Polyunsaturated fatty acids from Phyllocaulis boraceiensis mucus block the replication of influenza virus.

Influenza viruses cause worldwide outbreaks and pandemics in humans and animals every year with considerable morbidity and mortality. The molecular diversity of secondary metabolites extracted from mollusks is a good alternative for the discovery of novel bioactive compounds with unique structures and diverse biological activities. Phyllocaulis boraceiensis is a hermaphroditic slug that exudes mucus, in which was detected hydroxy polyunsaturated fatty acids that exhibited potent antiviral activity against measles virus. The objective of this study was to evaluate this property against Influenza viruses. Cell viability and toxicity of the mucus were evaluated on Madin-Darby canine kidney (MDCK) cells by MTT assay. Antiviral activity from mucus against influenza viruses was carried out by determination of the virus infection dose and by immunofluorescence assays. The crude mucus and its fractions exhibited low cytotoxicity on MDCK cells. A significant inhibition of viral replication, reduced by the order of eight times, was observed in influenza-induced cytopathic effect. In immunofluorescence assay was observed a decrease of more than 80% of the viral load on infected MDCK cell treated with mucus and its fractions. The viral glycoproteins hemagglutinin and neuraminidase located on the surface of the virus are crucial for the replications and infectivity of the influenza virus. Some authors demonstrated that lipids, such as, polyunsaturated fatty acids exhibited multiple roles in antiviral innate and adaptive responses, control of inflammation, and in the development of antiviral therapeutics. As corroborated by other studies, hydroxy polyunsaturated fatty acids interfered with the binding of influenza virus on host cell receptor and reduced viral titers. The results obtained indicated that polyunsaturated fatty acids from P. boraceiensis crude mucus and fractions 39 exerted antiviral activity against influenza virus.

Effects of carbohydrate supplementation on competitive runners undergoing overload training followed by a session of intermittent exercise.

This study evaluated the effects of a micro cycle of overload training (1st-8th day) on metabolic and hormonal responses in male runners with or without carbohydrate supplementation and investigated the cumulative effects of this period on a session of intermittent high-intensity running and maximum-performance-test (9th day). The participants were 24 male runners divided into two groups, receiving 61% of their energy intake as CHO (carbohydrate-group) and 54% in the control-group (CON). The testosterone was higher for the CHO than the CON group after the overload training (694.0 +/- 54.6 vs. CON 610.8 +/- 47.9 pmol/l). On the ninth day participants performed 10 x 800 m at mean 3 km velocity. An all-out 1000 m running was performed before and after the 10 x 800 m. Before, during, and after this protocol, the runners received solution containing CHO or the CON equivalent. The performance on 800 m series did not differ in either group between the first and last series of 800 m, but for the all-out 1000 m test the performance decrement was lower for CHO group (5.3 +/- 1.0 vs. 10.6 +/- 1.3%). The cortisol concentrations were lower in the CHO group in relation to CON group (22.4 +/- 0.9 vs. 27.6 +/- 1.4 pmol/l) and the IGF1/IGFBP3 ratio increased 12.7% in the CHO group. During recovery, blood glucose concentrations remained higher in the CHO group in comparison with the CON group. It was concluded that CHO supplementation possibly attenuated the suppression of the hypothalamic-pituitary-gonadal axis and resulted in less catabolic stress, and thus improved running performance.

Patagonin-CRISP: Antimicrobial Activity and Source of Antimicrobial Molecules in Duvernoy's Gland Secretion (Philodryas patagoniensis Snake).

Snake venom contains a variety of toxins with a range of biological activity, among these toxins cysteine-rich secreted proteins (CRISPs) can be found. The proteins of this family have masses of 20-30 kDa and display homologous amino acid sequences containing 16 cysteine residues, forming eight disulfide bonds. Some of these proteins have been explored, characterized, and described in terms of their activity; however, little is known about their range of activities. A search for new antimicrobial molecules is ongoing, as the number of microbial strains resistant to available antibiotics is increasing. We identified antimicrobial activity in the secretion of Duvernoy's gland of the rear-fanged Philodryas patagoniensis. Fractions of this venom were subjected to reverse-phase high performance liquid chromatography and analyzed to determine their antimicrobial activity with a liquid broth inhibition assay. One of the fractions presented activity against a Gram-negative bacterium and a filamentous fungus. This fraction was analyzed with LC-MS/MS, and a protein of 24,848.8 Da was identified. Database searches allowed us to identify it as a CRISP due to the presence of some unique fragments in the molecule. We called it patagonin-CRISP, as the same protein in the venom of P. patagoniensis had previously been characterized as having a different biological activity. Patagonin-CRISP presented activity at very low concentrations and showed no cytotoxic activity. This is the first time that antimicrobial activity has been identified for P. patagoniensis venom or for a CRISP family protein.

Antiproliferative and antiangiogenic effect of Amblyomma sculptum (Acari: Ixodidae) crude saliva in endothelial cells in vitro.

Neovascularization, a process that includes vasculogenesis and angiogenesis, may be a physiological or pathologic event, but in any cases the phenomenon is related to the formation of vascular net and sprouting of endothelial cells from preexisting blood vessel. The tumor environment, which counts on the tumor cell proliferation, is plenty of proangiogenic factors, such as angiogenin, TGF (α and ß), FGF, VEGF, all of them playing a crucial role in angiogenesis, an important hallmark of cancer frequently related to a poor prognosis. Therefore, therapies focusing the inhibition of cancer neovasculogenesis have become an interesting strategy for the development of antitumor therapies. In this work, we investigate the effect of tick saliva on the human endothelial cells, in order to understand its inhibitory effects on angiogenesis. To this end, the HUVEC cells were used as model of angiogenesis in vitro and the anti-proliferative, anti-migratory, cytotoxicity was evaluated. Our data depicts that saliva impairs cell development by causing structural changes while precludes cell proliferation and migration, that are crucial events related to angiogenesis. Aiming the identification of the bioactive components related to antiangiogenic activity, saliva was analyzed through the Mass Spectrometry and among all molecules identified, disintegrins and cathepsin L seems to be primarily responsible for the antiangiogenic effects of saliva.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest.

The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks.

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

The antiviral effect of mollusk mucus on measles virus.

Measles is a viral disease highly contagious spread by respiratory transmission. Although infection can be controlled by vaccination, numerous cases of measles have been registered in many areas of the world, highlighting the need for additional interventions. Terrestrial gastropods exude mucus on their body surface when traveling, to protect the body from mechanical injury, desiccation or contact with harmful substances. The mucus of mollusks has been studied as a source of new natural compounds with diverse biological activities. In this study, the antiviral activity of the mucus of the land slug P. boraceiensis was demonstrated in vitro using Vero cells infected with measles virus. The crude sample and four fractions were tested in cultures infected with measles virus and the antiviral activity was assessed by the cytopathic effect in infected cell cultures as well as by immunofluorescence and qPCR. Fractions 39 and 50 of the mucus from P. boraceiensis were analyzed by HPLC-DAD-ESI-MS/MS and infrared spectroscopy. A mixture of polyunsaturated fatty acids was found in the two fractions. A reduction in the growth of the measles virus was observed, measured by qPCR, with a protection index of 80% in Vero cells infected with measles and treated with fraction 39. Fraction 39 exhibited the best antiviral action in vitro and high contents of hydroxy-tritriacontapentaenoic acid and hydroxy-pentatriacontapentaenoic acid were found in this fraction.

Co-infection between influenza virus and flagellated bacteria.

Trypsin is required in the hemagglutinin (HA) cleavage to in vitro influenza viruses activation. This HA cleavage is necessary for virus cell entry by receptor-mediated endocytosis. Bacteria in the respiratory tract are potential sources of proteases that could contribute to the cleavage of influenza virus in vivo. From 47 samples collected from horses, pigs, and from humans, influenza presence was confirmed in 13 and these samples demonstrated co-infection of influenza with flagellated bacteria, Stenotrophomonas maltophilia from the beginning of the experiments. Despite treatment with antibiotics, the bacteria remained resistant in several of the co-infected samples (48.39%). These bacteria, considered opportunistic invaders from environmental sources, are associated with viral infections in upper respiratory tract of hosts. The protease (elastase), secreted by Stenotrophomonas maltophilia plays a role in the potentiation of influenza virus infection. Proteolytic activity was detected by casein agar test. Positive samples from animals and humans had either a potentiated influenza infectivity or cytopathic effect (CPE) in MDCK and NCI H292 cells, Stenotrophomonas maltophilia were always present. Virus and bacteria were observed ultrastructurally. These in vitro findings show that microbial proteases could contribute to respiratory complications by host protease activity increasing inflammation or destroying endogenous cell protease inhibitors.

Antiviral Action of Hydromethanolic Extract of Geopropolis from Scaptotrigona postica against Antiherpes Simplex Virus (HSV-1).

The studies on chemical composition and biological activity of propolis had focused mainly on species Apis mellifera L. (Hymenoptera: Apidae). There are few studies about the uncommon propolis collected by stingless bees of the Meliponini tribe known as geopropolis. The geopropolis from Scaptotrigona postica was collected in the region of Barra do Corda, Maranhão state, Brazil. The chemical analysis of hydromethanolic extract of this geopropolis (HMG) was carried out through HPLC-DAD-ESI-MS/MS and the main constituents found were pyrrolizidine alkaloids and C-glycosyl flavones. The presence of alkaloids in extracts of propolis is detected for the first time in this sample. The antiviral activity of HMG was evaluated through viral DNA quantification experiments and electron microscopy experiments. Quantification of viral DNA from herpes virus showed reduction of about 98% in all conditions and concentration tested of the HMG extract. The results obtained were corroborated by transmission electron microscopy, in which the images did not show particle or viral replication complex. The antiviral activity of C-glycosyl flavones was reported for a variety of viruses, being observed at different points in the viral replication. This work is the first report about the antiviral activity of geopropolis from Scaptotrigona postica, in vitro, against antiherpes simplex virus (HSV).

Discovery of a new antiviral protein isolated Lonomia obliqua analysed by bioinformatics and real-time approaches.

This study presents a new recombinant protein that acts as a powerful antiviral (rAVLO-recombinant Antiviral protein of Lonomia obliqua). It was able to reduce the replication by 10(6) fold for herpes virus and by 10(4) fold for rubella virus. RT-PCR of viral RNA rAVLO treated infected cells also showed similar rate of inhibition in replication. The analysis of this protein by bioinformatics suggests that this protein is globular, secreted with a signal peptide and has the ability to bind to MHC class I. It was found that there are several protein binding sites with various HLA and a prevalence of α-helices in the N-terminal region (overall classified as a α/ß protein type). BLAST similarity sequence search for corresponding cDNA did not reveal a similar sequence in Genbank, suggesting that it is from a novel protein family. In this study we have observed that this recombinant protein and hemolymph has a potent antiviral action. This protein was produced in a baculovirus/Sf-9 system. Therefore, these analyses suggest that this novel polypeptide is a candidate as a broad spectrum antiviral.

Effects of Lonomia obliqua caterpillar venom upon the proliferation and viability of cell lines.

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology, the caterpillar Lonomia obliqua has become the focus of toxicological studies due to recent findings about its venom constituents. The objective of this study was to investigate the effects of L. obliqua venom upon the viability and the proliferation of different cell lineages and to propose mechanisms for the herein observed induction of cell proliferation in glioma cell lines. MTT analyses indicate that L. obliqua venom increases the viability of tumor cell lines U138-MG and HT-29; on the other hand, it inhibits the viability of V-79 nontumor cells. Cell count based on the trypan blue exclusion method suggests a proliferating activity of the venom upon U138-MG cells. Exposure of U138-MG to crude venom extract led to a decrease in the production of nitric oxide, and activation of the cAMP signaling pathway inhibited the effects of the venom, indicating that these mechanisms may influence cell proliferation triggered by the venom. Despite the proliferative effects of crude venom on U138-MG and HT-29 cell cultures, a protein purified from L. obliqua hemolymph previously shown to have cytoprotective activity had no effect on U138-MG and HT-29; however, this same protein increased the viability of V-79 cells that had previously been exposed to the cytotoxic activity of the crude venom extract. This study indicates that the venom and the antiapoptotic protein act differently and have different effects on cell cultures, depending on the cell line analyzed. Biomolecules displaying either mitogenic or cytotoxic activities are of great biotechnological interest. Further studies encompassing the purification of active principles from L. obliqua venom are necessary to further elucidate its effects on different cell types.

Transient expression of rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells.

The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10µg rather than 5µg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15µg decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.

Antiviral effect of the egg wax of Amblyomma cajennense (Acari: Ixodidae).

The control of viral infections, especially those caused by influenza viruses, is of great interest in Public Health. Bio prospection has shown the presence of active principles in the hemolymph of arthropods, and in the salivary gland of ticks, and some of these are of interest for the development of new pharmacological drugs. Ticks lay their eggs in the environment, and to protect them from desiccation and microbial attack they involve the eggs in a waxy layer produced by an organ known as Gené's Organ. In this study, the eggs wax from tick Amblyomma cajennense (Fabricius) was extracted using ice cold phosphate buffer. The antiviral activity was evaluated with picornavirus and influenza virus. In both cases egg wax was able to inhibit virus replication. For influenza virus, an amount as small as 12 µg/mL of crude egg wax suspension neutralized 128 UHA (hemaglutinant unit) of H(1)N(1) influenza virus. With picornavirus, egg wax led to a 256-fold reduction in virus production by L929 cells. Egg wax was not cytotoxic to VERO, MDCK and L929 cell, being observed that the cell morphology was preserved with concentration as high as 2 mg/mL. In addition no genotoxic effect was observed for Vero cells, suggesting a very interesting potential antiviral activity.

Conventional and volatile salts as precipitating agents for recombinant antiviral protein / Sais convencionais e voláteis como agentes de precipitação para proteína recombinante com ação antiviral

Recombinant proteins expressed in cell culture have been shown to be relevant in the biopharmaceutical production focusing human health. The current work investigated the precipitation process of recAVLOEc protein, synthesized by E. coli BL21 (DE3) pLysS cells. The system is used for the AVLO expression that shown antiviral activity and it was found in the hemolymph of Lonomia obliqua caterpillar. The precipitation was conducted by the use of conventional salts (ammonium sulfate and sodium sulfate) and the volatile ammonium carbamate salt. Initially, the precipitated protein obtained from bacterial lysate was added to L929 cells to evaluate the cytotoxic effect; and besides Vero cells were infected with measles virus to verify the antiviral action of the precipitated recombinant protein. Toxic effect on the culture of L929 cells was observed for the precipitate obtained by the use of ammonium sulfate and sodium sulfate. In addition, tests in L929 cell cultures infected with EMC virus showed that samples of precipitated protein by salts did not show antiviral action. In Vero cell cultures, the precipitated protein by sodium sulfate showed antiviral action for measles virus.

Proteínas recombinantes expressas em culturas celulares têm se mostrado importantes na produção de fármacos de interesse para a saúde humana. Este estudo investigou a precipitação da proteína recAVLOEc, sintetizada por células de E. coli BL21 (DE3) pLysS, utilizadas como sistema de expressão da AVLO, proteína com atividade antiviral, originalmente encontrada na hemolinfa da lagarta Lonomia obliqua. A precipitação foi conduzida por meio do uso de sais convencionais (sulfato de amônio e de sódio) e do sal volátil carbamato de amônio. Inicialmente o precipitado proteico obtido do lisado bacteriano foi administrado em culturas de células L929 para avaliar o efeito citotóxico e posteriormente em células Vero infectadas com o vírus do sarampo, para a verificação da ação antiviral. Um efeito tóxico em culturas de L929 foi observado para os precipitados obtidos pelo uso de sulfato de amônio e de sódio. Testes em culturas de L929 infectadas com o vírus EMC foram também efetuados e as amostras de proteínas precipitadas com os sais convencionais e o sal volátil não resultaram em ação antiviral. Em culturas de células Vero, o uso do sulfato de sódio como agente de precipitação das proteínas contidas no lisado bacteriano resultou em ação antiviral para o sarampo.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.

Effect of bioactive peptides isolated from yeastolate, lactalbumin and NZCase in the insect cell growth.

In this study, we have described the biological activity of various hydrolysates and its effect on cell growth, growth rate and doubling time. A potent cell culture enhancer factor was observed in the yeastolate hydrolysates, mainly in the protein fractions with low molecular weight. In this case, a growth enhancer of 60.66% was obtained. Despite a lower efficiency of crude lactalbumin hydrolysates (14%), when lactalbumin and yeastolate were added together to the culture, the cell yields were of 102%, showing a synergic effect. Nevertheless, sub fraction from LMW, of lactalbumin, obtained by Sephadex G-10 gel filtration chromatography showed a higher positive effect (23.3%) than low molecular weight fraction of lactalbumin without this chromatography step (11.3%). It is suggested that low molecular weight lactalbumin could have some inhibitory protein. On the other hand, NZCase low molecular weight showed a positive effect of 29.33%, while its sub fractions showed a negative effect of 5.5%. With these data we can suggest that these hydrolysates could be an important element to design new media, serum free, being helpful in protein recombinant production.

Comparative study on PCR and microbiological culture performance for identifying Mycobacterium tuberculosis complex and Mycobacterium bovis specie in bovine samples

The present study aimed at evaluating the concordance between PCR and microbiological culture techniques for analysing organs samples from cattle with suspected lesions of tuberculosis. Fifty-twosamples collected from slaughter houses were analyzed by microbiological culture, and the extracted DNA was amplified by PCR using NZ1 and NZ2 primers. These primers identify the mycobacteria belongingto M. tuberculosis complex, and the primers pair pncA differentiate the M . bovis from M. tuberculosis species. The colonies isolated from 30 samples were suspended, and the extracted DNA was amplifiedby PCR using the same primer pairs. Although the agreement has been considered weak (k = 0.175) between microbiological culture and PCR performed directly in clinical samples using NZ1 and NZ2 primers, the two pairs of primers could amplify the target genes when 100% of the extracted DNA from 30 isolated colonies were used. Thus, PCR employing pncA primer pair enabled to identify M.bovis in the isolated colonies at a short time when compared with the biochemical assays. The concomitant use of PCR and bacteriologic culture techniques hastens the confirmation of detected agent, which is essential inconducting the epidemiological studies and in taking preventive control measures.

Co-infection between influenza virus and flagellated bacteria

Tripsina é necessária na ativação da clivagem do vírus influenza A in vitro. Esta clivagem é importante para entrada do vírus na célula por endocitose mediada pelo receptor celular. Bactérias presentes no trato respiratório são fontes de proteases que podem contribuir na replicação do vírus influenza in vivo. Entre 47 amostras coletadas de cavalos, suínos e humanos, a influenza foi isolada e confirmada em 13 que estavam co-infectadas com bactéria flagelada: Stenotrophomonas maltophilia desde o início destes experimentos. Apesar do tratamento das amostras com antibióticos, as bactérias resistiram em diversas delas (48.39%). A protease (elastase), secretada pela Stenotrophomonas maltophilia, desenvolveu papel decisivo na potencialização da infecção pelo vírus influenza. Essa atividade proteolítica foi detectada pelo teste de ágar-caseína. Amostras positivas para o vírus influenza isolado em animais, bem como em humanos tiveram potencialização da infectividade (ECP) em células MDCK e NCI-H292, sempre que a Stenotrophomonas maltophilia esteve presente. Os referidos microorganismos, bactéria e vírus foram observados ultra-estruturalmente. Esses achados in vitro demonstram como complicações respiratórias podem ocorrer in vivo, através da contribuição de protease microbiana, provocando aumento da inflamação ou destruição dos inibidores celulares de proteases endógenas, nos hospedeiros susceptíveis à influenza.

Performance evaluation of CHO-K1 cell in culture medium supplemented with hemolymph

Desenvolvimento de meios de cultura isentos de soro fetal bovino (SFB) é uma das grandes prioridades de pesquisa em desenvolvimento de processos com célula animal. O objetivo do presente trabalho foi realizar uma análise do potencial de uso da hemolinfa como suplemento do meio utilizado no cultivo da célula animal ancorante CHO-K1. Para isso, foi adicionado 1% v/v de extrato de hemolinfa ao meio DMEM contendo 10% v/v de SFB e 1,0 ou 4,5 g/L de glicose. O cultivo foi realizado em frascos tipo spinner em um ambiente de 10% v/v de CO2, a 37ºC, utilizando o microcarregador Cytodex 1. Comparando os resultados obtidos no ensaio com hemolinfa com um sem hemolinfa pode-se notar uma influência positiva da hemolinfa no cultivo, já que o ensaio com hemolinfa apresentou uma concentração máxima de células 52% maior e uma produtividade máxima de até 40% maior.