High frequency of Lamivudine and Telbivudine resistance mutations in hepatitis B virus isolates from human immunodeficiency virus co-infected patients on highly active antiretroviral therapy in Bucaramanga, Colombia.

Hepatitis B virus (HBV) antiviral Resistance-Associated Mutations (RAMs) in human immunodeficiency virus (HIV) coinfected patients undergoing highly active antiretroviral therapy (HAART) are complex and incompletely understood. We aimed to determine the prevalence of HBV coinfection, HBV genotypes, and RAMs in a cohort of people living with HIV (PLWH) in the northeastern region of Colombia. This cross-sectional study was carried out between February 2013 and February 2014. Virological, immunological and HAART data were collected from clinical records. In-house nested PCR and Sanger sequencing of the HBV pol gene were used to identify coinfections, genotypes, RAMs and HBV s antigen (HBsAg) escape mutants. Among 275 PLWH, HBV coinfection was confirmed in 32 patients (11.6%), of whom nine (28.2%) were HBsAg positive (active hepatitis B), and 23 (71.8%) were occult hepatitis B infections (OBI). All HBV sequences (n = 23) belonged to the genotype F3. Among HIV/HBV coinfections, 71.9% had CD4+ T cell counts above 200 cells/mm3 and 37.5% had undetectable HIV viral loads. The RAMs rtL80I, rtL180M, and rtM204V, which confer resistance to Lamivudine/Telbivudine and partially resistant to Entecavir, were found in all HBV isolates. An unknown rt236Y mutation to Tenofovir was also identified. Most patients under HAART received first-generation HBV antiviral therapy with a low genetic barrier to resistance. Antiviral Drug-associated Potential Vaccine-escape Mutations (ADAPVEMs) in the S gene were observed in all isolates ranging from 1-20 amino acid substitutions. However, no vaccine escape mutants were detected. In Conclusion, these findings highlight the importance of HBV molecular screening, antiviral resistance monitoring and new guidelines for PLWH to overcome RAMs and prevent HBV-related liver disease.

The effects of genetic polymorphisms on benzene-exposed workers: A systematic review.

BACKGROUND AND AIMS: Benzene is a group I carcinogen, which has been associated with leukemia and myelodysplastic syndrome. Moreover, it has been proposed that polymorphisms in benzene metabolizing genes influence the outcomes of benzene exposure in the human body. This systematic review aims to elucidate the existent relationship between genetic polymorphisms and the risk of developing adverse health effects in benzene-exposed workers. METHODS: Three databases were systematically searched until April 2020. The preferred reporting items for systematic reviews and meta-analyses method was used to select articles published between 2005 and 2020. Quality assessment and risk of bias were evaluated by the Newcastle-Ottawa scale. RESULTS: After full-text evaluation, 36 articles remained out of 645 initially screened. The most studied health effects within the reviewed papers were chronic benzene poisoning, hematotoxicity, altered urinary biomarkers of exposure, micronucleus/chromosomal aberrations, and gene methylation. Furthermore, some polymorphisms on NQO1, GSTT1, GSTM1, MPO, and CYP2E1, among other genes, showed a statistically significant relationship with an increased risk of developing at least one of these effects on benzene-exposed workers. However, there was no consensus among the reviewed papers on which specific polymorphisms were the ones associated with the adverse health-related outcomes, except for the NQO1 rs1800566 and the GSTT1 null genotypes. Additionally, the smoking habit was identified as a confounder, demonstrating worse health outcomes in exposed workers that smoked. CONCLUSION: Though there is a positive relationship between genetic polymorphisms and detrimental health outcomes for benzene-exposed workers, broader benzene-exposed cohorts that take into account the genetic diversity of the population are needed in order to determine which specific polymorphisms incur in health risks.

A novel method for isolation and culture of primary swine gastric epithelial cells.

BACKGROUND: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. RESULTS: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William's E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. CONCLUSIONS: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William's E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

Antifungal activity of phytotherapeutic preparation of Baccharis species from argentine Puna against clinically relevant fungi.

ETHNOPHARMACOLOGICAL RELEVANCE: B. boliviensis and B. tola are used in traditional medicine in the Argentine Puna to treat skin and soft tissue infections and inflammatory processes in humans and animals. AIM OF THE STUDY: To assess the potential of phytotherapeutic preparations of Baccharis species as antifungal agents against clinically relevant fungi and to determine the chemical composition of the extracts. MATERIAL AND METHODS: Phytotherapeutic preparations of B. boliviensis and B. tola collected in Argentine Puna were evaluated as an antifungal agent against clinically relevant fungi (yeast, non-dermatophytes, and dermatophytes) isolated of patients from a local Hospital, and reference strains, using macrodilution and microdilution assays. The bioactivity was supported by UHPLC-OT-MS metabolome fingerprinting. RESULTS: The results revealed that the plant preparations were active against most of evaluated fungal strains; B. boliviensis was more active than B. tola. Dermatophyte fungi strains were the most sensitive isolates. The phytotherapeutic preparation showed Minimal Inhibitory Concentration (MIC) values between 25 and 400 µg GAE/mL and Minimum Fungicidal Concentration (MFC) values between 50 and 400 µg GAE/mL. Regarding the phytochemical analysis, total phenolic and total flavonoid contents of hydroalcoholic preparation of B. boliviensis were greater than those of the B. tola extract. Both Baccharis species showed similar chromatographic patterns, fifty-two compounds were identified based on UHPLC-OT-MS including several terpenoids, flavonoids and phenolic acids that have been identified in this two endemic South American Baccharis species for the first time. Several identified compounds present antifungal properties, the presence of these compounds support the bioactivity of the Baccharis extracts. CONCLUSIONS: In this work the traditional use of both Baccharis species as an antimicrobial against commercial products resistant fungal strains was validate, principally against dermatophytes fungi such as T. rubrum, T. mentagrophytes, M. canis, and M. gypseum. These results indicate that the hydroalcoholic preparations could be used for the treatment of fungal infectious.

Electrosprayed chitosan microcapsules as delivery vehicles for vaginal phytoformulations.

The design of novel delivery systems to treat vaginal fungal infections is a topic of high interest. Chitosan, being itself antimicrobial and having good mucoadhesive properties, is an excellent candidate as a delivery matrix for active compounds. In this work, chitosan microcapsules containing dry extracts of Argentinean medicinal plants with proved biological properties (Larrea divaricata, L. cuneifolia, L. nitida, Zuccagnia punctata and Tetraglochin andina) were developed through electrospraying and compared with conventionally used tablets containing the same extracts. Total phenolics, loading efficacy, physical properties, morphology and particle size, molecular organization, water sorption capacity, release of bioactive compounds and biological properties were assessed. The encapsulation process or the inclusion in tablets did not degrade the bioactive compounds of the extracts. The release of phenolic compounds from chitosan microcapsules was faster than from tablets. The fingerprint of released phenolic compounds from microcapsules and tablets was similar to that from the dry extracts and the antioxidant and antifungal capacity remained unchanged. The FT-IR analysis suggested interactions between the chitosan and the extracts, which explained why the microcapsules kept the integrity in slightly acidic media. Increased solubility of the extracts when incorporated in the microcapsules was seen in simulated vaginal fluid, potentially increasing the bioavailability of bioactive compounds in the vaginal environment. This work highlights the potential of the chitosan-based delivery systems for phytomedicines with antifungal and antioxidant activity to be used in vulvovaginal candidiasis.

Tetraglochin andina Ciald.: A medicinal plant from the Argentinean highlands with potential use in vaginal candidiasis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Argentinean medicinal plant Tetraglochin andina Ciald, formerly classified as T. cristatum (Britton) Rothm is used in traditional medicine by inhabitants from Argentinean northwestern highlands (Puna) to treat candidiasis and as anti-inflammatory. AIM OF THE STUDY: To assess the potential of the crude drug as an anti-Candida agent with anti-inflammatory properties. The bioactivity and phytochemical composition of a dry extract of the plant was investigated. MATERIAL AND METHODS: The pharmacognostic description of the crude drug is carried out for the first time, including macroscopic and microscopic examinations of the different organs, physicochemical and extractive values (petroleum ether-, ethanol- and water-soluble). The dry extract from T. andina was evaluated as antifungal against pathogenic Candida sp. and Saccharomyces cerevisiae isolated from vaginal infections and reference strains, by the macrodilution and microdilution assays. The normal vaginal microbiome in women is characterized by the dominance of lactic acid-producing bacteria, mainly Lactobacillus spp. The effect of T. andina extract on Lactobacillus strains was also assayed. The inhibitory effect on proinflammatory enzymes (cyclooxygenase, lipoxygenase and phospholipase A2) and antioxidant capacity was studied. The chemical profile was analyzed by HPLC-ESI-MS. RESULTS: The hydroalcoholic extract inhibited the growth of all yeasts with Minimal Inhibitory Concentration (MIC) values between 12.5 and 400 µg GAE/mL and the MIC values on Lactobacillus were higher than the MIC values against Candida isolates ( > 400 µg GAE/mL). These results indicate that the hydroalcoholic extract could be used without affecting the normal microbiota of vaginal fluid. The extract showed antioxidant activity and could modulate the inflammatory process by three pathways (sPLA2, COX-2, LOX). The plant extract contained high total phenolic levels (386.9±1.7 mg GAE/g dry extract) and flavonoid levels (260.4±2.7 mg GAE/g dry extract). Fifty phenolic compounds were identified by HPLC-ESI-MS. They were mainly hydrolysable and condensed tannins. The dry extract was chemically and biologically stable during one year at room temperature or 4 °C. CONCLUSIONS: The presence of anti-Candida and anti-inflammatory activities in Tetraglochin andina extracts give support to their traditional use for treating conditions associated with microorganism infections and inflammatory process in humans. This plant preparation could be used to design phytopharmaceutical preparations to inhibit yeast growth and moderate the inflammatory and oxidative process.

Chemical and functional characterization of seed, pulp and skin powder from chilto (Solanum betaceum), an Argentine native fruit. Phenolic fractions affect key enzymes involved in metabolic syndrome and oxidative stress.

The aim of this work was to assess the nutritional and functional components of powder obtained by lyophilization of whole fruits, seeds, pulp and skin from chilto (Solanum betaceum Cav) cultivated in the ecoregion of Yungas, Argentina. The powders have low carbohydrate and sodium content and are a source of vitamin C, carotenoid, phenolics, potassium and fiber. The HPLC-ESI-MS/MS analysis of the fractions enriched in phenolics allowed the identification of 12 caffeic acid derivatives and related phenolics, 10 rosmarinic acid derivatives and 7 flavonoids. The polyphenols enriched extracts before and after simulated gastroduodenal digestion inhibited enzymes associated with metabolic syndrome, including α-glucosidase, amylase and lipase and exhibited antioxidant activity by different mechanisms. None of the analyzed fruit powders showed acute toxicity or genotoxicity. The powders from the three parts of S. betaceum fruit may be a potential functional food and the polyphenol enriched extract of seed and skin may have nutraceutical properties.

Anti-inflammatory, Antioxidant and Antimicrobial Activity Characterization and Toxicity Studies of Flowers of "Jarilla", a Medicinal Shrub from Argentina.

Zuccagnia punctata Cav. (Fabaceae) is an Argentine medicinal aromatic shrub (jarilla pispito, puspus, lata and jarilla macho). The chalcones were identified as pigments responsible for the yellow color of the flowers. Hydroethanolic extracts were obtained both from fresh flowers and from flowers dried by lyophilization. The extracts were standardized by their phenolic and flavonoids content. Their fingerprints by HPLC-DAD indicated the presence of two chalcones as major compounds (2',4'-dihydroxychalcone and 2',4'-dihydroxy-3'-methoxychalcone). Both extracts showed the same total phenolic, non-flavonoid phenolic and flavonoid phenolic content and their phenolic profiles were similar. The polyphenolic extracts exhibited antioxidant (free radical scavenging and inhibitory activity on lipoperoxidation) and anti-inflammatory (inhibition of lipoxygenase and cyclooxygenase enzymes) activities. The flower extracts were active against six Candida species with MIC values between 60 and 120 µg GAE x mL(-1) and were also active on methicillin-resistant Staphylococcus aureus (MIC: 250 µg GAE x mL(-1)) and Enterococcus faecalis (MIC: 500 µg GAE x mL(-1)). The extracts were neither toxic (Artemia salina test) nor mutagenic (Ames test). Jarilla flowers could be considered as a new dietary supplement that could help to prevent pathologies associated with oxidative stress and the polyphenolic extract obtained from them could be considered as a standardized phytotherapeutic product with antimicrobial, antioxidant and anti-inflammatory activities. The aim of this work was to determine the pigments responsible for the yellow color of the flowers of Z. punctata and to evaluate the functional properties of the polyphenolic extract of the flowers. The toxicity (Artemia salina) and mutagenic activity (Ames test) of the extract were also evaluated.

The effect of Zuccagnia punctata, an Argentine medicinal plant, on virulence factors from candida species.

Zuccagnia punctata Cav. has been used as a traditional medicine in Argentina for the treatment of bacterial and fungal infections. In this study, we evaluated the ability of Z. punctata extract (ZpE) and compounds isolated from it to inhibit the growth and virulence factors of Candida species. ZpE showed inhibitory activity against planktonic cells of all assayed Candida species with MIC values of 400 microg/mL and with MFC values between 400 and 1,200 microg/mL. The principal identified compounds by HPLC-MS/MS and UV-VIS were chalcones (2',4'-dihydroxy-3'-methoxychalcone, 2',4'- dihydroxychalcone), flavones (galangin, 3,7-dihydroxyflavone and chrysin) and flavanones (naringenin, 7-hydroxyflavanone and pinocembrine). These compounds were more effective as inhibitors than the extracts upon biofilm formation as well as on preformed Candida biofilm and yeast germ tube formation. Furthermore, ZpE and chalcones are able to inhibit exoenzymes, which are responsible for the invasion mechanisms of the pathogens. All these effects could moderate colonization, thereby suppressing the pathogen invasive potential. Our results indicate that ZpE and chalcones could be used in antifungal therapy.

Anti-inflammatory and antioxidant activities, functional properties and mutagenicity studies of protein and protein hydrolysate obtained from Prosopis alba seed flour.

Prosopis species are considered multipurpose trees and shrubs by FAO and their fruit constitute a food source for humans and animals. According to the "Código Alimentario Argentino", "algarrobo flour" is produced by grinding the whole mature pod, but in the traditional process most of the seeds are discarded. In this paper, the flour from seed was obtained. Then, the proteins were extracted and enzymatic hydrolysis was carried out. According to their amino acid profile and chemical score (>100%), the Prosopis alba proteins, are not deficient in essential amino acids considering the amount of amino acid necessary by adults. The protein isolate showed a good solubility (pH 7.4-9), emulsificant capacity, oil binding capacity and water adsorption capacity. The antioxidant ability of proteins was significantly increased with hydrolysis (SC50 values: 50-5µg/mL, respectively). Inhibitory activity of pro-inflammatory enzymes (lipoxygenase and phospholipase) was described. The mutagenicity/antimutagenicity of proteins and protein hydrolysates from seed flour were also analysed. The results suggest that P. alba cotyledon flour could be a new alternative in the formulation of functional foods not only for its high protein content but also by the biological and functional properties of its proteins and protein hydrolysates.

A colorimetric method to quantify endo-polygalacturonase activity.

We report a new colorimetric assay to quantify endo-polygalacturonase activity, which hydrolyzes polygalacturonic acid to produce smaller chains of galacturonate. Some of the reported polygalacturonase assays measure the activity by detecting the appearance of reducing ends such as the Somogyi-Nelson method. As a result of being general towards reducing groups, the Somogyi-Nelson method is not appropriate when studying polygalacturonase and polygalacturonase inhibitors in plant crude extracts, which often have a strong reducing power. Ruthenium Red is an inorganic dye that binds polygalacturonic acid and causes its precipitation. In the presence of polygalacturonase, polygalacturonic acid is hydrolyzed bringing about a corresponding gain in soluble Ruthenium Red. The described assay utilizes Ruthenium Red as the detection reagent which has been used previously in plate-based assays but not in liquid medium reactions. The new method measures the disappearance of the substrate polygalacturonic acid and is compared to the Somogyi-Nelson assay. The experimental results using lemon peel, a fern fronds and castor leaf crude extracts demonstrate that the new method provides a way to the quickly screening of polygalacturonase activity and polygalacturonase inhibitors in plant crude extracts containing high amounts of reducing power. On the other hand, the Ruthenium Red assay is not able to determine the activity of an exo-polygalacturonase as initial velocity and thus would allow the differentiation between endo- and exo-polygalacturonase activities.

Radical scavenging capacity and antimutagenic properties of purified proteins from Solanum betaceum fruits and Solanum tuberosum tubers.

In this study, antioxidant activities in free-radical-mediated oxidative systems and the genotoxic/antigenotoxic effects of two proteins with molecular mass around 17 kDa, purified from Solanum betaceum fruits (cyphomine) and Solanum tuberosum tubers (solamarine), were investigated. Both proteins inhibited uric acid formation with IC(50) values between 55 and 60 µg/mL, and both proteins were able to reduce oxidative damage by scavenging hydroxyl radicals and superoxide anion in a dose-dependent manner. Furthermore, the DPPH• reduction assay showed SC(50) values of 55-73 µg/mL. Cyphomine and solamarine were able to retain their antioxidant activity after heat treatment at 80 °C for 15 min. Allium cepa and Salmonella /microsome assays showed no genotoxic and mutagenic effects. Solamarine showed an antimutagenic effect against a direct mutagen (4-nitro-o-phenylenediamine). Consequently, the present study showed that the investigated proteins are promising ingredients for the development of functional foods with a beneficial impact on human health and an important source for the production of bioactive peptides.

Effect of seasonal variations and collection form on antioxidant activity of propolis from San Juan, Argentina.

Propolis was included in the Argentine Food Code as a functional food. The chemical parameters and antioxidant properties of propolis samples from the same colonies of Apis mellifera in San Juan (Cuyo region, Western Argentine) were compared every month for 1 year using two collection methods. Chemical parameters were analyzed by the spectrophotometric method and fingerprinting using high-performance liquid chromatography with ultraviolet detection. The antioxidant activities of propolis samples were measured using model systems including the analysis of the scavenging activities for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and the beta-carotene bleaching assay. The results showed that propolis had a higher free radical scavenging and lipid peroxidation inhibitory capacity than butylated hydroxytoluene and quercetin, antioxidants used in the pharmaceutical and food industries. The concentration required to scavenge 50% of free radicals (SC(50)) values differed depending on the sample collection month. Samples collected in November had the highest antioxidant capacity. In all cases, SC(50) values of propolis samples obtained by scraping were similar to those collected from a wire mesh (5 microg/mL for ABTS and 20-30 microg/mL for DPPH radicals). A significant positive correlation was found between the antioxidant capacity and flavonoid content of each analyzed extract. The chemical profiles were very similar. Galangin (3,5,7-trihydroxyflavone), an antioxidant compound, was detected in all samples as a major compound. The chromatographic profile suggests that of Baccharis sp., which would be one of the botanical sources of propolis from western Argentina, and the content of galangin can be used as a parameter for evaluating propolis quality. Our results suggest that Argentine propolis from Cuyo is a promising source of bioactive compounds as ingredients for developing functional foods with a beneficial impact on oxidative stress.

Antimicrobial activity of glycosidase inhibitory protein isolated from Cyphomandra betacea Sendt. fruit.

Broad-spectrum antimicrobial activity of an invertase inhibitory protein (IIP) isolated from Cyphomandra betacea ripe fruits is documented. Minimal inhibitory concentration (MIC) values were determined by agar macrodilution and broth microdilution assays. This IIP inhibited the growth of xylophagous and phytopatogenic fungi (Ganoderma applanatum, Schizophyllum commune, Lenzites elegans, Pycnoporus sanguineous, Penicillium notatum, Aspergillus niger, Phomopsis sojae and Fusarium mango) and phytopathogenic bacteria (Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae pv. syringae and Erwinia carotovora var carotovora). The IIP concentration required to completely inhibit the growth of all studied fungi ranged from 7.8 to 62.5 microg/ml. Phytopatogenic bacteria were the most sensitive, with MIC values between 7.8 and 31.25 microg/ml. Antifungal and antibacterial activities can be associated with their ability to inhibit hydrolytic enzymes. Our results indicate the possible participation of IIP in the plant defense mechanism and its potential application as a biocontrol agent against phytopathogenic fungi and bacteria.