Comparison of Transforming Growth Factor 1 (TGF-β 1) Expression in Various Lysate Platelet-Rich Fibrin (L-PRF) Concentrations on Human Dental Pulp Stem Cell Differentiation

ABSTRACT Objective: To compare Transforming growth factor-β1 (TGF-β1) expression in various L-PRF concentrations on the hDPSC differentiation process. Material and Methods: hDPSC cell cultures were subjected to serum starvation by reducing FBS levels in the hDPSC culture media. Lysate PRF was obtained from the PRF gel, which was then incubated at 4°C for 24 h. The supernatant was dried, transferred to a 2-ml Eppendorf tube, and stored at −20°C. The evaluation of TGF-β1 expression in 1%, 5%, 10%, and 25% L-PRF samples and 10% FBS (control) during the process of hDPSC differentiation was quantified using an ELISA reader on day 7. The expression of TGF-β1 was subjected to a one-way ANOVA test, followed by Bonferroni's post hoc test with significant values (p<0.05). Results: Significant differences were noted in TGF-β1 expression between 1%, 5%, 10%, and 25% L-PRF and the control group (10% FBS). The highest TGF-β1 expression occurred with 25% L-PRF (0.645 ± 0.048), followed by 10% L-PRF (0.461 ± 0.035), 10% FBS (0.374 ± 0.013), 5% L-PRF (0.275 ± 0.045), and the lowest expression was with 1% L-PRF (0.160 ± 0.045). Conclusion: The best result of TGF-B1 expression in hDPSC differentiation was in the 25% L-PRF group.

Culture media to detect and criteria to evaluate and report the activity of extracellular enzymes produced by phytopathogenic fungi / Meios de cultura para detectar e critérios para avaliar e divulgar atividade de enzimas extracelulares produzidas por fungos fitopatogênicos

Extracellular enzymes are involved in the fungal pathogenesis in plants. Currently, culture media, data analyses, and data report related to extracellular enzymes produced in vitro conditions are different and therefore, lack standardization. This work aimed to compare the culture media cited on the literature (normal) with the potato-dextrose-agar (PDA) medium combined with a specific compound to produce extracellular enzymes through three soilborne phytopathogenic fungi (F. solani f. sp. passiflorae, S. rolfsii, and R. solani AG-4 HGI), as well as to analyze and report enzyme data based on five different criteria. The assay was randomized, with three factors (culture media, isolates, and enzymes) and six repetitions. The studied enzymes were amylase (AM), carboxymethylcellulase (CMCase), lipase (LP), laccase (LC), catalase (CT), and gelatinase (GT). The normal media detected more enzymes and was more precise compared to the PDA medium plus specific compound. The criteria that calculated the area of the circular crown of AM, CMCase, LP, and LC and measured the intensity (0 = absence, up to 4 = intense) of CT and GT adopting note scale were the best to evaluate and report the results of the enzymes. We suggest the normal media culture to study enzyme production, as well as the criteria mentioned to assess and report the data related to enzyme activities.(AU)

As enzimas extracelulares estão envolvidas na patogênese de fungos em plantas. Atualmente, não há uma padronização de meios de cultura, formas de analisar e divulgar os dados de enzimas extracelulares produzidas em condições in vitro. Assim, o presente trabalho objetivou comparar os meios de cultura específicos relatados na literatura (normal) com o meio de batata-dextrose-ágar mais adição do substrato específico para produção de enzimas extracelulares por três diferentes fungos fitopatogênicos habitantes de solo (Fusarium solani f. sp. passiflorae, Sclerotium rolfsii e Rhizoctonia solani AG-4 HGI), bem como avaliar os dados das enzimas por cinco critérios diferentes. O delineamento experimental adotado foi o inteiramente ao acaso, em esquema de três fatores (meios, isolados e enzimas), com seis repetições. As enzimas investigadas foram amilase, carboximetilcelulase, lipase, lacase, catalase e gelatinase. Os meios normais detectaram mais enzimas, e essa detecção foi mais precisa em comparação com os meios de batata-dextrose-ágar mais o substrato específico. Os critérios que calcularam a área da coroa circular para as enzimas amilase, carboximetilcelulase, lipase e lacase e adotaram a escala de notas para medir a intensidade (0=ausência até 4=intensa) de catalase e gelatinase foram os melhores para avaliar e divulgar os resultados das enzimas. Assim, sugere-se padronizar os meios normais para estudos de produção de enzimas, bem como os critérios citados para avaliar e divulgar os dados das atividades das referidas enzimas.(AU)

Evaluación del rendimiento del sistema Rapid™ Yeast Pluz (Remel) a partir de 2 medios de cultivo diferentes / Performance evaluation of Rapid™ Yeast Plus (Remel) system from two different culture media

Dentro del género Candida, la especie más frecuentemente aislada de materiales clínicos es Candida albicans. Debido a la emergencia de otras especies que pueden presentar mayor índice de resistencia a los antifúngicos, se hace necesaria la identificación rápida de aquellas. El objetivo del presente trabajo fue evaluar el rendimiento del sistema RapID™ Yeast Plus a partir de subcultivos en 2 formulaciones diferentes, agar Sabouraud dextrosa modificado por Emmons (medio indicado en el inserto del equipo) y agar Sabouraud glucosado, que es el utilizado habitualmente en los laboratorios de la Ciudad Autónoma de Buenos Aires. Se estudiaron 166 cepas de muestras clínicas provenientes de los distintos hospitales que integran la Red de Micología de la Ciudad de Buenos Aires. De los resultados obtenidos se concluye que se deben mantener las condiciones y el medio de cultivo indicado por el fabricante

Within the genus Candida, Candida albicans is the most commonly isolated species from clinical samples. Due to the emergence of other species which can show a higher index of antifungal resistance, a fast identification of these species is necessary. The aim of this work was to evaluate the performance of the RapID Yeast Plus system from two different subculture media formulations: Sabouraud dextrose agar adjusted by Emmons (the medium is indicated in the equipment insert) and Sabouraud glucose agar, which is the most frequently used in Buenos Aires City laboratories. One hundred and sixty-six clinical sample strains coming from different hospitals belonging to the Mycology Network of Buenos Aires City were studied. From the obtained results, we conclude that the conditions and culture medium indicated by the manufacturer should be followed

Variation of Spirulina maxima biomass production in different depths of urea-used culture medium

Fewer studies have assessed the outdoor cultivation of Spirulina maxima compared with S. platensis, although the protein content of S. maxima is higher than S. platensis. Spirulina growth medium requires an increased amount of NaHCO3, Na2CO3, and NaNO3, which increases the production cost. Therefore, the current study used a low-cost but high-efficiency biomass production medium (Medium M-19) after testing 33 different media. The medium depth of 25 cm (group A) was sub-divided into A1 (50% cover with a black curtain (PolyMax, 12 oz ultra-blackout), A2 (25% cover), and A3 (no cover). Similarly the medium depths of 30 and 35 cm were categorized as groups B (B1, B2, and B3) and C (C1, C2, and C3), respectively, and the effects of depth and surface light availability on growth and biomass production were assessed. The highest biomass production was 2.05 g L-1 in group A2, which was significantly higher (p < 0.05) than that in all other groups and sub-groups. Spirulina maxima died in B1 and C1 on the fifth day of culture. The biochemical composition of the biomass obtained from A2 cultures, including protein, carbohydrate, lipid, moisture, and ash, was 56.59%, 14.42%, 0.94%, 5.03%, and 23.02%, respectively. Therefore, S. maxima could be grown outdoors with the highest efficiency in urea-enriched medium at a 25-cm medium depth with 25% surface cover or uncovered.

Growth of Haematococcus pluvialis Flotow in alternative media / Crescimento de Haematococcus pluvialis Flotow em meios alternativos

Abstract Current study investigates the effect of two alternative media NPK (20-5-20) fertilizer and NPK plus macrophyte (M+NPK) compared to the commercial medium (WC) under growth rate and physiological parameters in batch culture mode (2-L), and verifies whether the use of fertilizer (NPK) and macrophyte (Eichhornia crassipes) would be a good tool for Haematococcus pluvialis culture in the laboratory. The highest number of cells of H. pluvialis has been reported in NPK medium (5.4 × 105cells.mL–1) on the 28th day, and in the M+NPK and WC media (4.1 × 105 cells.mL–1 and 2.1 × 105 cells.mL–1) on the 26th day, respectively. Chlorophyll-a contents were significantly higher (p<0.05) in NPK medium (41-102 µg.L–1) and lower in WC and M+NPK media (14-61 µg.L–1). The astaxanthin content was less than 0.04 mg.L–1. Production cost of 10-L of H. pluvialis was low in all media, and NPK and M+NPK media had a cost reduction of 65% and 82%, respectively when compared with commercial medium (WC). The use of a medium based on commercial fertilizer and macrophyte (E. crassipes) produced a new medium formulation that proved to be efficient, at least in batch culture mode, in promoting high density culture of H. pluvialis. NPK and macrophyte (E. crassipes) medium seems to be an adequate alternative to replace the conventional one (WC).

Resumo O presente estudo investigou o efeito de dois meios alternativos, NPK (20-5-20) e NPK mais macrófita (M+NPK), em relação ao meio comercial (WC) para avaliar a taxa de crescimento e parâmetros fisiológicos em cultivo estático (2-L), a fim de verificar se o fertilizante (NPK) e a macrófita (Eichhornia crassipes) podem ser utilizados no cultivo de Haematococcus pluvialis em laboratório. O maior número de células de H. pluvialis foi observado no meio NPK (5,4 × 105 células.mL–1) no vigésimo oitavo dia, e nos meios M+NPK e WC foi de 4,1 × 105 células.mL–1 e 2,1 × 105.celulas.mL–1 no vigésimo sexto dia, respectivamente. Os teores de clorofila-a foram significativamente maiores (p<0,05) em meio NPK (41-102 g.L–1) quando comparado aos meios WC e M+NPK (14-61 g.L–1). O teor de astaxantina foi menor que 0,04 mg.L–1. O custo de produção de 10-L de H. pluvialis foi baixo em todos os meios sendo que os meios NPK e M+NPK apresentaram uma redução de custos de 65% e 82%, respectivamente, quando comparados ao meio comercial. O meio contendo fertilizante e o de macrófita (E. crassipes) obtiveram resultados eficientes em cultivo estático, com alta densidade celular de H. pluvialis. O meio NPK e o de macrófita (E. crassipes) demonstraram ser uma alternativa adequada para substituir o meio comercial (WC).

Utilización de espectrometría de masas para la identificación precoz de hemocultivos positivos: Validación de un algoritmo local / Use of mass spectrometry for rapid diagnosis of positive blood cultures: Validation of a local algorythm

Mass spectrometry (MS) is used in identification of positive blood culture, a contribution in the clinical management of septic patients. The protocol is labor intensive and disrupts the normal workflow in a clinical laboratory. We intended to make rapid diagnosis by using MS directly from shortly incubated blood agar plates (4 to 6 hours) comparing with results of the conventional method (MC). We worked in parallel 145 positive blood cultures, with correct identification by short method in 79% of cases. We observed better yield with non carbon bottles and with gramnegative rods. With this information we designed a rapid identification algorithm using MS, which allows advancing diagnosis in 12-16 hours, without increasing to the costs or work load, since extraction protocol is not used.

Espectrometría de masas (EM) es utilizada en identificación de hemocultivo positivo (HMP), constituyendo un aporte en el manejo clínico de paciente séptico. El protocolo de identificación directa es ultra laborioso, interrumpiendo el flujo de trabajo normal del laboratorio. Con el objetivo de hacer identificación rápida, utilizamos EM a partir de placas de agar sangre con incubación breve (4 a 6 h), comparando resultados con el método convencional en HMP. Se trabajó 145 frascos de HMP, identificando correctamente por método acortado 79% de los microorganismos. El rendimiento fue mejor en frascos sin carbón activado y en la identificación de bacilos gramnegativo. Con esta información, diseñamos algoritmo para la identificación precoz de hemocultivo positivo mediante EM, procesando a ciegas a las 4 a 6 h de incubación, lo que permite adelantar el diagnóstico en 12-16 h respecto del método tradicional, sin aumentar los costos ni la carga de trabajo, ya que no se utiliza protocolo de extracción.

Estudo do reaproveitamento de meio no cultivo de Arthrospira (Spirulina) platensis / Study of reuse of Arthrospira (Spirulina) platensis cultivation medium

Arthrosphira (Spirulina) platensis apresenta substâncias de interesse nas indústrias alimentícia, farmacêutica e cosmética. A produção industrial envolve uma quantidade muito grande de água e sua viabilidade deve contemplar o reuso do meio, visando uma diminuição de custos com nutrientes, bem como da poluição ambiental, tornando-se assim um processo sustentável. O presente trabalho teve como objetivo principal a avaliação do reaproveitamento do meio no cultivo de A. platensis usando tratamentos físico-químicos de floculação e adsorção. Para tanto, tal cianobactéria foi cultivada em fotobiorreator (FBR) tubular em processos de batelada alimentada e contínuo em intensidade luminosa de 120 µmol fótons m-2 s-1, sob controle de pH. Foram desenvolvidas técnicas de tratamento de meio de cultivo proveniente de processo descontínuo alimentado de A. platensis para a remoção de matéria orgânica (MO) e pigmentos (60 - 96 %), permitindo assim seu reuso em novos cultivos. A. platensis foi cultivada nos meios tratados utilizando frascos Erlenmeyer, com avaliação de parâmetros como concentração celular máxima (Xm), conteúdo de clorofila-a (Chl) e conteúdo de proteína na biomassa seca (PTN). No processo simultâneo de floculação e adsorção com carvão ativado em pó (CAP), foram testados dois agentes floculantes, cloreto férrico (F) e sulfato férrico (S) bem como diferentes tempos de contato. No processo simultâneo de floculação com F e adsorção com CAP, as condições ótimas foram: CAP = 24,4 mg L-1 e F = 20,3 mg L-1durante 30,4 min de tempo de contato; com obtenção de: Xm = 4893 ± 33 mg L-1, Chl = 24,3 ± 0,1 mg g-1, PTN = 36,1 ± 0,6 %. As condições ótimas de tratamento simultâneo de floculação com S e adsorção com CAP foram: CAP = 40,0 mg L-1 e S = 32,8 mg L-1 durante 36,1 min de tempo de contato, com obtenção de: Xm = 4863 ± 64 mg L-1, Chl = 24,5 ± 0,6 mg g-1, PTN = 60,1 ± 0,6 %. No processo sequencial de floculação com F seguido de adsorção com carvão ativado granulado (CAG), as condições ótimas foram atingidas com: CAG = 108,4 g e F = 10,0 mg L-1 durante 30,8 min de tempo de residência; obtendo-se: Xm = 3140 ± 77 mg L-1, Chl = 35,4 ± 0,2 mg g-1, PTN = 44,9 ± 0,0 %. Adicionalmente, os meios tratados nessas condições ótimas de cada tratamento, também foram testados em FBR tubulares, atingindo valores de Xm, Chl e PTN maiores do que os obtidos com meio padrão. Além disso, o processo simultâneo de cultivo celular em FBR tubulares e adsorção contínua do meio de cultivo exaurido em coluna de CAG removeu 51 - 79 % de MO e pigmentos. Foi demonstrado que uma proporção de 75 % de meio tratado no meio de alimentação não produz diminuição significativa de produtividade celular (PX) e os resultados foram: concentração celular em estado estacionário (Xs) de 1568 ± 15 mg L-1, PX = 941 mg L-1 d-1, PTN = 42,0 ± 0,6 %, com diminuição de 65 % no custo de meio de cultivo. Por fim, conclui-se que é viável a utilização de processos físico-químicos no tratamento de meio a ser reaproveitado no cultivo de A. platensis, inclusive em FBR tubulares, com apreciável incremento de clorofila-a e proteínas na biomassa obtida em meio tratado

Arthrospira (Spirulina) platensis have compounds of interest in the food, pharmaceutical and cosmetic industries. Industrial production involves high volumes of water and its viability should contemplate medium reuse, aiming to reduce not only nutrient costs, but also environmental pollution, thus becoming a sustainable process. This work had as main objective the evaluation of A. platensis culture medium reuse through the physicochemical treatments flocculation and adsorption. Thus, this cyanobacterium was cultivated in tubular photobioreactor (PBR) by fed-batch and continuous processes at light intensity 120 µmol photons m-2 s-1 under pH control. Treatment techniques were developed for culture medium from fed-batch process to properly removal of organic matter (OM) and pigments (60 - 96 %), thus allowing its reuse in new cultures. A. platensis was cultivated in treated medium using Erlenmeyer flasks, with the evaluation of parameters such as maximum cell concentration (Xm), chlorophyll content (Chl) and protein content in dry biomass (PTN). For simultaneous flocculation and adsorption with powdered activated carbon (PAC), two flocculants were used: ferric chloride (F) and ferric sulfate (S), as well as different contact times. In the simultaneous process of F flocculation and PAC adsorption, optimum conditions were: PAC = 24.4 mg L-1 and F = 20.3 mg L-1 for 30.4 min contact time; results were: Xm = 4893 ± 33 mg L-1, Chl = 24.3 ± 0.1 mg g-1, PTN = 36.1 ± 0.6 %. Optimal conditions in the simultaneous process of S flocculation and PAC adsorption were: PAC = 40.0 mg L-1 and S = 32.8 mg L-1 for 36.1 min contact time; results were: Xm = 4863 ± 64 mg L-1, Chl = 24.5 ± 0.6 mg g-1, PTN = 60.1 ± 0.6 %. In the sequential process of F flocculation followed by adsorption with granular activated carbon (GAC), optimal conditions were reached at GAC = 108.4 g and F = 10.0 mg L-1 for 30.8 min of residence time, at which Xm = 3140 ± 77 mg L-1, Chl = 35.4 ± 0.2 mg g-1 and PTNPTN = 44.9 ± 0.0 % were obtained. Moreover, medium treated at each optimal condition were also tested in tubular PBRs, reaching values of Xm, Chl and PTN higher than those obtained with standard medium. Furthermore, the simultaneous process of cell cultivation in tubular PBR and continuous adsorption of spent cultivation medium through GAC column removed 51 - 79 % of OM and pigments. It was showed that 75 % of treated medium in the feed medium does not cause significant decrease in cell productivity (PX) and results were: steady-state cell concentration (Xs) = 1568 ± 15 mg L-1, PX = 941 mg L-1 d-1, PTN = 42.0 ± 0.6 %, with 65 % reduction in medium price. At last, it can be inferred that the use of physicochemical processes in medium treatment is feasible for reuse in A. platensis cultivation, including that in tubular PBR, leading to considerable increase in chlorophyll and protein contents of the biomass obtained with treated medium

A new spectrophotometric method for quantification of potassium solubilized by bacterial cultures.

A new spectrophotometric method was developed for the quantification of potassium in the culture broth supernatant of K-solubilizing bacteria. The standard curve of potassium with the new method, which is based on the measurement of cobalt, showed a regression coefficient (R2) of 0.998. The quantification values of potassium obtained with flame photometric method and the newly developed method showed a significant correlation (r) of 0.978. The new method depends on the precipitation of sodium cobaltinitrite with solubilized potassium in liquid medium as potassium sodium cobaltinitrite, which develops bluish green colour by the addition of conc. HCl. The intensity of developed colour can be recorded at 623 nm. This method involves less number of steps, is easy and time saving, and can be used for the reliable estimation of available potassium in culture broth supernatant of K-solubilizing bacteria.

Efeito de diferentes meios nutritivos e fitorreguladores visando à otimização da calogênese de Annona mucosa (Jacq. ) / Effect of different culture media and plant growth regulators for the optimization of callus induction of Annona mucosa (Jacq. )

Annona mucosa é uma árvore frutífera da família Annonaceae, produtora de importantes metabólitos secundários de interesse medicinal, como lignanas, acetogeninas e alcaloides. A cultura in vitro de calos representa um importante recurso para a produção contínua de metabólitos, viabilizando a conservação da biodiversidade química e a obtenção controlada de material para estudos biológicos e fitoquímicos. O objetivo deste trabalho foi otimizar a produção de calos friáveis de A. mucosa, avaliando o efeito de diferentes meios nutritivos e fitorreguladores. Segmentos de folha e de hipocótilo de plântulas germinadas in vivo foram utilizados como explantes e inoculados nos meios de cultura MS, WPM e B5 suplementados com picloram (2 - 20µM) isolado ou combinado com as citocininas BAP, KIN ou TDZ (0,2 - 1µM). As culturas foram mantidas a 26±2ºC, no escuro, com subcultivos mensais. A produção de calos foi avaliada por aferição do peso dos calos, após 90 dias. Em todos os tratamentos na presença da auxina picloram, o cultivo de hipocótilos resultou em maior porcentagem de formação de calos, sobretudo no meio de cultura WPM. A associação com TDZ produziu massa calogênica friável altamente proliferativa e ausente de oxidação, alcançando valores superiores àqueles obtidos em trabalhos prévios com a espécie. Os resultados viabilizam o uso do material em suspensões celulares e posterior caracterização fitoquímica para a exploração da produção in vitro de metabólitos da espécie.

The Annona mucosa is a fruit tree of the Annonaceae family that produces a range of secondary metabolites of medicinal interest, such as lignans, acetogenins and alkaloids. The callus culture represents a renewable source of valuable medicinal compounds and controlled supply of material for biological and phytochemical studies. Therefore, this study was carried out to investigate the effects of three nutrient media, different concentrations of picloram and cytokinin types, in order to optimize the biomass yield and friability of calluses of A. mucosa. Leaf and hypocotyl segments from seedlings produced from in vivo seed germination were used as explants, which were inoculated in MS, WPM and B5 culture media supplemented with picloram (2-20µM) only or in addition to the cytokinins BAP, KIN or TDZ (0,2 - 1µM ). Cultures were maintained at 26±2ºC in the dark, with monthly subcultures. After 90 days, biomass production was evaluated. In all treatments, hypocotyl explants provided the highest percentage of callus formation, particularly in WPM. The association with TDZ produced highly proliferative friable callus, with no oxidation, reaching higher values than the previous works with this species. The results enable the use of the calluses produced in cell suspensions and the subsequent phytochemical characterization, in order to explore the in vitro production of metabolites of the species.

Estabilidad de la adenosina deaminasa en diferentes medios de transporte del líquido pleural / Adenosine deaminase stability in pleural effusions defferent means of transport

La determinación de los niveles de la adenosina deaminasa en el líquido pleural es sensible y específica para la tuberculosis pleural. La adenosina deaminasa en el líquido pleural disminuye con el tiempo a temperatura ambiente. El objetivo de este estudio es demostrar si existe diferencia en los valores de la adenosina deaminasa en líquidos pleurales en cuatro medios diferentes de transporte (hielo, citrato de sodio, heparina y ninguna sustancia, química añadida). Se determinaron los niveles de la enzima en ochenta y ocho (88) muestras de líquido pleural procedentes de 22 pacientes con derrames pleurales no diagnosticados. Se demostró la concordancia diagnóstica entre los diferentes medios de transporte. No se demostró diferencia significativa entre los niveles de la adenosina deaminasa en cada una de los diferentes medios de transporte hasta dos (2) horas posterior a su recolección. Se recomienda enviar las muestras de líquido pleural con el conservativo adecuado o con ácido etilen diamino tetracético de rutina en nuestro país

The determination of the levels of adenosine deaminase in pleural fluid is sensitive and specific for pleural tuberculosis. Adenosine deaminase in pleural fluid decreases over time at room temperature. The objective of this study is to demostrate if there is difference on the average values of adenosine deaminasa in pleural fluids in four different means of transport (ice, sodium citrate, heparin and no added chemical substance). The levels of the enzyme in eighty-eight (88) pleural fluid samples from 22 patients with diagnosed pleural effusions were determined. We demonstrated diagnostic concodance between the differents modes of transport. No significant difference is between the levels of adenosine deaminase in each of the different means of transport up to two (2) hours after collection. It is recommended to send by routine in our country samples of pleural fluid with the right conservative or Acid etilen diamino tetracetic

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

Diversity of leaf endophytic fungi in mangrove plants of Northeast Brazil

With the aim of increasing the knowledge about endophytic fungi, a group of microorganisms with high biotechnological potential and a valuable source of useful metabolites, a survey in leaves of mangrove plants (Avicennia schaueriana, Laguncularia racemosa, and Rhizophora mangle) was performed at the Itamaracá Island, PE, Brazil. Leaves were collected, during two seasons, dry and rainy, superficially sterilized and fragments maintained in Petri dishes with Potato dextrose agar (PDA) at 28º ± 2º C until isolation of the fungi. Fourty taxa were isolated: 25 species representing 19 genera and 15 morphotypes determined as Mycelia sterilia. Leaves of L. racemosa hosted the highest number of colony forming units (CFU) and taxa. Guignardia sp. and Colletotrichum gloeosporioides were the most frequently isolated, while Glomerella cingulata was the only species found in association with the three host plants. The proportional importance of each fungus differed among hosts. The similarity of fungi species between the two seasons reached only 4.2%, and that between the hosts was also low, with the maximum (A. schaueriana x L. racemosa) reaching 24.2%. Sphaerosporium, as well as Chloridium virescens var. virescens, Microsphaeropsis arundinis, Penicillium pinophilum, Periconia cambrensis, Phoma herbarum, P. diachenii, P. obscurans, Sordaria prolifica and Torula elisii are reported for the first time as endophytic in tropical regions.

Análisis de patrones morfológicos y anatómicos en la embriogénesis somática del banano Williams (AAA) / Analysis of the morphological and anatomical patterns in the somatic embryogenesis of Williams banana (AAA)

La embriogénesis somática representa una herramienta esencial en el mejoramiento genético y en la micropropagación clonal masiva de bananos mejorados. En el presente trabajo se analizaron los patrones morfológicos y anatómicos que ocurren durante la embriogénesis somática del banano Williams, dirigidos a conocer y mejorar este proceso. En la investigación se establecieron suspensiones celulares embriogénicas (SCE) a partir de callo embriogénico obtenido de manos florales inmaduras masculinas, las cuales originaron abundantes embriones que regeneraron plantas. Hacia los tres meses de cultivo se detectaron embriones somáticos (ES) primarios color blanco-crema en las manos florales de los nudos nueve a doce, contados a partir del ápice floral. Al cuarto mes estos ES primarios dieron origen al callo embriogénico, de color blanco crema, estructura granular, con abundantes ES torpedo en su periferia y con una organización celular en tres diferentes zonas. De este callo se cultivaron porciones pequeñas con ES torpedo en medio de multiplicación durante dos meses, dando origen a la SCE I. La misma se tamizó (250 µm) para establecer la SCE II. El sedimento de células y los agregados celulares embriogénicos de ambas SCE se trasladó a medio de maduración. Transcurridos dos meses los embriones maduros se transfirieron a medio de conversión de embriones, lográndose regenerar plantas completas a partir de las dos semanas. Las SCE produjeron numerosos embriones somáticos maduros y mostraron una buena conversión de embriones a plantas y regeneración de plantas. Este sistema de embriogénesis somática permitió la obtención de plantas funcionales en nueve meses.

Somatic embryogenesis represents an essential tool for the genetic improvement and for the mass clonal micropropagation of the improved banana plant. In this present work morphological and anatomical patterns were analyzed in the somatic embryogenesis of Williams banana, to know and enhance this process. In the investigation embryogenic cell suspensions (ECS) were established from embryogenic callus obtained from floral immature male hands, which gave rise to many somatic embryos that regenerated plants. Towards the three months of culture white-cream primary somatic embryos (SE) were detected in the floral hands of the nodes nine to twelve, counted from the floral apex. At the fourth month this primary SE gave origin to a creamy-white embryogenic callus, with granular structure and abundant SE torpedo on its periphery. Cell organization with three different zones was observed in callus. Small portions of this callus were cultivated in the multiplication medium for two months, to originate ECS I. This ECS was filtered through a mesh (250 µm pore size) to establish the ECS II. The sediment of embryogenic cells and cell clusters of the ECS were moved to maturation media. After two months the mature embryos were transferred to conversion medium, and two weeks later, whole plants were developed. The ECS produced numerous mature SE, which showed good conversion of embryos into plants and plant regeneration. This system of somatic embryogenesis permitted the mass production of functional plants in nine months.

Interspecific variation of the bacterial community structure in the phyllosphere of the three major plant components of mangrove forests

Mangrove forests encompass a group of trees species that inhabit the intertidal zones, where soil is characterized by the high salinity and low availability of oxygen. The phyllosphere of these trees represent the habitat provided on the aboveground parts of plants, supporting in a global scale, a large and complex microbial community. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonizers, which is influenced by numerous environmental factors in addition to leaf physical and chemical properties. Here, a combination of culture-base methods with PCR-DGGE was applied to test whether local or plant specific factors shape the bacterial community of the phyllosphere from three plant species (Avicenia shaueriana, Laguncularia racemosa and Rhizophora mangle), found in two mangroves. The number of bacteria in the phyllosphere of these plants varied between 3.62 x 10(4) in A. schaeriana and 6.26 x 10³ in R. mangle. The results obtained by PCR-DGGE and isolation approaches were congruent and demonstrated that each plant species harbor specific bacterial communities in their leaves surfaces. Moreover, the ordination of environmental factors (mangrove and plant species), by redundancy analysis (RDA), also indicated that the selection exerted by plant species is higher than mangrove location on bacterial communities at phyllosphere.

In vitro antibacterial activity of some antihistaminics belonging to different groups against multi-drug resistant clinical isolates

Antihistaminics are widely used for various indications during microbial infection. Hence, this paper investigates the antimicrobial activities of 10 antihistaminics belonging to both old and new generations using multiresistant Gram-positive and Gram-negative clinical isolates. The bacteriostatic activity of antihistaminics was investigated by determining their MIC both by broth and agar dilution techniques against 29 bacterial strains. Azelastine, cyproheptadine, mequitazine and promethazine were the most active among the tested drugs. Diphenhydramine and cetirizine possessed weaker activity whereas doxylamine, fexofenadine and loratadine were inactive even at the highest tested concentration (1 mg/ml). The MIC of meclozine could not be determined as it precipitated with the used culture media. The MBC values of antihistaminics were almost identical to the corresponding MIC values. The bactericidal activity of antihistaminics was also studied by the viable count technique in sterile saline solution. Evident killing effects were exerted by mequitazine, meclozine, azelastine and cyproheptadine. Moreover, the dynamics of bactericidal activity of azelastine were studied by the viable count technique in nutrient broth. This activity was found to be concentration-dependant. This effect was reduced on increasing the inoculum size while it was increased on raising the pH. The post-antimicrobial effect of 100 fg/ml azelastine was also determined and reached up to 3.36 h.

Replacement of fetal calf serum by human serum as supplementation for human fibroblast culture / Substituição de soro bovino fetal por soro humano como suplemento para cultura de fibroblastos humanos

INTRODUCTION: Fetal calf serum (FCS) is commonly used as a supplement in the culture medium for fibroblast cells. This supplementation is far from ideal as sample quality varies from batch to batch and the composition of FCS is not completely known. In addition, FCS may be contaminated with viruses and/or prions and may also cause adverse immunologic responses in humans. Due to these facts, a worldwide effort is being made to find alternatives for xenobiotic elements in cell cultures. Human serum could be a safer alternative, especially for clinical application. METHODS: We investigated human serum as a substitute for FCS in human fibroblast culture. Fresh human serum was obtained from 10 healthy volunteers. Fibroblasts were cultivated in multiwell plates containing either Dulbecco's modified Eagle's medium (DMEM) plus 10 percent FCS (D10) or DMEM plus 10 percent human serum (D10H). Cell counts were obtained between 24 and 264 hours of cultivation; results were expressed as the mean number of cells ± standard error of the mean to create cell proliferation curves. RESULTS: There was no statistical difference in fibroblast proliferation between the two groups. Human serum supported human fibroblast growth and proliferation, suggesting that it may be a potential substitute for FCS in human cell culture. Cells cultivated with human serum presented a different morphology, appearing smaller and more rounded as compared to cells cultivated in D10. CONCLUSIONS: These results demonstrate that human serum can be substituted for FCS in human fibroblasts culture and that fibroblasts cultivated in the presence of human serum have a morphology that is similar to in vivo fibroblasts.

INTRODUÇÃO: Soro bovino fetal (SBF) é comumente usado como suplemento no meio de cultura para cultivar fibroblastos. Essa forma de suplementação, porém, não é ideal, pois a qualidade das amostras de SBF é variada e sua composição não é completamente conhecida. Além disso, o SBF pode apresentar contaminação por vírus e príons ou causar complicações imunológicas. Assim, a comunidade científica tem buscado alternativas ao uso de elementos xenobióticos em cultura celular. O soro humano pode ser uma dessas alternativas, principalmente para aplicação clínica. MÉTODO: Soro humano, obtido de sangue de 10 voluntários saudáveis submetidos a avaliação sorológica prévia, foi testado como substituto do SBF em cultura de fibroblastos humanos. As células foram cultivadas em placas multipoços, contendo Dulbecco's Modified Eagle's Medium (DMEM) mais 10 por cento de SBF (D10) ou DMEM mais 10 por cento de sroro humano (D10H). Entre 24 e 264 horas de exposição aos meios testados, as células foram contadas e os resultados foram expressos em média ± erro padrão da média, para obtenção de curvas de proliferação celular. RESULTADOS: Não houve diferença estatística entre os grupos de proliferação. Fibroblastos na presença de soro humano aparentavam ser menores e mais arredondados em comparação àqueles mantidos em D10. CONCLUSÕES: Os resultados permitem inferir que o soro humano pode substituir o SBF em cultura de fibroblastos e que fibroblastos cultivados em meio suplementado por soro humano apresentam morfologia mais semelhante àqueles in vivo.

Atividade antimicrobiana de produtos naturais frente a bactérias formadoras do biofilme dentário / Antimicrobial activity of herbal products against dental biofilm forming bacteria

Objeti vo: Verifi car, in vitro, o efeito anti microbiano do pólen e dos extratos alcoólico e aquoso da própolis em suas formas pura e diluídas sobre cepas de referência Streptococcus mutans ATCC25175, Streptococcus salivarius ATCC 7073, Streptococcus mitis ATCC 903 e Lactobacillus casei ATCC 9595 pela determinação da Diluição Inibitória Máxima (DIM). Método: Uti lizou-se a clorexidina como controle positivo e água desti lada e álcool de cereais 70% como controles negativos. Efetuou-se a diluição das soluções de 1:1 até 1:64dos extratos alcoólico e aquoso da própolis diluídos em álcool 70% e água desti lada, respecti vamente. O pólen foi diluído em álcool, por ser uma substancia apolar, nas concentrações de 5% (quanti dade presente na composição química da própolis) e 50%. Cada linhagem bacteriana foi reati vada em caldo nutritivo Brain Heart Infusion (BHI) e semeadas as placas com auxílio de swabs, procedendo-se com testes de susceti bilidade, em duplicata, por meio do método da difusão em ágar e técnicado ágar recortado. Em seguida, foram incubadas a 37°C, em microaerofilia, por 48h. Resultados: Constatou-se que todas as diluições da própolis alcoólica inibiram o crescimento bacteriano enquanto a própolis aquosa mostrou os menores resultados tendo efeitoapenas sobre S. miti s na forma pura e nas diluições de 1:1 até 1:4. O pólen a 5% foi efi ciente sobre todas as bactérias, porém o pólen a 50% teve ação apenas sobre S. mitis. Os controlesnegati vos não apresentaram ati vidade. Conclusão: Apesar da própolis e do pólen apresentarem atividade anti microbiana contra as cepas de referência superiorà do placebo, esta, porém, foi inferior à da clorexidina.

Objective: To evaluate, in vitro, the antimicrobial effect of pollen and alcoholic and aqueous propolis extracts in their pure and diluted forms against reference strains Streptococcus mutans ATCC 25175, Streptococcus salivarius ATCC 7073, Streptococcus mitis ATCC 903 and Lactobacillus casei ATCC 9595, by determination of Maximum Inhibitory Dilution (MID). Methods: Chlorhexidine was used as a positive control and distilled water and 70% grain alcohol as negative controls. The alcoholic and aqueous propolis extracts were subjected to dilutions from 1:1 to 1:64 in 70% alcohol and distilled water, respectively. For being an apolar substance, pollen was diluted in alcohol at the concentrations of 5% (amount present in the chemical composition of propolis) and 50%. Each bacterial strain was reactivated in Brain Heart Infusion (BHI) broth and seeded onto plates with swabs, and the susceptibility tests were performed in duplicate by the agar diffusion method using the agar well technique. Next, the plates were incubated at 37øC in microaerophilia during 48 hours. Results: All dilutions of alcoholic propolis extract inhibited the bacterial growth while the aqueous propolis extract showed less efficient results, being effective only against S. mitis in its pure form and in the 1:1 to 1:4 dilutions. Pollen at 5% was efficient against all bacteria, but pollen at 5% had action only against S. mitis. The negative controls did not present antimicrobial activity. Conclusion: The antimicrobial activity of propolis and pollen against the reference strains was higher than that of placebo but lower than that of chlorhexidine.

Determination of free cisplatin in medium by differential pulse polarography after ultrasound and cisplatin treatment of a cancer cell culture.

The in vitro study was carried out for detection of the cisplatin in free form and in culture medium, depending on various conditions of sonodynamic human ovarian cancer cells A2780 treatment by differential pulse polarography (DPP). For sonodynamic treatment, we used cisplatin alone and combined cisplatin/ultrasound treatments. The ultrasound exposure intensity of 1.0 and 2.0 W∙cm-2 in far field for incubation periods 1, 24 and 48 h was used. The parameters of DPP measurements were - 1 s drop time, 5 mV.s-1 voltage scan rate, 50 mV modulation amplitude and negative scanning direction; platinum wire served as counter electrode and Ag|AgCl|3 M KCl as reference electrode. The results showed the dependence of free platinum quantities in culture medium on incubation time and treatment protocol. We found difference in concentration of free cisplatin between conventional application of cisplatin and sonodynamic treatment. The sonodynamic combined treatment of cisplatin and ultrasound field showed a higher cisplatin content in the culture medium than cisplatin treatment alone; a difference of 20% was observed for incubation time 48 h. The results also showed the influence of a time sequence of ultrasound and cytostatics in the sonodynamic treatment. The highest amount of free cisplatin in the solution was found for primary application of cisplatin and the subsequent ultrasound exposure. The quantity of free cisplatin increased with time, namely for time intervals 1-24 h. There was no difference between the DPP signal of cisplatin in reaction mixture containing cells in small quantities and micro-filtered mixture without cells. Thus, the DPP method is suitable for the detection and quantification of free cisplatin in the culture medium of cell suspension. Ultrasound field can be important factor during cytostatic therapy.