Carotenóides totais em Pothomorphe umbellata (L.) Miq. cultivadas in vitro

The aim of the present work was to compare the content of carotenoids between callus and regenerated plants of Pothomorphe umbellate. Germinated seeds (40 days old) were inoculated in different concentrations and combinations of BAP (benzylaminopurine) and NAA (naphthalene acetic acid) in order to stimulate the callus' production. After 60 days of culture, the callus containing some shoots were transferred to organogenesis medium (GA 3 0.1 mg L -1, BAP 0.5 mg L -1) for 40 days. Next, they were subcultivated in a medium for seedling growth (without regulators) for 40 days. Callus (collected after 60 days) and seedlings (collected after 140 days) were frozen in liquid nitrogen and kept under -80°C for future carotenoids' analysis. The highest concentration of carotenoids was found in plants cultivated in medium without regulators. The callus did not showed difference concerning the culture medium; however, they presented lower content of carotenoids in relation to plants.

In vitro antimicrobial activity of acroseal, polifl and epiphany against Enterococcus faecalis

Using the agar diffusion method, this study evaluated the in vitro antimicrobial activity of the commercial endodontic sealers Acroseal and Epiphany, a castor-oil based experimental sealer, Polifl, and a primer agent (Epiphany self-etching primer), against Enterococcus faecalis. Zinc oxide and eugenol cement (ZOE) served as control. Five wells per dish were made at equidistant points and immediately flled with the test and control materials. After incubation of the dishes at 37°C for 24 h and 48 h, the diameter of the zones of microbial growth inhibition produced around the wells was measured (in mm) with a millimeter rule. After 48 h, the diameters of the zones of microbial growth inhibition were the same as those observed at 24 h, only the substances continued to diffuse. Epiphany and Polifl did not show antibacterial activity (no formation of zones of microbial growth inhibition). The primer produced the largest zones of inhibition (17.62 mm) followed by Acroseal (7.25 mm) and ZOE (7.12 mm). E. faecalis was resistant to Epiphany and Polifl, while the primer and Acroseal sealer were effective against this microorganism under the tested conditions.

Characterization and in vitro cytocompatibility of an acid-etched titanium surface

The aims of this study were to characterize the microstructure of a commercially pure titanium (cpTi) surface etched with HCl/H 2SO 4 (AE-cpTi) and to investigate its in vitro cytocompatibility compared to turned cpTi (T-cpTi). T-cpTi showed a grooved surface and AE-cpTi revealed a surface characterized by the presence of micropits. Surface parameters indicated that the AE-cpTi surface is more isotropic and present a greater area compared to T-cpTi. The oxide film thickness was similar between both surfaces; however, AE-cpTi presented more Ti and O and less C. Osteoblastic cell proliferation, alkaline phosphatase activity, and bone-like nodule formation were greater on T-cpTi than on AE-cpTi. These results show that acid etching treatment produced a surface with different topographical and chemical features compared to the turned one, and such surface modification affected negatively the in vitro cytocompatibility of cpTi as demonstrated by decreasing culture growth and expression of osteoblastic phenotype.

Utilização de extratos de plantas medicinais e óleo de Eucaliptus no controle in vitro de Microsporum canis

INTRODUCTION: Microsporum canis is the most common cause of canine and feline dermatophytosis and thus has an important zoonotic role. OBJECTIVES: the aim of this study was to determine the antifungal action of medicinal plant extracts and of eucalyptus oil against pathogenic fungus Microsporum canis. METHODS: the extracts were prepared by mixing 300 g of previously washed leaves with 450 mL of distilled water. Then the material was triturated, filtered, sterilized and conserved at 10 + 2 oC. Fifteen milliliters of sterilized medium Sabouraud dextrose (Difco) at a temperature of 55 + 1 oC was added in Petri dishes containing the extracts in one, two, three, four and five mm concentrations. The fungus was inoculated once the medium was solidified. The inoculated dishes were maintained in B.O.D. incubator at 36 ± 0,5 oC until the fungus developed in the controls. RESULTS: the extracts from Punica granatum, Mangifera indica and Eucalyptus spp reduced the growth of fungus, but the extracts from Cymgopogom nardus, Tagetes minuta, Ruta graviolens, Cyperus rotundus, Annona moricata and Calendula spp leaves and flowers boosted the growth of fungus. The other extracts and the eucalyptus oil neither show any fungicidal action nor encourage mycelium growth. CONCLUSIONS: the use of most tested extracts and eucalyptus oil is not suitable for the treatment of Microsporum canis dermatophytosis due to lack of inhibitory effects. The extracts from Cymgopogom nardus, Tagetes minuta, Ruta graviolens, Cyperus rotundus, Annona moricata and from of Calendula spp leaves and flowers help the development of the fungus making clear that phytotherapy should be properly used, otherwise it can worsen the problem. However; extracts from Mangifera indica, Punica granatum and Eucalyptus spp. can be used as fungistatic.

In vitro effect of low-level laser therapy on typical oral microbial biofilms

The aim of this study was to evaluate the effect of specific parameters of low-level laser therapy (LLLT) on biofilms formed by Streptococcus mutans, Candida albicans or an association of both species. Single and dual-species biofilms - SSB and DSB - were exposed to laser doses of 5, 10 or 20 J/cm 2 from a near infrared InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm, 0.04 W). After irradiation, the analysis of biobilm viability (MTT assay), biofilm growth (cfu/mL) and cell morphology (SEM) showed that LLLT reduced cell viability as well as the growth of biofilms. The response of S. mutans (SSB) to irradiation was similar for all laser doses and the biofilm growth was dose dependent. However, when associated with C. albicans (DSB), S. mutans was resistant to LLLT. For C. albicans, the association with S. mutans (DSB) caused a significant decrease in biofilm growth in a dose-dependent fashion. The morphology of the microorganisms in the SSB was not altered by LLLT, while the association of microbial species (DSB) promoted a reduction in the formation of C. albicans hyphae. LLLT had an inhibitory effect on the microorganisms, and this capacity can be altered according to the interactions between different microbial species.

Effects of ascorbic acid on in vitro culture of bovine preantral follicles

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 μg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 μg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles. © 2012 Copyright Cambridge University Press.

Fermentation characteristics of several carbohydrate sources for dog diets using the in vitro gas production technique

Fermentable carbohydrates are an important part of the canine diet. They can improve gastrointestinal health by modifying gut microbial population and metabolic activity. The present study compared the fermentation characteristics and kinetic patterns of 10 carbohydrate sources using the in vitro gas production technique (IVGPT) with dog faecal inoculum. The substrates tested were: pure cellulose (PC), carboxymethylcellulose (CMC), sugar-cane fibre (SCF), beet pulp (BP), wheat bran (WB), fructooligosaccharides (FOS), inulin, yeast cell wall (YCW), ground psyllium seed (PS), pea hulls (PH). All substrates were incubated at 39°C under anaerobic conditions with faeces collected from dogs as microbial inoculum. Gas production of fermenting cultures was recorded and after 48 h, pH, shortchain fatty acids (SCFA) and organic matter disappearance (OMD) were determined. The results confirm high fermentation by dog faecal bacteria of FOS and inulin that produced high amounts of propionate and that underwent very rapid fermentation. Three substrates (SCF, CMC and PC) were not able to support bacterial growth, with low gas and SCFA production, and high BCFA formation. The PH and BP showed moderate OMD and SCFA production. Wheat bran B underwent rapid fermentation and generated a high proportion of butyrate. Psyllium seeds underwent slow fermentation with delayed gas production, supporting a high formation of SCFA, with an adequate amount of butyrate for bacterial growth while YCW, which showed a delayed fermentation, gave moderate SCFA production. The fermentation characteristics of PS and YCW suggest their potential use in promoting a more distal fermentation on intestinal tract. © Copyright S. Calabrò et al., 2013 Licensee PAGEPress, Italy.

Biostimulatory effect of low-level laser therapy on keratinocytes in vitro

Epithelial cells play an important role in reparative events. Therefore, therapies that can stimulate the proliferation and metabolism of these cells could accelerate the healing process. To evaluate the effects of low-level laser therapy (LLLT), human keratinocytes were irradiated with an InGaAsP diode laser prototype (LASERTable; 780 ± 3 nm; 40 mW) using 0.5, 1.5, 3, 5, and 7 J/cm2 energy doses. Irradiations were done every 24 h totaling three applications. Evaluation of cell metabolism (MTT assay) showed that LLLT with all energy doses promoted an increase of cell metabolism, being more effective for 0.5, 1.5, and 3 J/cm2. The highest cell counts (Trypan blue assay) were observed with 0.5, 3, and 5 J/cm2. No statistically significant difference for total protein (TP) production was observed and cell morphology analysis by scanning electron microscopy revealed that LLLT did not promote morphological alterations on the keratinocytes. Real-time polymerase chain reaction (qPCR) revealed that LLLT also promoted an increase of type I collagen (Col-I) and vascular endothelial growth factor (VEGF) gene expression, especially for 1.5 J/cm2, but no change on fibroblast growth factor-2 (FGF-2) expression was observed. LLLT at energy doses ranging from 0.5 to 3 J/cm2 promoted the most significant biostimulatory effects on cultured keratinocytes. © 2012 Springer-Verlag London Ltd.

Avaliação in vitro de diferentes métodos de análises de fungitoxicidade de óleos essenciais

Environmental problems caused by synthetic fungicides have increased the search for alternative methods of control of plant diseases. The objective was to evaluate the effect of essential oil of citronella grass, on the fungus Rhizoctonia solani, in different methods of in vitro fungitoxicity. We used a randomized design in a factorial design with four replications, where the factors were composed of four methods for assessing the in vitro fungitoxicity of the essential oil of citronella grass (essential oil diluted in Tween 80 (0.5%) and embedded in the culture medium PDA (potato dextrose agar) still melting, essential oil diluted in Tween 80 (0.5%) and distributed on the surface of the PDA; oil essential diluted in Tween 80 (0.5%) and distributed on filter paper attached to the inner surface of the lid of the Petri dish, pure essential oil and distributed on the surface of the culture medium, and control) and five evaluation periods (2, 4, 6, 8 and 10 days of incubation). Was used 0.25μL mL-1 of citronella oil in all treatments. Of the treatments evaluated the use of pure oil distributed on the surface of the culture medium was more effective in reducing the mycelial diameter in all evaluations. In this method the rate of mycelial growth was 9,02 mm day-1, reaching in last evaluation 79,77 mm.

In Vitro and In Vivo Activities of Ruthenium(II) Phosphine/Diimine/Picolinate Complexes (SCAR) against Mycobacterium tuberculosis

Rifampicin, discovered more than 50 years ago, represents the last novel class of antibiotics introduced for the first-line treatment of tuberculosis. Drugs in this class form part of a 6-month regimen that is ineffective against MDR and XDR TB, and incompatible with many antiretroviral drugs. Investments in R&D strategies have increased substantially in the last decades. However, the number of new drugs approved by drug regulatory agencies worldwide does not increase correspondingly. Ruthenium complexes (SCAR) have been tested in our laboratory and showed promising activity against Mycobacterium tuberculosis. These complexes showed up to 150 times higher activity against MTB than its organic molecule without the metal (free ligand), with low cytotoxicity and high selectivity. In this study, promising results inspired us to seek a better understanding of the biological activity of these complexes. The in vitro biological results obtained with the SCAR compounds were extremely promising, comparable to or better than those for first-line drugs and drugs in development. Moreover, SCAR 1 and 4, which presented low acute toxicity, were assessed by Ames test, and results demonstrated absence of mutagenicity. © 2013 Pavan et al.

Methods for obtaining reliable and reproducible results in studies of Candida biofilms formed in vitro

Biofilm formation is one of the most important attributes for virulence in Candida species and contributes to increased resistance to antifungal drugs and host immune mechanisms. These features have led to the development of several methodologies to reproduce a sessile community in vitro that can be used to study the development of a biofilm, its interaction with other microorganisms and the environment, and its susceptibility to available antifungal agents and also to search for new therapy strategies. The purpose of this review is to describe the most commonly used methods to study Candida biofilms in vitro, to discuss the benefits and limitations of the different methods to induce biofilm formation, and to analyse the architecture, viability and growth kinetics of Candida biofilms. © 2013 Blackwell Verlag GmbH.

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1β, CXCL-8 and MCP-1) and anti-inflammatory (TGF-β and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions.Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-β than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-β was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.Conclusion: These findings reinforce the systemic state of inflamm-aging frequently reported in elderly and considered a factor of susceptibility to numerous diseases. Still, the cytokine production from just monocytes of the elderly showed alterations, while in the lymphocyte presence not, suggesting an immunomodulator role of lymphocytes on monocytes. In addition, the differences between the production patterns by LPS-stimulated PBMC between young and elderly volunteers can be related with an imbalance in response against Gram-negative bacteria in throughout life. © 2013 Pinke et al.; licensee BioMed Central Ltd.

In vitro targeting of Polo-like kinase 1 in bladder carcinoma: Comparative effects of four potent inhibitors

Despite the improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little over the past 30 years. In the present study we tested and compared the in vitro antitumor activities of four different inhibitors of Polo-like kinase 1 (PLK1) (BI 2536, BI 6727, GW843682X, and GSK461364), against 3 bladder carcinoma cell lines RT4, 5637 and T24. The impact on radiosensitivity and drug interactions in simultaneous treatments with cisplatin, methotrexate, and doxorubicin were also investigated. Our results showed that PLK1 inhibition prevented cell proliferation and clonogenicity, causing significant inhibition of invasion of tumor cells, though modest differences were observed between drugs. Moreover, all PLK1 inhibitors induced G2/M arrest, with the subsequent induction of death in all 3 cell lines. Drug interactions studies showed auspicious results for all PLK1 inhibitors when combined with the commonly used cisplatin and methotrexate, though combinations with doxorubicin showed mostly antagonistic effects. Comparably, the four PLK1 inhibitors efficiently sensitized cells to ionizing radiation. Our findings demonstrate that irrespective of the inhibitor used, the pharmacological inhibition of PLK1 constrains bladder cancer growth and dissemination, providing new opportunities for future therapeutic intervention. However, further laboratorial and preclinical tests are still needed to corroborate the usefulness of using them in combination with other commonly used chemotherapeutic drugs. © 2013 Landes Bioscience.

Atividade antimicrobiana in vitro de extrato de Punica granatum L. sobre staphylococcus aureus isolado em leite bovino

Bovine mastitis is considered important disease causing major economic losses in dairy herds. Antimicrobial drug can promote resistance, chemical residues in food and environmental contamination. The purpose of this study was to evaluate the antibacterial activity in vitro of pomegranate extract on Staphylococcus aureus isolated from bovine milk, evaluate its antioxidant activity, and quantify levels of total phenols and fravonoides of the different extracts used. Aqueous extracts were used in nature and dry, from the peel of the fruit (EAC) and leaves (EAF). Additionally, it was evaluated the antioxidant activity (AA%), total phenols and flavonoids. Milk samples were inoculated, incubated, and the colonies were characterized as Staphylococcus aureus were adjusted to 1.0x106 UFC/mL in the 6 standard of the MacFarland scale. The sensitivity of the microbial isolates were determined in quintuplicate, in disk diffusion test. The minimum inhibitory concentration was determined by visible inhibition zone greater than 15mm. The results were evaluated by ANOVA, Tukey 5%, using the program SISVAR 5.3 - DEX/UFLA. Results implied that the aqueous extract from the bark of dried fruit was capable of inhibiting bacterial growth at concentrations of 3%. The other treatments only showed this activity, from the concentrations of 15%, 20% and 30% for dry EAF, in nature EAC and in nature EAF, respectively. Regarding the action antioxidant of dry EAC, was not correlated with total phenols and flavonoids. Probably other alkaloids substances present in the extract studied, may have been responsible for this activity. It is concluded that extracts of Punica granatum L., especially those obtained by the shell of the fruit, showed inhibitory activity against S. aureus, indicating your use potential for the control of bovine mastitis.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells. © CSIRO 2013.