RT-PCR for detection of bovine parainfluenza virus type 3 (bPIV-3)

The RT-PCR technique has been frequentely used for detection of the human parainfluenza virus type 3 (hPIV-3) but the literature is scarce in relation to the bovine parainfluenza virus type 3 (bPIV-3). The aim of this study was to describe a reverse transcriptase polymerase chain reaction (RT-PCR) for detection of bovine parainfluenza virus type 3 (bPIV-3) using degenerate oligonucleotides targeting a conserved region of hemagglutinin-neuraminidase (HN) gene. Reference strain SF-4 and three different brazilian bPIV-3 isolates, besides five viral strains from different sources, were included in this study. Viruses were cultured in MDBK cells under standard conditions. Hemagglutination (HA) test was used for viral titration and a direct immunofluorescence test (DFAT) for isolate screening. In RT-PCR all bPIV-3 isolates showed amplification of an expected 1009 bp fragment of HN gene, as oposed to non PIV-3 viral samples where no amplification was detected. Using SF-4 as positive control, sensitivity of 95 pg cDNA wasachieved. In spite of the low number of bPIV-3 isolates tested, the results obtained in this study point out the potential use of this technique for detection of bPIV-3 in bovine clinical specimens

ANTIMICROBIAL PEPTIDE P34 INFLUENCES GENE EXPRESSION OF LISTERIA MONOCYTOGENES GROWING IN SOFT CHEESE

Objective: To evaluate whether antimicrobial substances produced by autochthonous lactic acid bacteria (LAB) from Minas Frescal cheese are able to enhance the activity of bacteriocin P34 against Listeria monocytogenes and investigate the influence of P34 in specific gene expression of this bacterium after the inoculation in Minas Frescal cheese.Methods: Bacillus sp. P34 and L. monocytogenes ATCC 7644 were used in this study. The antimicrobial peptide P34 was purified and applied (0, 800 or 6400 AU/ml) to cheese surface before inoculation with L. monocytogenes. Antimicrobial activity and synergism were detected using the agar diffusion technique. Expression levels of D-Alanine-D-alanyl carrier protein ligase (dltA), Putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) mRNAs in bacteriocin-treated L. monocytogenes growing in Minas Frescal cheese were determined using real-time PCR.Results: The peptide P34 showed increased antilisterial activity when combined with culture supernatants of some selected LAB isolated from Minas Frescal cheese. The addition of peptide P34 to cheese caused a decrease of up to 3 log cycles in viable counts of artificially inoculated L. monocytogenes. The influence of peptide P34 on the expression of genes associated with components of the cell surface of L. monocytogenes was investigated by real-time PCR. A significant increase in the expression of the genes dltA, Imo 1695 and mptA was observed after 96 h in the presence of peptide P34.Conclusion: These results suggest that the peptide P34 influences the expression of genes involved in D-alanylation of teichoic acids and lipoteichoic acids and lysination of the cell membrane of phospholipids

Nanocapsules With Naringin And Naringenin Affect Hepatic and Renal Energy Metabolism Without Altering Serum Markers of Toxicity in Rats

Naringin and naringenin are flavonoids found in citrus fruits and have several health benefits, however these compounds are susceptible to degradation, limiting their therapeutic application. To solve this problem, an alternative is to incorporate them into nanocapsules. The aim of this work was to evaluate the toxicity of these nanocapsules against renal and hepatic serum markers and also on the activities of pyruvate kinase, Mg2+-ATPase, and creatine kinase. Nanocapsules containing naringin and naringenin, nanocapsules without the active compounds and the compounds in their free form were administered orally, once a day, for 28 days. After treatment, the serum levels of hepatic and renal markers were not altered, nor the activities of pyruvate kinase tissue, however, the treatment of nanocapsules with flavonoids increased the activities of mitochondrial creatine kinase in the kidney and hepatic Mg2+-ATPase. Thus, renal and hepatic serum markers, which are normally used as indicators of toxicity, did not change after the period of administration of the nanoparticles. However, the activities of important enzymes of the energy metabolism in these organs were affected. Our findings reinforce that nanomaterial testing for toxicity needs to go beyond traditional methods to ensure the safe use of nanoparticles for therapeutic purposes

Tucumã extracts decreases PML/RARΑ gene expression in NB4/APL cell line

Acute promyelocytic leukemia (APL) is a cancer pharmacologically treated with all-trans retinoic acid (ATRA), although well tolerated by most patients, some develop toxicity to ATRA, Differentiation Syndrome. The Amazon Biome has several fruits and oil plants rich in micronutrients, particularly carotenoids as the fruit tucumã (Astrocaryum aculeatum). This study analyzed the antitumor and cytoprotective activity of tucumã with and without concomitant exposure of ATRA in high concentration mimicking the toxicity of differentiation syndrome, as the potential cytotoxic effect of chemotherapeutic in an APL cell line. The cultured NB4 cells were exposed to ethanolic extracts of tucumã and to synergism with extracts and ATRA. Determination of proliferation, cell viability, caspases 1, 3, 8 and cell differentiation by nested RT-qPCR. The ATRA control had a strong inhibitory effect and toxicity as expected. The extracts also reduced cell proliferation by triggering apoptosis in concentration-dependent and reversing chromosome translocation, especially the lowest tested concentration of tucumã pulp extract. In the synergism, extracts act to maintain the levels of viability and apoptosis equal to the ATRA control but in contrast to drug that causes death and destruction of the genetic material, tucumã demonstrated a reduction of the gene expression indicating a possible protection against the toxicity of high concentrations of ATRA. These results suggest that fruits rich in retinoid molecules may have a cytotoxic effect against APL cells and reduced concentrations of carotenoids may act as cytoprotectors in APL cells treated with high concentrations of ATRA promoting cellular/molecular differentiation

INFLUENCE OF PEPTIDE P34 ON GENE EXPRESSION OF LISTERIA MONOCYTOGENES AND LISTERIA SEELEGERI

Objective: Investigate the influence of the antimicrobial peptides P34 and nisin on the expression of genes associated with components of the cell surface of Listeria monocytogenes and Listeria seeligeri.Methods: Antimicrobial activity was determined by addition of peptide P34 and nisin (12.5 µg/ml) onto Brain Heart Infusion agar (BHI) plates previously inoculated with indicator strains (L. monocytogenes ATCC 7644 or L. seeligeri AC 82/4) after incubation for 24 h at 37 °C or 240 h at 4 °C. Ribonucleic acid (RNA) was directly extracted from bacterial colonies at the border of the inhibition zones, and the expression levels of genes D-alanine-D-alanyl carrier protein ligase (dltA), putative phospholipid lysinylation (Imo 1695) and EIIABMan of mannose-specific PTS (mptA) were determined using real-time PCR.Results: A non-significant increase in the levels of transcription of genes dltA, Imo1695 and mptA was observed for L. monocytogenes treated with peptide P34 or nisin. Both peptides caused a similar decrease in dltA gene expression in L. seeligeri. The expression of gene Imo1695 significantly decreased (about 2000-fold) after treatment with the peptide P34 at 37 °C, while at 4 °C a reduction of 12-fold and 5-fold was detected for P34 and nisin, respectively. A significant decrease in mptA gene expression was observed by exposition to peptide P34 (31.872-fold) and nisin (16.047-fold) for 24 h at 37 °C.Conclusion: The results suggest that both peptide P34 and nisin influence the expression of genes related with the cell-surface/cell-membrane structure of L. seeligeri and in lesser extent L. monocytogenes

ANTIMYCOBACTERIAL, ANTIMICROBIAL AND ANTIFUNGAL ACTIVITIES OF GERANIUM OIL-LOADED NANO CAPSULES

Objective: The aim of this study was to perform the first ever investigation of the effect of activities in the nano capsules containing Geranium oil (NC1) against different species of pathogens such as Mycobacterium genus (both fast growing and slow growing), bacterial, and yeasts.Methods: The GO was analyzed by GC and GC/MS. Nano capsule suspensions (NC) were prepared by interfacial deposition of a preformed polymer method and the MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities.Results: GO-loaded nano capsules (NC1) presented nano metric mean diameters (188 nm), polydispersity indices below 0.149, pH (5.5), and zeta potentials (about-10.8 mV). The MICs were determined for the antimycobacterial, antimicrobial, and antifungal activities. The NC1 was effective to Mycobacterium smegmatis (149.7 µg ml-1), M. abscessos (35.9 µg ml-1), M. massiliense (35.9 µg ml-1), M. avium (71.8 µg ml-1), Enterococcus faecalis, Streptococcus sp. (149.7 µg ml-1) and Listeria monocytogenes (35.9 µg ml-1). The NC1 was able to significantly reduce the number of cells of C. albicans (by approximately 5 log), 4 log the number of cells of C. dublinensis, C. glabrata, and C. krusei, and 2 log the number of cells of C. parapsilosis compared to the control group.Conclusion: Our study showed that the geranium oil-loaded nano capsules have antimycobacterial activities similar to free oil. The GO was effective in inhibiting the formation of germ tubes of Candida albicans, yet the nano capsule containing GO failed to inhibit the formation of this important virulence factor.Â

DEVELOPMENT OF NANOEMULSION CONTAINING PELARGONIUM GRAVEOLENS OIL: CHARACTERIZATION AND STABILITY STUDY

Objective: To develop, characterize and evaluate the stability of nanoemulsions containing geranium oil (NEG) at different temperatures (4 °C, 25 °C and 45 °C) for 90 d.Methods: The quantification of oil in the nanostructure was performed by gas chromatography–mass spectrometry (GC-MS). The NEG was prepared in Ultra-Turrex and characterized by determining the particle size, polydispersity indices and pH. The thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM) to evaluate the thermal stability of the compounds, the thermal events and morphological analyses of NEG, respectively.Results: The results allow us to suggest that the use the ultra-turrax method is a strategy good to NEG preparation. The stability of the NEG was strongly influenced by storage temperature, with droplet size increasing rapidly at higher temperatures (45 °C), which was attributed to coalescence near the phase inversion temperature. The NEG submitted the low temperatures (4±2 °C) remained with the same particle size value (164 nm). However, the citronellol and geraniol showed a significant reduction throughout the test even in these conditions of temperature. Thermogram of NEG shows the crystallization peak at the cooling cycle in-20.1 °C and a melting was observed at 1.5 °C. TEM images indicated that NEG was spherical and nanometric.Conclusion: The proposed Ultra-Turrax method is simple which prevents volatilization of GO for the production of NEG. The formulations presented good physicochemical characteristics and stability for 90 d was only achieved in 4 °C

Resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin complex recovered the changes of creatine kinase and Na+, K+-ATPase activities found in the spleen from streptozotocin-induced diabetic rats

Type 1 diabetes (T1D) is the result of the selective destruction of the pancreatic β-cells by T cells of the immune system. Although spleen is a secondary lymphoid organ, it is also involved in the T1D pathogenesis. However, the alterations in a variety of cellular processes of this disease need to be further understood. We aimed to analyze the benefits of resveratrol, and its complexed form on diabetic complications in the spleen of rats. To this end, we investigated important enzymes of phosphoryl transfer network, and Na+, K+-ATPase activity. Wistar rats were divided into non-diabetic groups: Control, Ethanol, Resveratrol, Hydroxypropyl-β-cyclodextrin, Resveratrol-hydroxypropyl-β-cyclodextrin, and diabetic groups with the same treatments. Diabetes was induced by a single dose of 60 mg/kg of streptozocin intraperitoneally, and treatments by intragastric gavage once daily for 60 days. Hyperglycemia reduced creatine kinase activity, which was reversed by the administration of resveratrol. Na+, K+-ATPase activity was greatly affected, but it was reversed by resveratrol and resveratrol-hydroxypropyl-β-cyclodextrin. This suggest an energetic imbalance in the spleen of diabetic rats, and in case this also occurs in the diabetic patients, it is possible that resveratrol supplementation could be beneficial to the better functioning of the spleen in diabetic patients

Development of RT-PCR techniques for hemagglutinin-neuraminidase (HN) gene sequencing and detection of bovine type 3 Parainfluenza virus

Existem diversos trabalhos publicados sobre a utilização de diferentes métodos imunológicos para diagnosticar infecções do trato respiratório causadas por vírus parainfluenza bovino tipo 3 (bPIV-3). Entretanto, é escassa a literatura sobre a utilização da técnica de isolamento viral. Até o presente momento não havia sido relatada a utilização da Transcrição Reversa - Reação em Cadeia da Polimerase (RT-PCR), na detecção de bPIV-3. O objetivo deste estudo foi contribuir para uma melhor caracterização dos bPIV-3 através do desenvolvimento de técnicas de RTPCR para a sua detecção. Utilizando-se uma amostra referência de bPIV-3 (SF-4) e uma amostra de bPIV-3 isolada no Rio Grande do Sul (amostra DIO) foram desenvolvidas técnicas de RT-PCR para a amplificação de diferentes regiões do gene da hemaglutinina-neuraminidase (HN). Após seqüenciamento parcial do gene HN e alinhamento das seqüências da amostra DIO, os resultados revelaram homologia de 99% em relação à amostra referência, 98% a 91% quando comparada com outras amostras de bPIV-3 previamente publicadas na rede e 79% a 80% quando comparada com as amostras de hPIV-3. Foi desenvolvida, também, uma técnica de RT-PCR para amplificação de parte do gene da HN do bPIV-3 e do hPIV-3. Nos experimentos de otimização, a técnica de RT-PCR, comparada com o isolamento viral, apresentou sensibilidade de 140 DICC50, boa especificidade e reprodutibilidade. Os resultados, após seqüenciamento da amostra de vírus bPIV-3 isolada no RS, apresentaram, grande homologia com os das amostras de bPIV-3 comparadas, especialmente a amostra referência. Os dados obtidos neste estudo mostraram que a RT-PCR desenvolvida para a detecção de PIV-3 foi capaz de amplificar um fragmento de 1009 bp do gene da HN de amostras de PIV-3 isoladas de bovinos e humanos, possibilitando a sua utilização em diagnóstico e em estudos epidemiológicos.There are several published studies on the application of different immunological methods for diagnosis of respiratory infections caused by bovine type 3 Parainfluenza virus (bPIV-3). However, the literature on viral isolation procedure is very scarce. At present there is no report about the utilization of reverse transcription-polymerase chain reaction (RT-PCR) for bPIV-3 detection. The aim of this study was to contribute to a better caracterization of bovine type 3 Parainfluenza virus by developing RT-PCR techniques to its detection. A reference sample (SF-4) and a sample of bPIV-3 isolated in Rio Grande do Sul (DIO sample) were used to develop RT-PCR techniques by amplification of different regions of hemagglutinin-neuraminidase gene (HN). After HN gene partial sequencing of DIO sample and sequence alignment, the results revealed 99% homology when compared to reference sample, 98% the 91% homology in relation to bPIV-3 samples previously published and 79% the 80% if compared to hPIV-3 samples. It was also developed a RT-PCR for amplification of a part of bPIV-3 and hPIV-3 NH gene. In optimization experiments, compared to viral isolation procedure, the RTPCR displayed a sensitivity of 140 DICC50, good specificity and reproductibility. Results after sequencing of bPIV-3 sample isolated in RS displayed strong homology with those of bPIV-3 tested samples, specially the reference sample. Data obtained in this study showed that the RT-PCR technique developed for PIV- 3 detection was able to amplify an 1009 bp HN gene fragment of bovine and human PIV-3 samples which enables its utilization in diagnostic and epidemiological studies