Supposedly identical microplastic particles substantially differ in their material properties influencing particle-cell interactions and cellular responses.

Microplastics and its putative adverse effects on environmental and human health increasingly gain scientific and public attention. Systematic studies on the effects of microplastics are currently hampered by using rather poorly characterised particles, leading to contradictory results for the same particle type. Here, surface properties and chemical composition of two commercially available nominally identical polystyrene microparticles, frequently used in effect studies, were characterised. We show distinct differences in monomer content, ζ-potentials and surface charge densities. Cells exposed to particles showing a lower ζ-potential and a higher monomer content displayed a higher number of particle-cell-interactions and consequently a decrease in cell metabolism and proliferation, especially at higher particle concentrations. Our study emphasises that no general statements can be made about the effects of microplastics, not even for the same polymer type in the same size class, unless the physicochemical properties are well characterised.

Can the essential oil of rosemary (Rosmarinus officinalis Linn.) protect rats infected with itraconazole-resistant Sporothrix brasiliensis from fungal spread?

INTRODUCTION: Itraconazole is the first-choice option to treat human and animal sporotrichosis. However, the emergence of itraconazole-resistant strains has encouraged research on new active antifungals. Among them, the essential oil of rosemary (Rosmarinus officinalis Linn., Lamiaceae) has shown antifungal activity in vitro. OBJECTIVE: Assessing, for the first time, the effectiveness of rosemary essential oil in vivo in experimental cutaneous sporotrichosis, as well as its chemical composition and action mode. METHODS: Itraconazole-resistant Sporothrix brasiliensis was inoculated in the left foot pad of 30 Wistar rats, which were randomized (n=10) for treatment with saline solution (control, CONT), itraconazole (ITRA, 10 mg/kg) and rosemary oil (ROSM, 250 mg/kg) for 30 days at an oral dose of 1 mL, daily. Clinical evolution, histopathology and fungal burden were investigated. GC-MS was used for chemical analysis; sorbitol protection and ergosterol effect were used to evaluate the action mechanism of rosemary oil. RESULTS: ROSM was the only group evolving to skin lesion remission, lack of edema and exudate, and mild-to-absent yeast cells. Rosemary oil delayed fungal spreading and protected systemic organs, mainly liver and spleen. The ROSM group presented lower fungal load than that observed for the CONT and ITRA groups (p<0.05). Antifungal action took place at complexation level after ergosterol application. Most compounds were 1,8-cineole/eucalyptol (47.91%), camphor (17.92%), and α-pinene (11.52%). CONCLUSIONS: These findings have evidenced that rosemary oil is a promising antifungal to treat sporotrichosis, since it protects systemic organs from fungal spread.

Isolation of primary human B lymphocytes from tonsils compared to blood as alternative source for ex vivo application.

B lymphocytes ('B cells') are components of the human immune system with obvious potential for medical and biotechnological applications. Here, we discuss the isolation of primary human B cells from both juvenile and adult tonsillar material using a two-step procedure based on gradient centrifugation followed by separation on a nylon wool column as alternative to the current gold standard, i.e., negative immunosorting from buffy coats by antibody-coated magnetic beads. We show that the nylon wool separation is a low-cost method well suited to the isolation of large amounts of primary B cells reaching purities ≥ 80%. More importantly, this method allows the preservation of all B cell subsets, while MACS sorting seems to be biased against a certain B cell subtype, namely the CD27+ B cells. Importantly, compared to blood, the excellent recovery yield during purification of tonsillar B cells provides high number of cells, hence increases the number of subsequent experiments feasible with identical cell material, consequently improving comparability of results. The cultivability of the isolated B cells was demonstrated using pokeweed mitogen (PWM) as a stimulatory substance. Our results showed for the first time that the proliferative response of tonsillar B cells to mitogens declines with the age of the donor. Furthermore, we observed that PWM treatment stimulates the proliferation of a dedicated subpopulation and induces some terminal differentiation with ASCs signatures. Taken together this indicates that the proposed isolation procedure preserves the proliferative capability as well as the differentiation capacity of the B cells.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae / Ação anti-helmítica do óleo essencial de Anethum graveolens sobre ovos e larvas de Haemonchus contortus

Abstract Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (�� <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Resumo Os nematoides gastrintestinais são responsáveis ​​por grandes perdas econômicas na ovinocultura, e seu controle tem sido realizado quase exclusivamente pela administração de anti-helmínticos, que levaram a sérios problemas de resistência. Na busca de medidas alternativas de controle, destaca-se a pesquisa fitoterápica. O objetivo deste trabalho foi avaliar a ação do óleo essencial de Anethum graveolens (endro) em diferentes estágios de Haemonchus contortus, bem como testar a viabilidade celular para o óleo. Larvas e ovos de H. contortus foram obtidos de fezes de ovinos infectados e óleo essencial extraído de sementes de plantas através do aparelho de Clevenger. As concentrações avaliadas foram 9,4, 4,7, 2,35, 1,17, 0,58 e 0,29 mg/mL. Verificou-se a Inibição de eclosão dos ovos (IE), Inibição de Desenvolvimento Larval (IDL) e Inibição de Migração Larval (IML). Tiabendazol 0,025 mg/mL em IE e Levamisole 0.02 mg/mL nos testes IDL e IML foram usados ​​como controles positivos, enquanto água destilada e uma solução Tween 80 foram usados como controles negativos. Os resultados de inibição obtidos para a maior concentração de óleo foram: IE 100%, IDL 98,58% e IML 63,7%, diferindo (�� <0,05) dos controles negativos. Os principais componentes presentes em 95,93% do óleo total de A. graveolens foram Di-hidrocarvona (39,1%), Carvona (22,24%), D-Limoneno (16,84%), Apiol (10,49%) e Trans-di-hidrocarvona (7,26%). As concentrações mínimas de óleo essencial de A. graveolens necessárias para inibir 50% (IC50) de eclosão dos ovos, desenvolvimento larval e migração larval foram de 0,006 mg/mL, 2,536 mg/mL e 3,963 mg/mL, respectivamente. A viabilidade celular nas células MDBK (rim bovino Madin-Darby), quando incubadas com o óleo essencial de A. graveolens, foi de 86% para a maior (9,4 mg/mL) e 99% para a menor concentração (0,29 mg/mL). O óleo essencial de A. graveolens mostrou ser uma alternativa promissora no controle de nematoides gastrintestinais de ovinos.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae.

Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing (𝑝 <0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Megabenthic assemblages at the southern Central Indian Ridge - Spatial segregation of inactive hydrothermal vents from active-, periphery- and non-vent sites.

Active hydrothermal vents are small-scale habitats hosting endemic fauna in a well-defined zonation around fluid effluents. The fauna of inactive hydrothermal vents and its relation to active vents and non-vent area is poorly known. Characterizing inactive areas is prerequisite to establish protected areas, especially in the context of potential seafloor massive sulfide mining, which targets inactive sites. Hierarchical clustering and Distance-based Redundancy Analysis revealed five assemblages, with significantly associated substrate types: I) active hydrothermal vent, II) periphery, III) inactive hydrothermal vent and IV) soft- and V) hard-substrate within the non-vent area. For the first time, a unique inactive faunal assemblage could be identified within the hydrothermally extinct inactive Gauss field and on adjacent hard substrates. The spatial separation from the active Edmond field and periphery and the non-vent area indicates the existence of an inactive assemblage.

Chemical composition of essential oils from the apiaceae family, cytotoxicity, and their antifungal activity in vitro against candida species from oral cavity / Composição química de óleos essenciais da família apiaceae, citotoxicidade e sua atividade antifúngica in vitro contra espécies de candida da cavidade oral

Abstract The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.

Resumo Os objetivos desta pesquisa foram: avaliar a composição química e a citotoxicidade dos óleos essenciais de Cuminum cyminum (cominho), Anethum graveolens (endro), Pimpinella anisum (erva-doce) e Foeniculum vulgare (funcho), bem como sua atividade antifúngica in vitro contra dez isolados de Candida spp.. A composição química dos óleos foi analisada por meio de cromatografia gasosa acoplada à espectrometria de massa (GC / MS). Os ensaios de citotoxicidade foram realizados, utilizando o reagente de proliferação celular WST-1 em fibroblastos de ratinho L929 (20x103 poço-1). A determinação da Concentração Inibitória Mínima (MIC) foi realizada através da técnica de microdiluição em caldo (CLSI). Os principais componentes químicos foram o cuminaldeído (32.66%) para cominho, carvona (34.89%) para o endro, trans-anetol (94.01%) para erva-doce e anetol (79.62%) para a funcho. O endro e a erva-doce não foram citotóxicos em todas as concentrações testadas, no entanto, o óleo de cominho foi citotóxico na concentração de 20 mg.mL-1 e o endro nas concentrações de 20 e 8 mg.mL-1. Todas as leveduras foram suscetíveis aos óleos essenciais avaliados. O cominho apresentou a menor CIM contra as leveduras. Concluímos que todos os óleos essenciais apresentaram ação inibidora contra Candida spp., e C. cyminum, P. anisum e F. vulgare não foram citotóxicos nas mesmas concentrações inibitórias mínimas para os fungos.

Torquetenovirus viremia for early prediction of graft rejection after kidney transplantation.

OBJECTIVES: New biomarkers reflecting the degree of immunosuppression in transplant recipients are needed to provide an optimal personalized balance between rejection and infection risks. METHODS: For this purpose, we investigated TTV viremia dynamics in 66 kidney transplant recipients followed up for two years after transplantation, in relation to BK virus infection and graft rejection. RESULTS: After transplantation, TTV viremia rose by ≥2 log10 copies/mL from baseline to month 3, then declined by ≥1 log10 copies/mL thereafter. Higher TTV viremia was associated with recipients of a deceased donor, a lower count of CD8+ T cells and a higher BKV viremia. Importantly, TTV loads were significantly lower in KTR who would later display graft rejection; indeed, patients with TTV viremia lower than 3.4 log10 copies/mL at transplantation or lower than 4.2 log10 copies/mL at month 1 had a higher risk of developing graft rejection in the two following years (hazard ratio (HR) at D0 = 7.30, p = 0.0007 and HR at M1 = 6.16, p = 0.001). CONCLUSIONS: TTV viremia measurement at early times post transplantation predicts graft rejection and would represent a useful tool to improve kidney transplant monitoring.

Atividade in vitro do óleo essencial de Origanum vulgare L. em isolados clínicos de Aspergillus spp / In vitro activity of essential oil of Origanum vulgare L. in clinical isolates from Aspergillus spp

Aspergillus fumigatus, A. flavus e A. niger são os mais importantes agentes etiológicos da aspergilose, relevante micose aviária, com tratamento ineficaz e altas taxas de mortalidade. Em vista da importância da aspergilose, da necessidade de prospectar novos fármacos e do potencial terapêutico do óleo essencial de Origanum vulgare L. (OEO), o orégano, objetivou-se avaliar a sensibilidade in vitro de isolados clínicos de Aspergillus spp. em relação ao OEO. O óleo foi obtido por hidrodestilação em Clevenger, e a análise química realizada por cromatografia de massa (GC/MS). Observaram-se 15 diferentes compostos ativos, sendo 4-terpineol, hidrato de sabinene e timol os majoritários. Nos testes de microdiluição em caldo (Reference..., 2008), todos os isolados (n= 23) foram sensíveis ao OEO: A. fumigatus teve CIM entre 28,125mg/mL (0,1875%) e 450mg/mL (3,0%), A. flavus entre 112,5mg/mL (0,75%) e 450mg/mL, e A. niger 112,5mg/mL. CFM variou de 112,5mg/mL a 450mg/mL nos isolados de A. fumigatus, de 225mg/mL (1,5%) a 450mg/mL em A. flavus, e foi de 450mg/mL em A. niger. CIM e CFM foram idênticos em 6/14 isolados, o que demonstra que o óleo com a mesma concentração pode ter capacidade fungistática e fungicida. CIM 90 correspondeu à CIM máxima. Os resultados demonstram a atividade anti-Aspergillus do OEO, com CIM 90 de 450mg/mL (3%).(AU)

Aspergillus fumigatus, A. flavus and A. niger are the most important etiological agents of aspergillosis, a relevant avian mycosis, with innefective treatment and high mortality rates. Due the importance of aspergillosis, the necessity of prospection of new drugs and the therapeutic potential of the essential oil of Origanum vulgare L. (OEO), popularly known as oregano, aims to evaluate the in vitro sensitivity of Aspergillus spp. opposing to OEO. The oil was obtained by hydrodistillation in Clevenger, and the chemical analysis performed by mass chromatography (GC/MS). 15 different active compounds were observed, being 4-terpineol (18.4%), sabinene hydrate (15.6%) and thymol (13.6%), the majority components. In the in vitro susceptibility test, all strains showed sensitivity to OEO, MIC of Aspergillus fumigatus ranged from 28,125mg/mL (0,1875%) to 450mg/mL (3,0%), A. flavus 112,5mg/mL (0,75%) to 450mg/mL, and A. niger 112,5mg/mL. MFC ranged from 112,5mg/mL to 450mg/mL in the A. fumigatus isolates, 225mg/mL (1,5%) to 450mg/mL in A. flavus, and 450mg/mL for A. niger. The MIC and FMC values were identical in 6/14 of the isolated subjects, demonstrating that the oil with the same concentration can have fungistatic and fungicidal capacity. The results demonstrates anti-Aspergillus activities of OEO with CIM90 de 450mg/mL (3%).(AU)

Chemical composition of essential oils from the apiaceae family, cytotoxicity, and their antifungal activity in vitro against candida species from oral cavity.

The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.

Evaluation of anti-enzyme properties of Origanum vulgare essential oil against oral Candida albicans.

This study aimed to evaluate the anti-enzymatic activity of Origanum vulgare (oregano) essential oil against 15 strains of Candida albicans. Candida albicans samples were isolated from the oral mucosa of patients with denture stomatitis treated in a Dentistry school on a public university. Preparation of the inoculum was performed with a suspension of C. albicans reactivated 24h earlier in 5mL of sterile phosphate buffer saline (PBS) adjusted to a 0.5-turbidity on the MacFarland scale (1,5×108UFC/mL). The essential oil was obtained by hydrodistillation in a Clevenger-type machine and analyzed by gas chromatography. Enzymatic assay was performed to test phospholipase anti-enzymatic properties. Chromatography analysis revealed that the main compounds present in the essential oil were 4-terpineol (41.17%), thymol (21.95%), γ-terpinene (5.91%) and carvacrol (4.71%). For the anti-enzymatic test, the statistical analysis showed that there was found statistically significant interactions between the factors time and concentration (P≤0,001). Thus, essential oil of oregano at 1%, 5% and 10% presented significant reductions in the production of the phospholipase enzyme produced by Candida albicans strains. However, the longer the incubation time of the essential oil, there is a relatively moderate reduction in its anti-enzymatic activity.

Chemical composition of essential oils from the apiaceae family, cytotoxicity, and their antifungal activity in vitro against candida species from oral cavity

Abstract The aims of this research were: evaluate the chemical composition and the cytotoxicity of the Cuminum cyminum (cumin), Anethum graveolens (dill), Pimpinella anisum (anise) and Foeniculum vulgare (fennel) essential oils, as well as their antifungal activity in vitro against ten Candida spp. isolates. The chemical composition of the oils was analyzed by means of gas chromatography coupled with mass spectrometry (GC/MS). The cytotoxicity assays were performed, using the cell proliferation reagent WST-1 in L929 mouse fibroblasts (20x103 well-1). The determinate the Minimum Inhibitory Concentration (MIC), was performed through the Broth Microdilution technique (CLSI). The chemical main components were the cuminaldehyde (32.66%) for cumin, carvone (34.89%) for the dill, trans-anethole (94.01%) for the anise and anethole (79.62%) for the fennel. Anise and fennel did not were cytotoxic in all the tested concentrations, however the cumin oil was cytotoxic in the concentration of 20 mg.mL-1 and the dill in the concentrations of 20 and 8 mg.mL-1. All yeasts were susceptible against the evaluated essential oils. Cumin presented the lowest MIC against yeasts. We concluded that all the essential oils presented inhibitory action against Candida spp., and C . cyminum, P. anisum and F. vulgare were not cytotoxic in the same minimum inhibitory concentrations for the fungi.

Resumo Os objetivos desta pesquisa foram: avaliar a composição química e a citotoxicidade dos óleos essenciais de Cuminum cyminum (cominho), Anethum graveolens (endro), Pimpinella anisum (erva-doce) e Foeniculum vulgare (funcho), bem como sua atividade antifúngica in vitro contra dez isolados de Candida spp.. A composição química dos óleos foi analisada por meio de cromatografia gasosa acoplada à espectrometria de massa (GC / MS). Os ensaios de citotoxicidade foram realizados, utilizando o reagente de proliferação celular WST-1 em fibroblastos de ratinho L929 (20x103 poço-1). A determinação da Concentração Inibitória Mínima (MIC) foi realizada através da técnica de microdiluição em caldo (CLSI). Os principais componentes químicos foram o cuminaldeído (32.66%) para cominho, carvona (34.89%) para o endro, trans-anetol (94.01%) para erva-doce e anetol (79.62%) para a funcho. O endro e a erva-doce não foram citotóxicos em todas as concentrações testadas, no entanto, o óleo de cominho foi citotóxico na concentração de 20 mg.mL-1 e o endro nas concentrações de 20 e 8 mg.mL-1. Todas as leveduras foram suscetíveis aos óleos essenciais avaliados. O cominho apresentou a menor CIM contra as leveduras. Concluímos que todos os óleos essenciais apresentaram ação inibidora contra Candida spp., e C. cyminum, P. anisum e F. vulgare não foram citotóxicos nas mesmas concentrações inibitórias mínimas para os fungos.

Antihelminthic action of the Anethum graveolens essential oil on Haemonchus contortus eggs and larvae

Abstract Gastrointestinal nematodes are responsible for great economic losses in sheep raising, and their control has long been carried out almost exclusively by the administration of anthelmintics, which have led to serious resistance problems. In the search for alternative control measures, phytotherapic research is highlighted. The aim of this study was to evaluate the action of Anethum graveolens (dill) essential oil on different stages of Haemonchus contortus life cycle, as well its cytotoxicity MDBK (Madin-Darby bovine kidney) cells. H. contortus larvae and eggs were obtained from infected sheep feces, and essential oil extracted from plant seeds through the Clevenger apparatus. 9.4, 4.7, 2.35, 1.17. 0.58 and 0.29 mg/mL concentrations were evaluated. The Egg Hatch Inhibition (HI), Larval Development Inhibition (LDI) and Larval Migration Inhibition (LMI) techniques were used. Thybendazole 0.025 mg/mL in HI and Levamisole 0.02 mg/mL in the LDI and LMI tests were used as positive controls, while distilled water and a Tween 80 solution were used as positive negative controls. The inhibition results obtained for the highest oil concentration were: HI 100%, LDI 98.58% and LMI 63.7%, differing ( 0.05) from negative controls. Main A. graveolens oil components present in 95.93% of the total oil were Dihydrocarvone (39.1%), Carvone (22.24%), D-Limonene (16.84%), Apiol (10.49%) and Trans-dihydrocarvone (7.26%). Minimum A. graveolens essential oil concentrations required to inhibit 50% (IC50) of egg hatching, larval development and larval migration were 0.006 mg/mL, 2.536 mg/mL and 3.963 mg/mL, respectively. Cell viability in MDBK (Madin-Darby bovine kidney) cells, when incubated with A. graveolens essential oil, was 86% for the highest (9.4 mg/mL) and 99% for the lowest concentration (0.29 mg/mL). A. graveolens essential oil, according to the results obtained in this study, is a promising alternative in sheep gastrointestinal nematode control.

Resumo Os nematoides gastrintestinais são responsáveis por grandes perdas econômicas na ovinocultura, e seu controle tem sido realizado quase exclusivamente pela administração de anti-helmínticos, que levaram a sérios problemas de resistência. Na busca de medidas alternativas de controle, destaca-se a pesquisa fitoterápica. O objetivo deste trabalho foi avaliar a ação do óleo essencial de Anethum graveolens (endro) em diferentes estágios de Haemonchus contortus, bem como testar a viabilidade celular para o óleo. Larvas e ovos de H. contortus foram obtidos de fezes de ovinos infectados e óleo essencial extraído de sementes de plantas através do aparelho de Clevenger. As concentrações avaliadas foram 9,4, 4,7, 2,35, 1,17, 0,58 e 0,29 mg/mL. Verificou-se a Inibição de eclosão dos ovos (IE), Inibição de Desenvolvimento Larval (IDL) e Inibição de Migração Larval (IML). Tiabendazol 0,025 mg/mL em IE e Levamisole 0.02 mg/mL nos testes IDL e IML foram usados como controles positivos, enquanto água destilada e uma solução Tween 80 foram usados como controles negativos. Os resultados de inibição obtidos para a maior concentração de óleo foram: IE 100%, IDL 98,58% e IML 63,7%, diferindo ( 0,05) dos controles negativos. Os principais componentes presentes em 95,93% do óleo total de A. graveolens foram Di-hidrocarvona (39,1%), Carvona (22,24%), D-Limoneno (16,84%), Apiol (10,49%) e Trans-di-hidrocarvona (7,26%). As concentrações mínimas de óleo essencial de A. graveolens necessárias para inibir 50% (IC50) de eclosão dos ovos, desenvolvimento larval e migração larval foram de 0,006 mg/mL, 2,536 mg/mL e 3,963 mg/mL, respectivamente. A viabilidade celular nas células MDBK (rim bovino Madin-Darby), quando incubadas com o óleo essencial de A. graveolens, foi de 86% para a maior (9,4 mg/mL) e 99% para a menor concentração (0,29 mg/mL). O óleo essencial de A. graveolens mostrou ser uma alternativa promissora no controle de nematoides gastrintestinais de ovinos.

Effects of essential oils of Rosmarinus officinalis Linn. and Origanum vulgare Linn. from different origins on Sporothrix brasiliensis and Sporothrix schenckii complex / Efeitos dos óleos essenciais de Rosmarinus officinalis Linn. e Origanum vulgare Linn. de diferentes origens em Sporothrix brasiliensis e complexo Sporothrix schenckii

Rosmarinus officinalis L. (rosemary) and Origanum vulgare L. (oregano) are known to have antimicrobial properties, but studies on sporotrichosis are scarce. This study aimed to evaluate the anti-Sporothrix spp. activity of essential oils from commercial products and oils extracted from aerial parts of these plants and analyze their chemical constituents. S. schenckii complex and S. brasiliensis (n: 25) isolated from humans, cats, dogs, and environmental soil were tested through M27-A3 guidelines of CLSI with modification for phytotherapics. The essential oils of R. officinalis L. were similar for MIC50 and MFC50 ≤2.25mg/mL for extracted oil; and 4.5mg/mL and 9mg/mL, respectively, for commercial oil. Both products showed MIC90 of 18mg/mL and MFC90 of 36mg/mL. In O. vulgare L., the extracted oil had better activity with MIC50 and MFC50 ≤2.25mg/mL, and MIC90 and MFC90 of 4.5mg/mL, whereas the commercial oil showed MIC50 and MFC50 of 9mg/mL and MIC90 18mg/mL, respectively, and MFC90 of 36mg/mL. Through gas chromatography (CG/FID), thymol and α-terpinene were majority for extracted oil of O. vulgare L., and carvacrol and γ-terpinene made up the majority of the commercial oil. Both essential oils of R. officinalis L. showed 1,8-cineole and α-pinene as major. The fungal isolates were susceptible to all tested essential oils, including in itraconazole-resistant S. brasiliensis isolates. The extracted and commercial oils of the plants presented in vitro anti-Sporothrix spp. activity, and they are promising for treatment of sporotrichosis, including in cases refractory to itraconazole. More studies should be performed about toxicity and in vivo efficacy for its safe use.(AU)

Rosmarinus officinalis L. (alecrim) e Origanum vulgare L. (orégano) são conhecidos pelas propriedades antimicrobianas, entretanto seus estudos na esporotricose são escassos. Este trabalho objetivou avaliar a atividade anti-Sporothrix spp. de óleos extraídos e comerciais dessas plantas e analisar seus constituintes químicos. Isolados do complexo S. schenckii e S. brasiliensis (n: 25) de humanos, gatos, cães e solo, foram testados pela diretriz M27-A3 do CLSI com modificações para fitoterápicos. Os óleos de R. officinalis L. foram similares com CIM50 e CFM50 ≤2.25mg/mL para extraído; e 4.5mg/mL e 9mg/mL, respectivamente, para comercial. Ambos os produtos demonstraram CIM90 de 18mg/mL e CFM90 de 36mg/mL. Em O. vulgare L., o óleo extraído apresentou melhor atividade com CIM50 e CFM50≤2.25mg/mL e CIM90 e CFM90 de 4.5mg/mL, ao passo que o óleo comercial mostrou CIM50 e CFM50 de 9mg/mL; e CIM90 de 18mg/mL e CFM90 de 36mg/mL. Por meio da cromatografia gasosa (CG/FID), timol e α-terpineno foram majoritários para o óleo extraído de O. vulgare L., e carvacrol e γ-terpineno para o comercial. Ambos os óleos de R. officinalis L. apresentaram 1,8-cineol e α-pineno como prevalentes. Os isolados foram sensíveis a todos os óleos essenciais testados, inclusive S. brasiliensis, resistentes ao itraconazol. Os óleos extraídos e comerciais de R. officinalis L. e O. vulgare L. apresentaram atividade anti-Sporothrix spp. in vitro e são promissores para o tratamento da esporotricose, inclusive em casos refratários ao itraconazol. Mais estudos devem ser realizados sobre toxicidade e eficácia in vivo para seu uso seguro.(AU)

Potencial cicatricial da Bixa orellana L. em feridas cutâneas: estudo em modelo experimental / Therapeutic potential of Bixa orellana L. in skin wounds: a study in the rat model of open wound healing

O uso de fitoterápicos é uma alternativa de baixo custo e de fácil acesso para o tratamento de feridas cutâneas. Objetivou-se avaliar a ação do extrato oleoso de urucum na cicatrização de feridas cutâneas abertas. Inicialmente, identificaram-se os principais ácidos graxos do óleo de urucum. Foi realizado ensaio citotóxico para determinar as concentrações a serem utilizadas no ensaio in vivo. No experimento, feridas cutâneas em ratos Wistar foram diariamente tratadas com: extrato de urucum 0,1% (U 0,1%), extrato de urucum 0,01% (U 0,01%), vaselina (V) e solução fisiológica (SF), por até 21 dias. Aos quatro, sete, 14 e 21 dias, foi avaliada clinicamente a presença de exsudato, crosta e epitelização. Determinaram-se as áreas da lesão, e amostras de pele, fígado e rins foram coletadas para avalição histológica. Aos 21dias, amostras de pele foram coletadas para análise tensiométrica. Clinicamente, todos os grupos de tratamento apresentaram evolução cicatricial fisiológica. Os grupos U 0,1% e U 0,01% apresentaram maior presença de epitelização aos sete dias e maior retração cicatricial aos quatro dias. Na histologia, U 0,1% e U 0,01% apresentaram aos quatro e sete dias maior quantidade de fibrina e inflamação que V e SF, e, nos demais momentos, não houve diferenças entre os grupos. Quanto à fase cicatricial, aos quatro dias todos os grupos encontravam-se na fase inflamatória, aos sete dias U 0,1% e U 0,01% permaneciam na fase inflamatória, diferindo de SF e V, que se caracterizavam na fase proliferativa. Aos 14 dias, os grupos apresentavam-se em transição de fase proliferativa para maturação e, aos 21dias, estavam todos na fase de maturação. Os grupos tratados com urucum expressaram menor resistência à tensão que V e SF. Concluiu-se com este estudo que o extrato oleoso de urucum acelera o processo cicatricial nos primeiros dias, mas proporciona uma cicatriz de baixa qualidade.

Phytotherapies are a low cost, easily accessible alternative to traditional medicines in wound healing management. The purpose of this study was to assess the oil extract of Bixa orellana L. as a healing agent in the rat model of open wound healing. Initially, the oil was obtained and characterized through gas chromatography. Furthermore, the cytotoxic potential of the oil was verified in cell cultures to determine the doses used in animal experiments. Wounds were surgically produced in Wistar rats, these were treated with the oil extract at 0.1% (U 0.1%), 0.01% (U 0.01%), petrol jelly (V) and saline (SF) for up to 21 days. At four, seven and 14 days of treatment the wounds were assessed clinically regarding the presence of exudate, crust and epithelialization. The wound area was also determined and skin, kidney and liver tissues were harvested for histopathology. At 21 days of treatment the skins were also harvested for tension resistance assessment. Clinically, all groups evolved similarly, however, those treated with U 0.1% and U 0.01% had a greater amount of epithelialized wounds by day seven, and grater shrinkage by day four. Histopathologicaly, the skin samples of oil treated wounds had more lesions in the inflammatory phase at seven days, when compared to the controls, which were majorly in the proliferation phase. By 14 days no difference was observed among groups, which were all in the transition from the proliferation to the maturation phase. By day 21, all wounds were in the maturation phase. Oil treated wounds also had more fibrin in the first two assessment dates, when compared to the controls. Tension resistance of the oil treated wounds was, however, inferior to that of the controls. This study shows that B. orellana L. oil will hasten the onset of the healing process and its initial phases, but will ultimately produce a scar of poorer quality.

Use of Triticum aestivum in open wound healing: a clinical, pathological, and tensiometric assessment in the rabbit model / Uso de Triticum aestivum na cicatrização de feridas: um ensaio clínico, histopatológico e tensiométrico em coelhos

While Triticum sp. has been shown to act in wound healing, stimulating collagen synthesis by fibroblasts, the use of this plant extract has yet to be assessed in vivo, in commercially viable presentations. This study used rabbits and assessed, on days seven, 14, and 21, the presence or absence of granulation tissue and epithelialization, histopathological structures, and scar quality through the breaking and tension strength. Treatments, performed for 21 days, were aqueous extract of T. aestivum at a concentration of 2mg/mL (group I) and 10mg/mL (group II) and a nonionic cream (control group). We demonstrate that the formation of granulation tissue was not significantly different between treatments. In the analysis of epithelial tissue, wounds in group II differed from other treatments by day 7. On days 14 and 21 there was no significant clinical difference between groups. In the histopathological evaluation, scar quality and rupture strength did not differ between the groups in the studied period. In the tension strength evaluation, group I differed from the others, presenting a higher tension strength overall. The studied treatments did not differ regarding healing evolution of the skin wounds, but T. aestivum extract, at 2mg/mL, presents better results in the tension strength evaluation...

O extrato de trigo (Triticum sp.) vem sendo usado na cicatrização de feridas por estimular a síntese de fibroblastos, entretanto a sua aplicabilidade in vivo em apresentações comercialmente viáveis ainda tem de ser demonstrada. Neste estudo, avaliaram-se feridas cutâneas de coelhos tratadas com extrato aquoso de T. aestivum quanto à presença de tecido de granulação e epitelização, estruturas histológicas, qualidade cicatricial, além de ensaio tensiométrico. As feridas foram tratadas diariamente, por 21 dias, com diferentes concentrações do extrato (grupo I = 2mg/mL; grupo II = 10mg/mL) ou apenas o veículo (grupo controle = creme não iônico), e avaliadas nos dias sete, 14 e 21. A formação de tecido de granulação não diferiu entre os tratamentos. A epitelização aconteceu em menor tempo em feridas do grupo II, mas aos 14 dias já não havia diferença neste parâmetro. Na avaliação histopatológica, a qualidade cicatricial e a força de ruptura não diferiram no período estudado, entretanto a resistência tensiométrica das feridas do grupo I foi maior que a dos demais tratamentos. Dessa forma, conclui-se que, mesmo não havendo diferença na evolução cicatricial de feridas tratadas ou não com extrato aquoso de T. aestivum, o uso desse composto, a 2mg/mL, resultou em tecidos cicatriciais mais resistentes à tração...

Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6) cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6) cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

Atividade antifúngica do óleo essencial de Origanum vulgare frente a Malassezia pachydermatis / Antifungal activity of Origanum vulgare essential oil against Malassezia pachydermatis

Objetivou-se com este estudo avaliar a atividade antifúngica in vitro do óleo essencial de Origanum vulgare frente a isolados clínicos de Malassezia pachydermatis. As folhas secas de O. vulgare foram adquiridas de distribuidor comercial com certificado de qualidade e origem e encaminhadas para extração do óleo essencial e cromatografia. Para realização do teste in vitro, foi utilizada a técnica de microdiluição em caldo (CLSI M27A3) com modificações para fitofármacos e M. pachydermatis. O óleo essencial de orégano foi testado nas concentrações de 28 a 0,87mg/mL diluído em caldo Sabouraud com 1% de tween 80. Todos os isolados foram testados em duplicata. Na análise cromatográfica do óleo essencial, foram identificados 12 compostos, sendo timol, a-terpineno e 4-terpineol os compostos majoritários. A CIM e a CFM dos 42 isolados de M. pachydermatis variaram de <0,87 a 7mg/mL, com valores de CIM50 e CIM90 de 1,18 e 3,28mg/mL, respectivamente. Com este estudo foi possível concluir que M. pachydermatis é sensível ao óleo essencial de orégano mesmo em concentrações baixas. Dessa maneira, o óleo essencial de orégano apresenta-se como promissor na bioprospecção de novos fármacos para o tratamento das otites e dermatites na clínica de pequenos animais...

The aim of this study was to evaluate the in vitro antifungal activity of essential oil of Origanum vulgare against clinical isolates of Malassezia pachydermatis. The dried leaves of O. vulgare were purchased from a commercial distributor with certified quality and origin and referred for essential oil extraction and chromatography. The technique for in vitro testing was microdilution (CLSI M27A3) with modifications to phytochemicals and M. pachydermatis. The essential oil of O. vulgare was tested at concentrations from 28 to 0.87mg/mL in Sabouraud broth diluted with 1% of tween 80. All isolates were tested in duplicate. In the chromatographic analysis of the essential oil 12 compounds were identified, and thymol, α-terpinene, 4-terpineol were the major compounds. The MIC and the MFC of the 42 isolates of M. pachydermatis ranged from <0.87 to 7mg/mL with MIC50 and MIC90 values of 1.18 and 3.28 mg/mL, respectively. With this study it was concluded that M. pachydermatis is sensible to O. vulgare essential oil even at low concentrations. Thus, the essential oil of O. vulgare is presented as bioprospecting in the promising new drugs for the treatment of otitis and dermatitis in small animal clinic...

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells.

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L-lysine) (Ca-alg-PLL), and a novel calcium alginate-poly(ethylene glycol) hybrid material (Ca-alg-PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca-alg-PLL capsules, while a resazurin-based assay was used for comparison in case of the Ca-alg-PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter-assay relative standard deviations below 10%.

Direct detection of formaldehyde in air by a novel NAD+- and glutathione-independent formaldehyde dehydrogenase-based biosensor.

An amperometric enzyme-based sensor-system for the direct detection of formaldehyde in air is under investigation. The biosensor is based on a native bacterial NAD(+)- and glutathione-independent formaldehyde dehydrogenase as biorecognition element. The enzyme was isolated from Hyphomicrobium zavarzinii strain ZV 580, grown on methylamine hydrochloride in a fed-batch process. The sensor depends on the enzymatic conversion of the analyte to formic acid. Released electrons are detected in an amperometric measurement at 0.2V vs. Ag/AgCl reference electrode by means of a redox-mediator. To optimize the sensing device, Ca(2+) and pyrroloquinoline quinone (PQQ) were added to the buffer solution as reconstitutional substances. At this stage, the sensor shows linear response in the tested ppm-range with a sensitivity of 0.39 microA/ppm. The signal is highly reproducible with respect to sensitivity and base line signal. Reproducibility of sensitivity is more than 90% within the same bacterial batch and even when enzyme of different bacterial batches is used.