Nanostructured lipid carriers loaded with essential oils: a strategy against SARS-CoV-2.

This work aimed to investigate the effectiveness of Lippia sidoides and Syzygium aromaticum essential oils (EOs) encapsulated in nanostructured lipid carriers (NLCs) as SARS-CoV-2 inhibitors through virucidal activity assessment. We developed anionic and cationic NLCs loaded with the EOs and assessed their physicochemical properties and SARS-CoV-2 virucidal activity, focusing on the effects of EO type and the NLCs composition. The NLCs exhibited particle sizes of 141.30 to 160.53 nm for anionic and 109.30 to 138.60 nm for cationic types, with PDIs between 0.16 and 0.25. High zeta potentials (>29.0 in modulus) indicated stable formulations. The NLCs effectively encapsulated the EOs, achieving encapsulation efficiencies between 84.6 to 100% w/w of marker compound. The EOs-loaded NLCs reduced the SARS-CoV-2 virion count, exceeding 2 logs over the control. NLCs loaded with Lippia sidoides and Syzygium aromaticum EOs represent an innovative strategy for combating SARS-CoV-2.

Renal developmental disturbances and their long-term consequences in female pups from vitamin D-deficient mothers: involved mechanisms.

The mechanisms involved in kidney disturbances during development, induced by vitamin D3 deficiency in female rats, that persist into adulthood were evaluated in this study. Female offspring from mothers fed normal (control group, n=8) or vitamin D-deficient (Vit.D-, n=10) diets were used. Three-month-old rats had their systolic blood pressure (SBP) measured and their blood and urine sampled to quantify vitamin D3 (Vit.D3), creatinine, Na+, Ca+2 and angiotensin II (ANGII) levels. The kidneys were then removed for nitric oxide (NO) quantification and immunohistochemical studies. Vit.D- pups showed higher SBP and plasma ANGII levels in adulthood (P<0.05) as well as decreased urine osmolality associated with increases in urinary volume (P<0.05). Decreased expression of JG12 (renal cortex and glomeruli) and synaptopodin (glomeruli) as well as reduced renal NO was also observed (P<0.05). These findings showed that renal disturbances in development in pups from Vit.D- mothers observed in adulthood may be related to the development of angiogenesis, NO and ANGII alterations.

Transdentinal photobiostimulation of stem cells from human exfoliated primary teeth.

AIM: To evaluate the effects of infrared light-emitting diode (LED) irradiation on stem cells from human exfoliated deciduous teeth (SHEDs). METHODOLOGY: Exfoliated primary teeth were obtained (n = 3), and SHEDs obtained from the teeth were seeded on the pulpal surface of 0.2-mm-thick dentine discs produced from permanent molars. The cells were incubated for 24 h by placing the discs in plain Dulbecco's modified Eagle's medium (DMEM). The DMEM was then replaced with new culture medium formulated for odontoblast differentiation. After 12 h in the second medium, SHEDs were irradiated through the dentine discs using an infrared LED (850 nm) with a power density of 80 mW cm-2 . Energy doses (EDs) delivered to the occlusal surface of the dentine discs were 0 (control), 2 and 4 J cm-2 (n = 6). Subsequent tests were performed 72 h after irradiation. These tests included cell viability (MTT), alkaline phosphatase activity (ALP), total protein production (TP), scanning electron microscopy (SEM), as well as gene expression for ALP, Col I, DSPP and DMP-1. Data were analysed using Kruskal-Wallis and Mann-Whitney t-tests (α = 0.05). RESULTS: Both EDs (2 and 4 J cm-2 ) significantly increased cell viability and ALP activity. For TP, ALP and Col I gene expression, only the 4 J cm-2 group had significantly higher values compared to the control group. Cell morphology was not affected by irradiation. CONCLUSION: Infrared LED irradiation was capable of biostimulating SHEDs through a 0.2 mm thickness of dentine, especially at the 4 J cm-2 level.

Nutritional deprivation and LPS exposure as feasible methods for induction of cellular - A methodology to validate for vitro photobiomodulation studies.

Previous studies have demonstrated that high biostimulation takes place when cells under stress are subjected to phototherapy by laser or light-emitting-diode (LED) devices. Several studies selected nutritional deprivation by reducing the concentration of fetal bovine serum (FBS) in the culture medium or the exposure of cultured cells to lipopolysaccharide (LPS) as an in vitro cellular stress condition. However, there are no data certifying that these stimuli cause stressful conditions for cultured cells. This investigation assessed the induction of cellular stress by decreasing the concentration of FBS or adding LPS to culture medium. Odontoblast-like cells (MDPC-23) were cultured in complete culture medium (DMEM) containing 10% FBS. After a 12-hour incubation period, the DMEM was replaced by fresh medium containing 10% FBS (control), low concentrations of FBS (0, 0.2, 0.5, 2, or 5%) or LPS from Escherichia coli (10µg/ml). After an additional 12-hour incubation, cell viability, total cell-counting, total protein production, and gene expression of heat shock protein 70 (HSP70) were assessed. Data were statistically analyzed by ANOVA complemented by the Tukey test, with 5% considered significant. Cell viability was negatively affected only for 0% FBS, while reduced viable cell numbers and total protein production were detected for FBS concentrations lower than 2%. Higher HSP70 gene expression was also observed for FBS concentrations lower than 2% and for cells exposed to LPS. The nutritional deprivation model with culture medium lower than 2% of FBS can be safely used to induce cellular stress for in vitro photobiomodulation studies.

Atividade antibacteriana de Óleos Essenciais sobre Streptococcus mutans e Staphylococcus aureus / Antibacterial Activity of Essential Oils against Strains of Streptococcus and Staphylococcus

Este trabalho objetivou avaliar a ação antimicrobiana in vitro dos óleos essenciais de Ocimum basilicum (Manjericão Exótico), Thymus vulgaris (Tomilho Branco), e de Cinnamomum cassia (Canela da China) sobre cepas bacterianas de Streptococcus mutans (ATCC 25175) e Staphylococcus aureus (ATCC 25923). A atividade antibacteriana dos óleos essenciais foi determinada pela Concentração Inibitória Mínima (CIM) e a Concentração Bactericida Mínima (CBM) através da técnica de microdiluição e do esgotamento. Para a CIM, foram utilizadas placas de 96 poços e inseriu-se 100µL de caldo BHI, 100µL da diluição dos óleos essenciais no primeiro poço e 10µL da suspensão bacteriana (1,5x10(6) microrganismos/mL). Realizou-se a diluição seriada partindo-se da concentração inicial de 8% até 0,0625%. A CIM correspondeu à última diluição na qual não foi verificada a presença de bactérias. Para obter a CBM, realizou-se a semeadura em Ágar Miller-Hinton das diluições correspondentes a CIM, 2CIM e 4CIM. As placas foram incubadas a 37º C em estufa bacteriológica por 24 horas. Os testes foram realizados em triplicata tendo a clorexidina como controle positivo. Para S. aureus (ATCC 25923) a CIM e CBM dos óleos essenciais de C. cassia, O. basilicum e T. vulgaris foram 0,0625%, 4% e 0,0625%, respectivamente. Para S. mutans a CIM e a CBM dos óleos essenciais de C. cassia e T. vulgaris foram 0,125% e 0,25%, respectivamente. Já a CIM do O. basilicum foi 4% e não apresentou ação bactericida. Conclui-se que os óleos essenciais avaliados apresentaram ação antibacteriana frente a cepas de S. mutans e S. aureus, sendo que os menores valores de CIM e de CBM foram provenientes dos óleos de C. cassia e T. vulgaris.

The aim of this study is to evaluate the in vitro antimicrobial activity of essential oils of Ocimum basilicum (basil), Thymus vulgaris (thyme) and Cinnamomum cassia (Chinese cinnamon) against strains of Streptococcus mutans (ATCC 25175) and Staphylococcus aureus (ATCC 25923). The antibacterial activity of the essential oils was determined by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The MIC was obtained by the microdilution technique using 96-well microplates, in which we inserted: 100µL of Brain Heart Infusion broth at double concentration, 100µL of the dilution of the essential oils and 10µL of bacterial suspension (1.5 x 10(6) organisms / mL). The products were diluted from the initial concentration of 8% up to 0.0625%. The MIC corresponded to the last dilution in which there was no presence of bacteria or turbidity in the culture medium. The MBC was obtained by seeding, in Mueller-Hinton agar, 10µL aliquots of dilutions corresponding to the MIC and the two immediately preceding ones (2MIC and 4MIC). The plates were incubated at 37°C in a bacteriological incubator for 24 hours. The tests were performed in triplicate, and 2% Chlorhexidine Digluconate was the control product. For S. aureus, the MIC and MBC of the essential oils of C. cassia, O. basilicum and T. vulgaris were 0.0625%, 0.0625% and 4%, respectively. For S. mutans, the MIC and MBC of the essential oils of C. cassia and T. vulgaris were 0.125% and 0.25%, respectively. The MIC of O. basilicum against S. mutans was 4% and the essential oil showed no bactericidal action. Chlorhexidine Digluconate presented antibacterial activity against all organisms. The evaluated essential oils presented antibacterial activity against the strains of S. mutans and S. aureus, and we highlight the essential oils from C. cassia and T. vulgaris with the lowest MIC and MBC.

Atividade antifúngica de óleos essenciais frente a amostras clínicas de Candida albicans isoladas de pacientes HIV positivos / Antifungal activity of essential oils against clinical samples of Candida albicans isolated from HIV-positive patients

Objetivou-se avaliar a atividade antifúngica dos óleos essenciais de Ocimum basilicum L. (manjericão), Cymbopogon martinii L. (palmarosa), Thymus vulgaris L. (tomilho) e Cinnamomum cassia Blume (canela da china) sobre cepas de Candida albicans isoladas de pacientes HIV positivos e cepa padrão (ATCC 76845). Quinze amostras clínicas de C. albicans (C1-C15) foram repicadas em ágar Sabouraud Dextrose, para confecção de suspensões em solução salina estéril (0,9%) contendo 1,5 x 10(6) UFC mL-1. As emulsões dos óleos essenciais foram preparadas em água destilada estéril e tween 80, com concentrações variando entre 1024 µg mL-1 e 4 µg mL-1. A ação antifúngica foi determinada por meio da Concentração Inibitória Mínima (CIM) utilizando-se a técnica da microdiluição. Foram utilizados como controles positivos a nistatina e o miconazol (50 µg mL-1). Os testes foram realizados em triplicata, sendo a CIM, a menor concentração capaz de inibir o crescimento das leveduras, observada por método visual de acordo com a turvação do meio de cultura. Para C. albicans (ATCC 76845), a CIM do óleo essencial de C. cassia foi 64 µg mL-1, enquanto para óleo de C. martinii foi 1024 µg mL-1. Para as cepas clínicas, verificou-se que a CIM de C. cassia para 80% das cepas foi 64 µg mL-1, sendo a variação dos valores da CIM entre 128 µg mL-1 e 64 µg mL-1. Observou-se que para 66,6% das amostras clínicas, a CIM de C. martinii foi 612 µg mL-1. Constatou-se que a nistatina não apresentou atividade frente às cepas clínicas (C1-C15), enquanto a atividade antifúngica do miconazol foi verificada em 100% das amostras. Não se constatou atividade antimicrobiana dos óleos essenciais de O. basilicum e T. vulgaris, nas concentrações avaliadas. Concluiu-se que os óleos essenciais de C. cassia e C. martinii, em diferentes concentrações, apresentam atividade antifúngica sobre cepas de C. albicans isoladas de pacientes HIV positivos e cepa padrão (ATCC 76845). Entretanto não foi observada inibição antimicrobiana para os óleos de O. basilicum e T. vulgaris.

The aim of this study was to evaluate the antifungal activity of essential oils from Ocimum basilicum L. (basil), Cymbopogon martinii L. (palmarosa), Thymus vulgaris L. (thyme) and Cinnamomum cassia Blume (Chinese cinnamon) against Candida albicans strains isolated from HIV-positive patients and the standard strain (ATCC 76845). Fifteen clinical samples of C. albicans (C1-C15) were subcultured in Sabouraud Dextrose agar to prepare suspensions in sterile saline solution (0.9%) containing 1.5 x 10(6) CFU mL-1. The emulsions of essential oils were prepared in sterile distilled water and Tween 80, with concentrations ranging between 1024 µg mL-1 and 4 µg mL-1. The antifungal action was determined by means of the Minimum Inhibitory Concentration (MIC), using the microdilution technique. Nystatin and miconazole (50 µg mL-1) were used as positive controls. The tests were performed in triplicate and the MIC was the lowest concentration capable of inhibiting the growth of yeasts, which was observed by the visual method, according to the turbidity of the culture medium. For C. albicans (ATCC 76845), the MIC of C. cassia essential oil was 64 µg mL-1, while the MIC for C. martini was 1024 µg mL-1. Considering the clinical strains, the MIC of C. cassia was 64 µg mL-1 for 80% of the strains, and the variation in MIC values was between 128 µg mL-1 and 64 µg mL-1. For 66.6% of the clinical samples, the MIC of C. matinii was 612 µg mL-1. Nystatin did not present activity against the clinical strains (C1-C15), while the antifungal activity of miconazole was noticed for 100% of the samples. The antimicobrial activity of essential oils from O. basilicum and T. vulgaris was not identified at the evaluated concentrations. It was concluded that the essential oils from C. cassia and C. martinii, at different concentrations, presented antifungal activity against C. albicans strains isolated from HIV-positive patients and the standard strain (ATCC 76845). However, antifungal activity was not observed for the essential oils from O. basilicum and T. vulgaris.

FLI1 polymorphism affects susceptibility to cutaneous leishmaniasis in Brazil.

Mapping murine genes controlling cutaneous leishmaniasis (CL) identified Fli1 as a candidate influencing resistance to L. major and enhanced wound healing. We examine FLI1 as a gene controlling CL and mucosal leishmaniasis (ML) caused by L. braziliensis in humans. Intron 1 single nucleotide polymorphisms tagging promoter and enhancer elements were analysed in 168 nuclear families (250 CL; 87 ML cases) and replicated in 157 families (402 CL; 39 ML cases). Robust case-pseudocontrol logistic regression analysis showed association between allele C (odds ratio (OR) 1.65; 95% confidence interval 1.18-2.29; P=0.003) of FLI1_rs7930515 and CL in the primary sample that was confirmed (OR 1.60; 95% confidence interval 1.10-2.33; P=0.014) in the replication set (combined P=1.8 × 10(-4)). FLI1_rs7930515 is in linkage disequilibrium with the functional GAn microsatellite in the proximal promoter. Haplotype associations extended across the enhancer, which was not polymorphic. ML associated with inverse haplotypes compared with CL. Wound healing is therefore important in CL, providing potential for therapies modulating FLI1.

Metodologia para germinação de sementes de Leonurus sibiricus L / Methodology for Leonurus sibiricus L. seed germination

O objetivo do trabalho foi investigar a influência de diferentes temperaturas, o comportamento fotoblástico e a absorção de água de sementes de Leonurus sibiricus L. Essa espécie medicinal é originária da Índia, distribuída pela Ásia, África e América, utilizada no tratamento de reumatismo, problemas dermatológicos e respiratórios. Para tanto, as sementes foram submetidas a temperaturas entre 5 a 40ºC, com intervalos de 5ºC, e alternadas de 20/30, 20/25 e 25/30ºC, com 5 repetições de 50 sementes cada, em condições de luz e escuro. No estudo da absorção de água as sementes foram colocadas para germinar na temperatura de 20ºC e na presença da luz e pesadas para avaliar o ganho de água durante todo o processo germinativo, até a protrusão da radícula. Pelos resultados verificou-se que os maiores porcentuais de germinação e índice de velocidade de germinação ocorreram na temperatura constante de 20ºC, e nas temperaturas alternadas 20/25, 25/30, 25/30ºC sob luz. Houve germinação na temperatura mínima de 10ºC e na máxima 40ºC. No ensaio de absorção de água verificou-se que as sementes iniciam a protrusão da radícula com 65 horas de exposição e seguem padrão trifásico na curva de absorção. O modelo estatístico ajustado para a espécie foi y = 1,869 (1 - 0,414 exp ( -0,201t) + exp [-2,397 + 0,037 (t - 65)], com R²= 0,9998.

The aim of this study was to investigate the influence of different temperatures on the photoblastic response and water uptake of Leonurus sibiricus L. seeds. This medicinal species is from India and has been distributed over Asia, Africa and America, where it is used in the treatment of rheumatic, dermatological and respiratory disorders. Thus, seeds were subjected to temperatures between 5 and 40ºC, at 5ºC intervals, and 20/30, 20/25 and 25/30ºC alternate temperatures, with five replicates of 50 seeds each, under light and dark conditions. In the study of water uptake, seeds were allowed to germinate at 20ºC under light and weighed throughout the germinative process until radicle protrusion. The highest germination percentage and speed index were detected at 20ºC constant temperature and at 20/25, 25/30 and 25/30ºC alternate temperatures under light. There was germination at 10ºC (minimum temperature) and at 40ºC (maximum temperature). The study on water uptake showed that seeds began radicle protrusion at 65h exposure and follow the triphasic pattern in the uptake curve. The statistical model fit for the species was y = 1.869 (1 - 0.414 exp (-0.201 t) + exp [-2.397 + 0.037 (t - 65)], R² = 0.9998.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys.

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys.

A chimeric yellow fever (YF)-dengue serotype 2 (dengue 2) virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

Phenotypic and molecular analyses of yellow fever 17DD vaccine viruses associated with serious adverse events in Brazil.

The yellow fever (YF) 17D virus is one of the most successful vaccines developed to data. Its use has been estimated to be over 400 million doses with an excellent record of safety. In the past 3 years, yellow fever vaccination was intensified in Brazil in response to higher risk of urban outbreaks of the disease. Two fatal adverse events temporally associated with YF vaccination were reported. Both cases had features similar to yellow fever disease, including hepatitis and multiorgan failure. Two different lots of YF 17DD virus vaccine were administered to the affected patients and also to hundreds of thousands of other individuals without any other reported serious adverse events. The lots were prepared from the secondary seed, which has been in continuous use since 1984. Nucleotide sequencing revealed minor variations at some nucleotide positions between the secondary seed lot virus and the virus isolates from patients; these differences were not consistent across the isolates, represented differences in the relative amount of each nucleotide in a heterogeneous position, and did not result in amino acid substitutions. Inoculation of rhesus monkeys with the viruses isolated from the two patients by the intracerebral (ic) or intrahepatic (ih) route caused minimal viremia and no clinical signs of infection or alterations in laboratory markers. Central nervous system histological scores of rhesus monkeys inoculated ic were within the expected range, and there were no histopathological lesions in animals inoculated ih. Altogether, these results demonstrated the genetic stability and attenuated phenotype of the viruses that caused fatal illness in the two patients. Therefore, the fatal adverse events experienced by the vaccinees are related to individual, genetically determined host factors that regulate cellular susceptibility to yellow fever virus. Such increased susceptibility, resulting in clinically overt disease expression, appears to be extremely rare.