manipulation, can also be observed; lane 2, crude culture medium. Asterisks indicate the chromatographic fractions and are shown in panels A and B (5 μg/lane). NG, non-glycosylated protein (soybean trypsin inhibitor) used as negative control for the periodic acid-Schiff staining. MM, molecular mass markers.</p

Complete amino acid sequence and location of Omp-28, an important immunogenic protein from Salmonella enterica serovar typhi

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-06-05T13:11:11Z No. of bitstreams: 1 Ferreira AGCN Complete amino acid....pdf: 248067 bytes, checksum: 7969c6615f973f5965ef74d4113187c7 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-06-05T13:26:26Z (GMT) No. of bitstreams: 1 Ferreira AGCN Complete amino acid....pdf: 248067 bytes, checksum: 7969c6615f973f5965ef74d4113187c7 (MD5)Made available in DSpace on 2017-06-05T13:26:26Z (GMT). No. of bitstreams: 1 Ferreira AGCN Complete amino acid....pdf: 248067 bytes, checksum: 7969c6615f973f5965ef74d4113187c7 (MD5) Previous issue date: 2004CNPq, FAPERJ, and PAPES-Fiocruz.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Fisiologia e Farmacodinâmica. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Escola Nacional de Saúde Pública. Departamento de Ciências Biológicas. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Laboratório de Microscopia Eletrônica. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Fisiologia e Farmacodinâmica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Química. Departamento de Bioquímica. Rio de Janeiro, RJ, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Química. Departamento de Bioquímica. Rio de Janeiro, RJ, Brasil / Universidade Federal Rural. Instituto de Ciências Exatas. Departamento de Química. Rio de Janeiro, RJ, Brasil /Omp-28 isolated from Salmonella enterica serovar typhi presented a subunit molecular mass of 9,632 Da by MALDI-TOF MS. It was denatured, S-alkylated, and 1) directly submitted to Edman sequencing, 2) cleaved with CNBr, and 3) hydrolyzed either with endoproteinase Glu-C or Asp-N. The major CNBr peptide containing the C-terminal portion of Omp-28 was isolated by tricine-SDS-PAGE and electroblotted whereas Omp-28 enzymatic peptides were isolated by C18-RP-HPLC. All peptides were sequenced. This approach allowed the elucidation of the complete primary structure of Omp-28. Its amino acid sequence is identical to that deduced from part of the DNA of the “putative periplasmic transport protein” of either S. enterica serovar typhimurium and a multiple drug resistant S. enterica serovar typhi. Omp-28 homologous protein sequences were also deduced from Escherichia coli and Yersinia pestis genomic DNA. All proteins had their secondary structures predicted. Immunogold cytochemistry indicated that Omp-28 is found on the bacterium outer membrane