In vitro fermentation parameters and biohydrogenation of vegetable oils without glycerol

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Desenvolvimento e avaliação das atividades antioxidante e despigmentante in vitro de emulsões múltiplas a/o/a contendo dipalmitato kójico

Currently, there has been a growing concern for men and women with the appearance of the face and body, driven primarily by aesthetic standards set by the media. For this, the pharmaceutical and cosmetic industries have conducted numerous research projects aiming at the development of formulations that mitigate the aging and some skin disorders such as hipercromies. One of the most frequent pathologies of skin is melasma, a manifestation of hyperpigmentation caused by hipermelanogenesis symmetrical and progressive, caused usually by hormonal irregularities, exposure to sunlight and genetic factors. In addition to sunscreen, the treatment is indicated the use of depigmenting substances, among them the kojic dipalmitate (DK), which is cleaved into kojic acid (5- hydroxy-2-hydroxy-methyl-4H-piran-4-one) by esterase after absorption by the skin cells. The kojic acid inhibits the action of tyrosinase as a chelator of ions and promotes the reduction of eumelanin and its precursor monomer. To promote a controlled release and improve the stability of the system, the DK can be incorporated into multiple emulsions, that is, complex systems composed of two emulsifications, where the two types of emulsions (W/O and O/W or O/W and W/O) exist simultaneously, forming emulsions of type W/O/W or O/W/O. This work aimed to incorporate the DK in emulsion W/O/W, physical-chemical systems obtained and to evaluate the antioxidant and depigmenting action in vitro of the developed formulations. The physico-chemical characterization was performed by microscopic analysis, quantification and size distribution, determination of pH, conductivity, zeta potential and bioadhesive test of the formulations. The droplet size in accordance with the use of light microscopy and dynamic light scattering is approximately 1μm. The pH, electrical conductivity and bioadhesion have not changed with the addition... (Complete abstract click electronic access below)

Atividade antibacteriana in vitro de extratos e substâncias isoladas de espécies de Croton frente Staphylococcus aureus causador de mastite bovina

Pós-graduação em Medicina Veterinária - FCAV

Fatores de virulência e infecção epitelial in vitro de biofilmes simples e mistos de Cndida albicans, Staphylococcus aureus sensível (MSSA) e resistente à meticilina (MRSA)

Pós-graduação em Reabilitação Oral - FOAR

Glycerol combined with oils did not limit biohydrogenation of unsaturated fatty acid but reduced methane production in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Neutrophil extracellular traps identification in tegumentary lesions of patients with paracoccidioidomycosis and different patterns of NETs generation in vitro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Screening da atividade antiproliferativa e investigação toxicológica in vitro e in vivo de compostos de coordenação

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Stability of Carotenoids, Total Phenolics and In Vitro Antioxidant Capacity in the Thermal Processing of Orange-Fleshed Sweet Potato (Ipomoea batatas Lam.) Cultivars Grown in Brazil

Intervention strategies regarding the biofortification of orange-fleshed sweet potato, which is a rich source of carotenoids for combating vitamin A deficiency, are being developed in Brazil. This study was conducted to evaluate the concentrations of individual carotenoids, total phenolic compounds and antioxidant capacity in the roots of four biofortified sweet potato cultivars that were raw or processed by four common heat treatments. HPLC, Folin-Ciocalteu, DPPH and ABTS assays were used. All cultivars showed high levels of carotenoids in raw roots, predominantly all-trans-beta-carotene (79.1-128.5 mg.100 g(-1) DW), suggesting a high estimated vitamin A activity. The CNPH 1194 cultivar reported carotenoids values highest than those of other cultivars (p < 0.05). The total phenolic compounds varied among cultivars and heat treatments (0.96-2.05 mg.g(-1) DW). In most cases, the heat treatments resulted in a significant decrease in the carotenoids and phenolic compounds contents as well as antioxidant capacity. Processing of flour presented the greatest losses of major carotenoids and phenolics. The phenolic compounds showed more stability than carotenoids after processing. There were significant correlations between the carotenoids and phenolic compounds and the antioxidant capacity.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Differential gene expression and developmental competence in in vitro produced bovine embryos

The embryonic developmental block occurs at the 8-cell stage in cattle and is characterized by a lengthening of the cell cycle and an increased number of embryos that stop development. The maternal-embryonic transition arises at the same stage resulting in the transcription of many genes. Gene expression studies during this stage may contribute to the understanding of the physiological mechanisms involved in the maternal-embryonic transition. Herein we identified genes differentially expressed between embryos with high or low developmental competence to reach the blastocyst stage using differential display PCR. Embryos were analysed according to developmental kinetics: fast cleavage embryos showing 8 cells at 48 h post insemination (hpi) with high potential of development (F8), and embryos with slow cleavage presenting 4 cells at 48 hpi (54) and 8 cells at 90 hpi (S8), both with reduced rates of development to blastocyst. The fluorescence DDPCR method was applied and allowed the recovery of 176 differentially expressed bands with similar proportion between high and low development potential groups (52% to F8 and 48% in S4 and S8 groups). A total of 27 isolated fragments were cloned and sequenced, confirming the expected primer sequences and allowing the identification of 27 gene transcripts. PI3KCA and ITM2B were chosen for relative quantification of mRNA using real-time PCR and showed a kinetic and a time-related pattern of expression respectively. The observed results suggest the existence of two different embryonic genome activation mechanisms: fast-developing embryos activate genes related to embryonic development, and slow-developing embryos activate genes related to cellular survival and/or death.

In vitro evaluation of the odontogenic potential of mouse undifferentiated pulp cells

The aim of this study was to evaluate the odontogenic potential of undifferentiated pulp cells (OD-21 cell line) through chemical stimuli in vitro. Cells were divided into uninduced cells (OD-21), induced cells (OD-21 cultured in supplemented medium/OD-21+OM) and odontoblast-like cells (MDPC-23 cell line). After 3, 7, 10 and 14 days of culture, it was evaluated: proliferation and cell viability, alkaline phosphatase activity, total protein content, mineralization, immunolocalization of dentin matrix acidic phosphoprotein 1 (DMP1), alkaline phosphatase (ALP) and osteopontin (OPN) and quantification of genes ALP, OSTERIX (Osx), DMP1 and runt-related transcription factor 2 (RUNX2) through real-time polymerase chain reaction (PCR). Data were analyzed by Kruskal-Wallis and Mann-Whitney U tests (p<0.05). There was a decrease in cell proliferation in OD-21 + OM, whereas cell viability was similar in all groups, except at 7 days. The amount of total protein was higher in group OD-21 + OM in all periods; the same occurred with ALP activity after 10 days when compared with OD-21, with no significant differences from the MDPC-23 group. Mineralization was higher in OD-21+OM when compared with the negative control. Immunolocalization demonstrated that DMP1 and ALP were highly expressed in MDPC-23 cells and OD-21 + OM cells, whereas OPN was high in all groups. Real-time PCR revealed that DMP1 and ALP expression was higher in MDPC-23 cell cultures, whereas RUNX2 was lower for these cells and higher for OD-21 negative control. Osx expression was lower for OD-21 + OM. These results suggest that OD-21 undifferentiated pulp cells have odontogenic potential and could be used in dental tissue engineering.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules – dexamethasone, triiodothyronine (T3) and insulin – on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system

STUDY DESIGN: Ex vivo in vitro study evaluating a novel intervertebral disc/endplate culture system. OBJECTIVES: To establish a whole-organ intervertebral disc culture model for the study of disc degeneration in vitro, including the characterization of basic cell and organ function. SUMMARY OF BACKGROUND DATA: With current in vivo models for the study of disc and endplate degeneration, it remains difficult to investigate the complex disc metabolism and signaling cascades. In contrast, more controlled but simplified in vitro systems using isolated cells or disc fragments are difficult to culture due to the unconstrained conditions, with often-observed cell death or cell dedifferentiation. Therefore, there is a demand for a controlled culture model with preserved cell function that offers the possibility to investigate disc and endplate pathologies in a structurally intact organ. METHODS: Naturally constrained intervertebral disc/endplate units from rabbits were cultured in multi-well plates. Cell viability, metabolic activity, matrix composition, and matrix gene expression profile were monitored using the Live/Dead cell viability test (Invitrogen, Basel, Switzerland), tetrazolium salt reduction (WST-8), proteoglycan and deoxyribonucleic acid quantification assays, and quantitative polymerase chain reaction. RESULTS: Viability and organ integrity were preserved for at least 4 weeks, while proteoglycan and deoxyribonucleic acid content decreased slightly, and matrix genes exhibited a degenerative profile with up-regulation of type I collagen and suppression of collagen type II and aggrecan genes. Additionally, cell metabolic activity was reduced to one third of the initial value. CONCLUSIONS: Naturally constrained intervertebral rabbit discs could be cultured for several weeks without losing cell viability. Structural integrity and matrix composition were retained. However, the organ responded to the artificial environment with a degenerative gene expression pattern and decreased metabolic rate. Therefore, the described system serves as a promising in vitro model to study disc degeneration in a whole organ.

A new optical detection method to assess the erosion inhibition by in vitro salivary pellicle layer

OBJECTIVES Application of the recently developed optical method based on the monitoring of the specular reflection intensity to study the protective potential of the salivary pellicle layer against early enamel erosion. METHODS The erosion progression was compared between two treatment groups: enamel samples coated by the 15 h-in vitro-formed salivary pellicle layer (group P, n=90) and the non-coated enamel surfaces (control group C, n=90). Different severity of the erosive impact was modelled by the enamel incubation in 1% citric acid (pH=3.6) for 2, 4, 8, 10 or 15 min. Erosion quantification was performed by the optical method as well as by the microhardness and calcium release analyses. RESULTS Optical assessment of the erosion progression showed erosion inhibition by the in vitro salivary pellicle in short term acidic treatments (≤ 4 min) which was also confirmed by microhardness measurements proving significantly less (p<0.05) enamel softening in the group P at 2 and 4 min of erosion compared to the group C. SEM images demonstrated less etched enamel interfaces in the group P at short erosion durations as well. CONCLUSIONS Monitoring of the specular reflection intensity can be successfully applied to quantify early erosion progression in comparative studies. In vitro salivary pellicle (2h) provides erosion inhibition but only in short term acidic exposures. CLINICAL SIGNIFICANCE The proposed optical technique is a promising tool for the fast and non-invasive erosion quantification in clinical studies.

In vitro reconstitution of light-harvesting POR-protochlorophyllide complex with protochlorophyllides a and b

NADPH:protochlorophyllide oxidoreductase (POR; EC1.1.33.1) is a key enzyme for the light-induced greening of angiosperms. In barley, two POR proteins exist, termed PORA and PORB. These have previously been proposed to form higher molecular weight light-harvesting complexes in the prolamellar body of etioplasts (Reinbothe, C., Lebedev, N., and Reinbothe, S. (1999)Nature 397, 80–84). Here we report the in vitro reconstitution of such complexes from chemically synthesized protochlorophyllides (Pchlides) a andb and galacto- and sulfolipids. Low temperature (77 K) fluorescence measurements revealed that the reconstituted, lipid-containing complex displayed the same characteristics of photoactive Pchlide 650/657 as the presumed native complex in the prolamellar body. Moreover, Pchlide F650/657 was converted to chlorophyllide (Chlide) 684/690 upon illumination of the reconstituted complex with a 1-ms flash of white light. Identification and quantification of acetone-extractable pigments revealed that only the PORB-bound Pchlide a had been photoactive and was converted to Chlide a, whereas Pchlide b bound to the PORA remained photoinactive. Nondenaturing PAGE of the reconstituted Pchlide a/b-containing complex further demonstrated a size similar to that of the presumed native complexin vivo, suggesting that both complexes may be identical.