Development of a cell-based reporter assay for detection of Human alphaherpesviruses.

Immunosuppressed patients can suffer from Human alphaherpesvirus (HSV) infection with fast evolution, severe atypical symptomatology, and often-fatal outcome. Thus, the development and validation of new methods in vitro and in vivo to promote an early diagnosis and effective treatment of these patients are crucial. Therefore, this work aimed to develop a cell-based reporter assay for the detection of HSV through the transfection of Vero cells with the ICP10 promoter from HSV-2 linked to the pZsGreen1-1 plasmid. The assay was evaluated on Vero cells infected with HSV-1 or HSV-2 and followed by treating them with anti-HSV agents (acyclovir, gallic acid, convallatoxin, and Uncaria sp. extract) or with no anti-HSV activity agents (Passiflora edulis extract and cardenolide derivatives). The GFP expression was increased by both HSV cellular infection, which was detected by flow cytometry and fluorescence microscopy. F2R Zsgreen1-1 cells infection with 200 and 600 PFU/mL of HSV-2 increased the fluorescence intensity, when compared to the controls, by approximately 30% and 60%, respectively. Infection with 100 and 600 PFU/mL of HSV-1 also increased the fluorescence intensity by approximately 20% and 35%, when compared to the controls, respectively. The F2R ZsGreen1-1 system revealed to be an efficient assay, which can be used for clinical diagnosis, antiviral resistance evaluation, HSV cycle studies, and new antiviral drug research.

Investigation of the cytotoxic activity of two novel digitoxigenin analogues on H460 lung cancer cells.

Cardiac glycosides (CGs) are natural compounds traditionally used for the treatment of heart disorders, and recently new therapeutic possibilities were proposed. Their antitumor reports and clinical trials have notably enhanced, including those targeted for lung cancer, the most lethal type that lacks of new treatment agents, instigating the research of these molecules. The CGs studied here, named C10 {3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin} and C18 (3ß-(aminoacetyl)amino-3-deoxydigitoxigenin), are semisynthetic derivatives prepared from digitoxigenin scaffold. Both compounds demonstrated high cytotoxicity for different cancer cell lines, especially H460 lung cancer cells, and their cytotoxic effects were deeply investigated using different methodological approaches. C10 induced cell death at lower concentrations and during shorter periods of treatment than C18, and increased the number of small and irregular nuclei, which are characteristics of apoptosis. This type of cell death was confirmed by caspase-3/7 assay. Both compounds reduced H460 cells proliferative potential by long-term action, and C10 showed the strongest potential. Moreover, these compounds induced a significant decrease of the area and viability of H460 spheroids providing preclinical favorable profiles to develop new chemotherapeutic agents.

Clerodane Diterpenes from the Marine Sponge Raspailia bouryesnaultae Collected in South Brazil.

The marine sponge Raspailia bouryesnaultae, collected in South Brazil, was selected for detailed investigation considering the results of a screening that pointed to an in vitro antiproliferative effect against non-small cells of human lung cancer (A549) and anti-herpes activity against Herpes Simplex virus type 1 (KOS and 29R strains) of ethanolic extracts. The fractionation and chemical investigation of the sponge's hexanic fraction led to the isolation and structural elucidation of six clerodane diterpenes. The main component was identified as the already-reported raspailol (1), isolated from a sponge of the same genus collected in New Zealand. The structure of a new diterpene (2) with a rearranged skeleton was established by high-resolution mass spectrometry (HRMS) and 1D and 2D Nuclear magnetic resonance spectroscopy (NMR) experiments, and named here as raspadiene. Furthermore, four diterpenes were elucidated as isomers of clerodane diterpenes previously obtained from plants, namely kerlinic acid (3), kerlinic acid methyl ester (4), annonene (5), and 6-hydroxyannonene (6). They differ in their stereochemistry, since these diterpenes are characterized by a trans ring fusion at the decalin moiety and the relative configuration of the two methyl groups at C-8 and C-9 in a cis relationship (type trans/cis). The Raspailia diterpenes have a cis ring fusion at the decalin moiety, and the two methyl groups at C-8 and C-9 are in a trans relationship (type cis/trans). The isolated compounds were evaluated for their potential antiproliferative effects on human cancer cell line A549, and it was observed that the diterpenes bearing a hydroxyl group at C-6 exhibited moderate cytotoxic activity, with 50% inhibitory concentration (IC50) values lower than 25 µM. The evaluation of the potential anti-herpes activity against Herpes Simplex Virus type 1 (HSV-1, KOS and 29R strains) showed that the more promising results were observed for the new compound 2, since it inhibited HSV-1 (KOS and 29R strains) replication by 83% and 74%, respectively.

Can Storage Stability and Simulated Gastrointestinal Behavior Change the Cytotoxic Effects of Concentrated Guava Leaves Extract against Human Lung Cancer Cells?

The influence of storage stability and simulated gastrointestinal behavior of different extracts of guava leaves extracts (NC: not concentrated, and C10 and C20: concentrated by nanofiltration) was evaluated based on their total phenolic compound (TPC) contents and antioxidant activity as well as on their cytotoxic effects on A549 and Vero cells. The results showed that C10 and C20 presented high stability for 125 days probably due to their high TPC contents and antioxidant activity. The simulated gastrointestinal behavior modified their TPC contents; however, after all digestion steps, the TPC values were higher than 70%, which means that they were still available to exert their bioactivities. Additionally, the cytotoxic effects of these extracts were evaluated before and after the simulated gastrointestinal behavior or under different storage conditions. C10 presented the best selectivity indices (SI) values (IC50 Vero cells/IC50 A549 cells) at both conditions suggesting that it can be considered a potential extract to be developed as a functional food due to its resistance to the gastrointestinal digestion and storage conditions tested.

Matrix Effects of the Hydroethanolic Extract of Calyces of Physalis peruviana L. on Rutin Pharmacokinetics in Wistar Rats Using Population Modeling.

Rutin is the rutinose conjugate of quercetin. It presents several biological activities and is the major flavonoid in the hydroalcoholic extract of the calyces of Physalis peruviana L. It also shows hypoglycemic activity after oral administration. The aim of this work was to study the matrix effects of the extract from P. peruviana calyces on the pharmacokinetics of rutin and its metabolites in Wistar rats, using non-compartmental and population pharmacokinetic analyses. A pharmacokinetic study was performed after intravenous and oral administration of different doses of pure rutin and the extract. In the non-compartmental analysis, it was found that rutin from the extract exhibited higher distribution and clearance, as well as an 11-fold increase in the bioavailability of its active metabolites. A population pharmacokinetic model was also carried out with two compartments, double absorption and linear elimination, in which the extract and the doses were the covariates involved. This model correctly described the differences observed between rutin as a pure compound and rutin from the extract, including the dose dependency.

Reviewing the mechanisms of natural product-drug interactions involving efflux transporters and metabolic enzymes.

The World Health Organization (WHO) and other worldwide health agencies have recently taken initiatives to encourage the use of traditional medicine and/or complementary/alternative medicine in order to promote well-being and public health. In this way, one of the WHO's concerns is the safe use of these therapies. Phytotherapy is a strategy consisting of the use of medicinal plants (MP) and/or herbal medicinal products (HMP) for medicinal purposes. The use of phytotherapy concomitantly with drugs may cause interactions compromising the expected pharmacological action or generating toxic effects. These interactions are complex processes that may occur with multiple medications targeting different metabolic pathways, and involving different compounds present in MP and HMP. Thus, the aim of this review was to summarize the main MP- and HMP-drug interactions that involve specific transporters (P-glycoprotein and BCRP) and CYP450 enzymes (CYP3A4 and CYP2D6), which play relevant roles in the mechanisms of interactions. Firstly, multiple databases were used to search studies describing in vitro or in vivo MP and HMP-drug interactions and, after that, a systematic note-taking and appraisal of the literature was conducted. It was observed that several MP and HMP, metabolic pathways and transcription factors are involved in the transporters and enzymes expression or in the modulation of their activity having the potential to provide such interactions. Thus, the knowledge of MP- and HMP-drug interaction mechanisms could contribute to prevent harmful interactions and can ensure the safe use of these products to help the establishment of the therapeutic planning in order to certify the best treatment strategy to be used.

Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-derivatives.

In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize the readily accessible 3ß-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of compounds were obtained from derivative (5): (i) O-glycosyl trizols through click chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different residues linked via an amino-group. All obtained derivatives have their chemical structures confirmed, and their anti-herpes (against HSV-types 1 and 2 replication) and cytotoxic (against PC3, A549, HCT-8 and LNCaP cell lines) activities evaluated. Compounds 10 and 11 exhibited the most promising results against HSV-1 (KOS and 29-R strains) and HSV-2 (333 strain) replication with SI values > 1000. Both compounds were also the most cytotoxic for the human cancer cell lines tested with IC50 values similar to those of paclitaxel. They also presented reduced toxicity toward non-cancerous cell lines (MRC-5 and HGF cells). Promising compounds were tested in regard to their ability to inhibit Na+/K+-ATPase. The inhibition rate correlates suitably with the bioactivity demonstrated by those both compounds against the different human cancer cells tested as well as against HSV replication. Moreover, the results showed that specific chemical features of compound 10 and 11 influenced the bioactivities tested. In summary, it was possible to obtain novel digitoxigenin-derivatives with remarkable cytotoxic and anti-herpes activities as well as low toxicity and high selectivity. In this way, they could be considered potential molecules for the development of new drugs.

Depuration dynamics of oysters (Crassostrea gigas) artificially contaminated by Salmonella enterica serovar Typhimurium.

The State of Santa Catarina produces the greatest quantity of edible mollusks in Brazil. To guarantee sanitary qualify, mollusk cultures should be monitored for contamination by pathogenic microorganisms. A self-purification or "depuration" system that eliminates Salmonella enterica serovar Typhimurium contamination from oysters has been developed and evaluated. The depuration process occurred within a closed system, in which 1000 L of water was recirculated for 24 h. The water was sterilized with ultraviolet (UV) light, chlorine, or both together. Oysters (Crassostrea gigas) artificially contaminated with S. typhimurium were harvested every 6 h. Samples of oyster tissue were excised and both the presence and numbers of bacteria were determined. Combined UV light and chlorine treatments resulted in total elimination of bacteria within 12 h. Polymerase chain reaction detected bacteria in water exposed to the three treatments. This pioneering study is the first of its kind in Brazil and represents a major contribution to commercial mollusk culture in this country.

A new semisynthetic cardenolide analog 3ß-[2-(1-amantadine)- 1-on-ethylamine]-digitoxigenin (AMANTADIG) affects G2/M cell cycle arrest and miRNA expression profiles and enhances proapoptotic survivin-2B expression in renal cell carcinoma cell lines.

Cardiac glycosides are well known in the treatment of cardiovascular diseases; however, their application as treatment option for cancer patients is under discussion. We showed that the cardiac glycoside digitoxin and its analog AMANTADIG can inhibit the growth of renal cell carcinoma (RCC) cell lines and increase G2/M cell cycle arrest. To identify the signaling pathways and molecular basis of this G2/M arrest, microRNAs were profiled using microRNA arrays. Cardiac glycoside treatment significantly deregulated two microRNAs, miR-2278 and miR-670-5p. Pathway enrichment analysis showed that all cardiac glycoside treatments affected the MAPK and the axon guidance pathway. Within these pathways, three genes, MAPK1, NRAS and RAC2, were identified as in silico targets of the deregulated miRNAs. MAPK1 and NRAS are known regulators of G2/M cell cycle arrest. AMANTADIG treatment enhanced the expression of phosphorylated MAPK1 in 786-O cells. Secondly, we studied the expression of survivin known to be affected by cardiac glycosides and to regulate the G2/M cell phase. AMANTADIG treatment upregulated the expression of the pro-apoptotic survivin-2B variant in Caki-1 and 786-O cells. Moreover, treatment with AMANTADIG resulted in significantly lower survivin protein expression compared to 786-O control cells. Summarizing, treatment with all cardiac glycosides induced G2/M cell cycle arrest and downregulated the miR-2278 and miR-670-5p in microarray analysis. All cardiac glycosides affected the MAPK-pathway and survivin expression, both associated with the G2/M phase. Because cells in the G2/M phase are radio- and chemotherapy sensitive, cardiac glycosides like AMANTADIG could potentially improve the efficacy of radio- and/or chemotherapy in RCCs.

Platelet-rich plasma influence on human osteoblasts growth.

OBJECTIVE: The influence of progressively high concentrations of platelet-rich plasma (PRP) on human osteoblast hFOB1.19 proliferation was evaluated. MATERIAL AND METHODS: The PRP was obtained from a human source. Two experiments were conducted. In the first one, PRP was diluted to 50%, 25%, 12.5% and 6.125% (v/v) with culture medium (Modified Eagle's Medium (MEM) : Ham's F12 Medium (HAM-F12%) and 1% antibiotics-antimicotic) supplemented with 10% of fetal bovine serum (FBS). In the second experiment, all conditions were identical except for the absence of FBS in the culture medium. RESULTS: The results of the osteoblast proliferation test were higher when stimulated by the 50% PRP dilution, with or without FBS. A further study is suggested to determine if concentrations above 50% could cause higher rates of osteoblast proliferation. In this study, the results were not statistically different (P<0.05) with 12.5% and 6.125% PRP dilutions. Additionally, it was shown that FBS is not necessary for PRP-mediated induction of osteoblast proliferation. CONCLUSION: This study concluded that PRP promotes osteoblast proliferation and suggested its clinical application to bone graft procedures in implant dentistry.