HIV Reverse Transcriptase Fidelity And Inhibition Are Modulated By Divalent Cations In A Concentration-Dependent Manner In Vitro

Human immunodeficiency virus (HIV) rapidly evolves through generation and selection of mutants that can escape drug therapy. This process is fueled, in part, by the presumably highly error prone polymerase reverse transcriptase (RT). Fidelity of polymerases can be influenced by cation co-factors. Physiologically, magnesium (Mg2+) is used as a co-factor by RT to perform catalysis, however, alternative cations including manganese (Mn2+), cobalt (Co2+), and zinc (Zn2+) can also be used. I demonstrate here that fidelity and inhibition of HIV RT can be influenced differently, in vitro, by divalent cations depending on their concentration. The reported mutation frequency for purified HIV RT in vitro is typically in the 10-4 range (per nucleotide addition), making the enzyme several-fold less accurate than most polymerases. Paradoxically, results examining HIV replication in cells indicate an error frequency that is ~10 times lower than the error rate obtained in the test tube. Here, I reconcile, at least in part, these discrepancies by showing that HIV RT fidelity in vitro is in the same range as cellular results, in physiological concentrations of free Mg2+ (~0.25 mM). At low Mg2+, mutation rates were 5-10 times lower compared to high Mg2+ conditions (5-10 mM). Alternative divalent cations also have a concentration-dependent effect on RT fidelity. Presumed promutagenic cations Mn2+ and Co2+ decreases the fidelity of RT only at elevated concentrations, and Zn2+, when present in low concentration, increases the fidelity of HIV-1 RT by ~2.5 fold compared to Mg2+. HIV-1 and HIV-2 RT inhibition by nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs) in vitro is also affected by the Mg2+ concentration. NRTIs lacking 3'-OH group inhibited both enzymes less efficiently in low Mg2+ than in high Mg2+; whereas inhibition by the “translocation defective RT inhibitor”, which retains the 3ʹ-OH, was unaffected by Mg2+ concentration, suggesting that NRTIs with a 3ʹ-OH group may be more potent than other NRTIs. In contrast, NNRTIs were more effective in low vs. high Mg2+ conditions. Overall, the studies presented reveal strategies for designing novel RT inhibitors and strongly emphasize the need for studying HIV RT and RT inhibitors in physiologically relevant low Mg2+ conditions.

Minerals and vitamin B9 in dried plants vs. infusions: assessing absorption dynamics of minerals by membrane dialysis tandem in vitro digestion

Vitamins and mineral elements are among the most important phytochemicals due to their important role in the maintenance of human health. Despite these components had already been studied in different plant species, their full characterization in several wild species is still scarce. In addition, the knowledge regarding the in vivo effects of phytochemicals, particularly their bioaccessibility, is still scarce. Accordingly, a membrane dialysis process was used to simulate gastrointestinal conditions in order to assess the potential bioaccessibility of mineral elements in different preparations of Achillea millefolium (yarrow), Laurus nobilis (laurel) and Taraxacum sect. Ruderalia (dandelion). The retention/passage dynamics was evaluated using a cellulose membrane with 34 mm pore. Dandelion showed the highest levels of all studied mineral elements (except zinc) independently of the used formulations (dried plant or infusion), but yarrow was the only species yielding minerals after the dialysis step, either in dried form, or as infusion. In fact, the ability of each evaluated element to cross the dialysis membrane showed significant differences, being also highly dependent on the plant species. Regarding the potential use of these plants as complementary vitamin B9 sources, the detected values were much lower in the infusions, most likely due to the thermolability effect.

Études in vivo et in vitro sur le potentiel néphroprotecteur de plantes médicinales anti-diabétiques de la pharmacopée traditionnelle des Cris de la Baie-James

Notre équipe a identifié le thé Labrador [Rhododendron groenlandicum L. (Ericaceae)] comme une plante potentiellement antidiabétique de la pharmacopée traditionnelle des Cris de la Baie James orientale. Dans la présente étude, nous avons évalué les effets néphroprotecteurs potentiels de la plante. De la microalbuminurie et de la fibrose rénale ont été développées chez des souris alimentées avec une diète grasse (DG). Le R. groenlandicum améliore d’une façon non-significative la microalbuminurie, avec des valeurs de l’aire sous la courbe (ACR) diminuant de 0.69 à 0.53. La valeur de la fibrose rénale qui était, à l’origine, de 4.85 unités arbitraires (UA) dans des souris alimentées à la DG, a chuté à 3.27 UA après avoir reçu un traitement de R. groenlandicum. Le R. groenlandicum a réduit la stéatose rénale de presque la moitié alors que l’expression du facteur de modification Bcl-2 (Bmf) a chuté de 13.96 UA à 9.43 UA. Dans leur ensemble les résultats suggèrent que le traitement avec R. groenlandicum peut améliorer la fonction rénale altérée par DG. Dans l’étude subséquente, notre équipe a identifié 17 espèces de la forêt boréale, de la pharmacopée traditionnelle des Cris de la Baie James orientale, qui ont présenté des activités biologiques prometteuses in vitro et in vivo dans le contexte du DT2. Nous avons maintenant examiné ces 17 extraits afin d’identifier lesquels possèdent un potentiel cytoprotecteur rénale en utilisant des cellules Madin Darby Canine Kidney (MDCK) mises à l’épreuve dans un médium hypertonique. Nous concluons que plusieurs plantes antidiabétiques Cris exercent une activité de protection rénale qui pourrait être pertinente dans le contexte de la néphropathie diabétique (ND) qui affecte une proportion importante des Cris. La G. hispidula et la A. balsamea sont parmi les plantes les plus puissantes dans ce contexte et elles semblent protectrices principalement en inhibant la caspase 9 dans la voie de signalisation apoptotique mitochondriale. Finalement, nous avons utilisé une approche de fractionnement guidée par un test biologique pour identifier les fractions actives et les composés de A. balsamea avec un potentiel de protection rénale in vitro dans des cellules MDCK mises au défi avec un médium hypertonique. La fraction d’hexane (Hex) possède le potentiel le plus élevé parmi toutes les fractions de solvant contre les dommages cellulaires induits par le stress hypertonique. Dans des études précédentes, trois composés purs ont été identifiés à partir de la fraction Hex, à savoir, l’acide abiétique, l’acide déhydroabiétique et le squalène. L’acide abiétique se distinguait par son effet puissant dans le maintien de la viabilité des cellules MDCK (AnnV-/PI-) à un niveau relativement élevé (augmentation de 25.48% relative au stress hypertonique, P<0.0001), ainsi qu’une réduction significative (diminution de 20.20% par rapport au stress hypertonique, P<0.0001) de l’apoptose de stade précoce (AnnV+/PI-). L’acide abiétique peut donc servir à normaliser les préparations traditionnelles d’A. balsamea et à trouver des applications potentielles dans le traitement de la néphropathie diabétique. Les trois études ont été intrinsèquement liées les unes aux autres, par conséquent, nous avons réussi à identifier R. groenlandicum ainsi que A. balsamea comme nouvelles plantes prometteuses contre la néphropathie diabétique. Nous croyons que ces résultats profiteront à la communauté crie pour la gestion des complications diabétiques, en particulier la néphropathie diabétique. En parallèle, nos données pourraient faire avancer l'essai clinique de certaines plantes médicinales de la pharmacopée traditionnelle des Cris de la Baie James orientale du Canada.

In vitro anti-Candida activity of Glycyrrhiza glabra L.

The severity and frequency of opportunistic fungal infections still growing, concomitantly to the increasing rates of antimicrobial drug’s resistance. Natural matrices have been used over years due to its multitude of health benefits, including antifungal potential. Thus, the present work aims to evaluate the anti-Candida potential of the phenolic extract and individual phenolic compounds of Glycyrrhiza glabra L. (licorice), by disc diffusion assay, followed by determination of the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for both planktonic cells and biofilms. Licorice extract evidenced inhibitory potential against the nineteen tested Candida strains, but no pronounced effect was observed by testing the most abundant individual phenolic compounds. Candida tropicalis strains were the most sensible, followed by Candida glabrata, Candida parapsilosis and, then, Candida albicans. Lower MIC and MFC values were achieved to C. glabrata and C. tropicalis, which confirms its susceptibility to licorice extract; however, for C. tropicalis strains a higher variability was observed. Anti-biofilm potential was also achieved, being most evident in some C. glabrata and C. tropicalis strains. In general, a twice concentration of the MIC was necessary for planktonic cells to obtain a similar potential to that one observed for biofilms. Thus, an upcoming approach for new antifungal agents, more effective and safer than the current ones, is stablished; notwithstanding, further studies are necessary in order to understand its mechanism of action, as also to assess kinetic parameters.

In vitro and in vivo delivery of the secretagogue diadenosine tetraphosphate from conventional and silicone hydrogel soft contact lenses

Purpose To evaluate the possible use of soft contact lenses (CL) to improve the secretagogue role of diadenosine tetraphosphate (Ap4A) promoting tear secretion. Methods Two conventional hydrogel CL (Omafilcon A and Ocufilcon D) and two silicone hydrogel (SiH) CL (Comfilcon A and Balafilcon A) were used. Ap4A was loaded into the lenses by soaking in a 1 mM Ap4A solution during 12 h. In vitro experiments were performed by placing the lenses in multi-wells during 2 h containing 1 ml of ultrapure water. 100 μl aliquots were taken at time zero and every minute for the first 10 min, and then every 15 min. In vivo experiments were performed in New Zealand rabbits and both the dinucleotide release from SiH and tear secretion were measured by means of Schirmer strips and high-pressure liquid chromatography (HPLC) analysis. Results Ap4A in vitro release experiments in hydrogel CL presented a release time 50 (RT50) of 3.9 ± 0.2 min and 3.1 ± 0.1 min for the non-ionic and the ionic CL, respectively. SiH CL released also Ap4A with RT50 values of 5.1 ± 0.1 min for the non-ionic and 2.7 ± 0.1 min for the ionic CL. In vivo experiments with SiH CL showed RT50 values of 9.3 ± 0.2 min and 8.5 ± 0.2 min for the non-ionic and the ionic respectively. The non-ionic lens Ap4A release was able to induce tear secretion above baseline tear levels for almost 360 min. Conclusion The delivery of Ap4A is slower and the effect lasts longer with non-ionic lenses than ionic lenses.

Screening natural resources for mineralogenic and osteogenic bioactivities using in vitro and in vivo fish systems

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Simplificação do processo de multiplicação in vitro da oliveira "Olea europaea L."

A oliveira tem sido multiplicada ao longo dos tempos por métodos convencionais de propagação vegetativa como a enxertia e a estacaria lenhosa e semilenhosa. No entanto, estes métodos revelam-se lentos ou ineficientes para determinadas cultivares. No caso da cv. ‘Galega Vulgar’, ainda com grande expressão no olival português, e de difícil enraizamento por estacaria semilenhosa, tem sido usada a micropropagação de modo a contornar essas limitações e assim obter um elevado número de plantas em curto período de tempo. O custo final de produção por este processo ainda é elevado, podendo comprometer a sua aplicação a nível comercial. Grande parte dos custos estão relacionados com a fase de enraizamento in vitro que carece de ambiente estéril e condições de assépsia para a sua execução. Com vista a uma redução de custos associados a esta fase de produção, pretendeu-se com este trabalho testar a viabilidade do enraizamento ex vitro, na ausência de condições de assépsia. Este método poderá permitir uma significativa redução da mão-de-obra, ao mesmo tempo que facilitará a aclimatização das plantas e a obtenção de um sistema radicular de melhor qualidade. Compararam-se as taxas de enraizamento in vitro (controlo), com as obtidas ex vitro. Foram utilizados explantes provenientes de dois clones da cv. ‘Galega Vulgar’, (cl. 1441 e cl. 2022) cultivados e mantidos in vitro há vários anos no Laboratório de Melhoramento e Biotecnologia da Universidade de Évora. Para além do clone foi avaliada a influência do tipo de estaca (basal e apical), da hormona de enraizamento (AIB e ANA), da sua concentração (540 e 3000 ppm) e ainda de dois substratos, Preformas Jiffy® e pastilhas de fibra coco. Os melhores resultados foram obtidos com o clone 1441 em pastilhas de fibra de coco prensada, com o uso de estacas basais. Quanto à auxina, não se observaram diferenças significativas entre a utilização de ANA na concentração de 540 ppm e AIB na concentração de 3000 ppm. A aclimatização das plantas foi conseguida com taxas elevadas de sucesso, independentemente do tratamento utilizado. Conclui-se que a aplicação do método de enraizamento ex vitro simplifica procedimentos e mantém taxas de enraizamento elevadas, conduzindo assim a uma efetiva redução de tempo e custos associados; Simplifying procedures for in vitro propagation of olive “Olea europaea L.” Abstract: The olive tree has been multiplied throughout the ages by conventional methods of vegetative propagation such as grafting and wood or softwood cuttings. These propagation methods are somehow inefficient for certain cultivars. For the CV. ‘Galega Vulgar‘, still with great expression in Portuguese olive orchards, propagation has been attempted by in vitro culture in order to circumvent these limitations and so obtain a large number of plants in short time period. The final production fees associated to this process are still high which may compromise its application to a commercial level. Most of this process fees are related to the in vitro rooting phase which lacks sterile and aseptic conditions for its implementation. Aiming to reduce the costs associated with this production phase, this work tested the feasibility of the ex vitro rooting in the absence of aseptic conditions, which can allow a significant reduction of the manpower involved and an easier plant acclimatization due to its transplant with a balled-root system. In vitro rooting rates (control) were compared with those obtained with the ex vitro experiments. Explants from two clones of the cv. ‘Galega Vulgar‘ (cl. 1441 and cl. 2022), grown and maintained in vitro for several years in the Laboratory of Biotechnology and Plant Breeding of the University of Évora, were used in the trials. In addition to the clone, the effect of the cutting type (basal and apical), the rooting hormone (AIB and ANA), their concentration (540 and 3000 ppm) and two substrates, Preformas Jiffy ® and pressed coco fiber pellets, were also evaluated. The best results were obtained with the clone 1441, when rooted in pressed coco fiber pellets, using basal cuttings. Under this conditions no significant differences were observed between the use of ANA at 540 ppm or AIB in the 3000 ppm. Acclimatization of plants was achieved with high rates of success, regardless of the treatment used. It can be concluded that the application of the ex vitro rooting method allows to maintain high rooting rates, contributing for an effective reduction of time and fees of the rooting process.

Synchronizing the in vitro germination of Psidium guineense Sw. seeds by means of osmotic priming.

The Brazilian guava (Psidium guineense Swartz) is seed-propagated and, being native to the Caatinga biome, may frequently have uneven germination.Thus, we aimed to evaluate the synchronization of the in vitro seed germination of three accessions of the Brazilian guava, using water, polyethyleneglycol (PEG 6000), and potassium nitrate (KNO3) at different potentials and times of osmotic priming. Seeds from three accessions of the Brazilian guava (Y85, Y93,and Y97) from the UNEB/BA Germplasm Active Bank were subjected to the following pretreatments: -0.6, -1.0, -1.4, and -1,8 MPa PEG 6000; 10 and 20% KNO3 for 24h; 10 and 20% KNO3 for 48h; water for 24 and 48h; and non-primed seeds as the control. The experimental design was therefore a 10x3+1 factorial scheme. We assessed the germination percentage (G), mean germination time (MGT), germination speed (GS), and germination speed index (GSI). Data was subjected to analysis of variance followed by a means test (Duncan at 5% probability) and regression. There was interaction between the priming treatments and accessions for all evaluated features, except G. PEG 6000 decreased the MGT (from 6 to 8 days) and increased GS and GSI of seeds from all three accessions at potentials -1.0 to -1.5 MPa.Water-priming had a positive effect on MGT, GS, and GSI of accession Y85 seeds. KNO3 negatively affected germination of seeds from all three accessions. Thereby, we could synchronize seed germination of accessions Y85 and Y97 with PEG 6000.