Efficient animal-serum free 3D cultivation method for adult human neural crest-derived stem cell therapeutics

Due to their broad differentiation potential and their persistence into adulthood, human neural crest-derived stem cells (NCSCs) harbour great potential for autologous cellular therapies, which include the treatment of neurodegenerative diseases and replacement of complex tissues containing various cell types, as in the case of musculoskeletal injuries. The use of serum-free approaches often results in insufficient proliferation of stem cells and foetal calf serum implicates the use of xenogenic medium components. Thus, there is much need for alternative cultivation strategies. In this study we describe for the first time a novel, human blood plasma based semi-solid medium for cultivation of human NCSCs. We cultivated human neural crest-derived inferior turbinate stem cells (ITSCs) within a blood plasma matrix, where they revealed higher proliferation rates compared to a standard serum-free approach. Three-dimensionality of the matrix was investigated using helium ion microscopy. ITSCs grew within the matrix as revealed by laser scanning microscopy. Genetic stability and maintenance of stemness characteristics were assured in 3D cultivated ITSCs, as demonstrated by unchanged expression profile and the capability for self-renewal. ITSCs pre-cultivated in the 3D matrix differentiated efficiently into ectodermal and mesodermal cell types, particularly including osteogenic cell types. Furthermore, ITSCs cultivated as described here could be easily infected with lentiviruses directly in substrate for potential tracing or gene therapeutic approaches. Taken together, the use of human blood plasma as an additive for a completely defined medium points towards a personalisable and autologous cultivation of human neural crest-derived stem cells under clinical grade conditions.

Rejection thresholds (RjT) of sweet likers and dislikers

Sweetness is generally a desirable taste, however consumers can be grouped into sweet likers and dislikers according to optimally preferred sucrose concentrations. Understanding the levels of sweetness in products that are acceptable and unacceptable to both consumer groups is important to product development and for influencing dietary habits. The concentrations at which sucrose decreases liking (the rejection threshold; RjT) in liquid and semi-solid matrices were investigated in this study. Thirty six consumers rated their liking of 5 sucrose aqueous solutions; this identified 36% sweet likers (SL) whose liking ratings increased with increasing sucrose and 64% sweet dislikers (SD) whose liking ratings decreased above 6% (w/v) sucrose. We hypothesized that SL and SD would have different RjT for sucrose in products. This was tested by preparing 8 levels of sucrose in orange juice and orange jelly and presenting each against the lowest level in forced choice preference tests. In orange juice, as sucrose increased from 33g/L to 75g/L the proportion of people preferring the sweeter sample increased in both groups. However, at higher sucrose levels, the proportion of consumers preferring the sweet sample decreased. For SD, a RjT was reached at 380 g/L, whereas a significant RjT for SL was not reached. RjT in jelly were not reached as the sweetness in orange jelly was significantly lower than for orange juice (p<0.001). Despite statistically significant differences in rated sweetness between SL and SD (p=0.019), the extent of difference between the two groups was minor. The results implied that sweet liker status was not substantially related to differences in sweetness perception. Self-reported dietary intake of carbohydrate, sugars and sucrose were not significantly affected by sweet liker status. However the failure to find an effect may be due to the small sample size and future studies within a larger, more representative population sample are justifiable from the results of this study.

Seeding of single crystal superalloys--Role of seed melt-back on casting defects

Single crystal seeds of the nickel-base superalloy CMSX-4 have been partially melted in a temperature gradient and then quenched. Small islands of random orientation are observed throughout the melted-back semi-solid. These random orientations appear to be pinched-off secondary dendrite arms, but there is no evidence that they are transported ahead of the dendrite tips to nucleate stray grains during directional solidification.

Palma forrageira (Opuntia ficus indica e Nopalea cochenillifera) como matéria-prima para produção de etanol celulósico e enzimas celulolíticas

The need for new sources of energy and the concern about the environment have pushed the search for renewable energy sources such as ethanol. The use of lignocellulosic biomass as substrate appears as an important alternative because of the abundance of this raw material and for it does not compete with food production. However, the process still meets difficulties of implementation, including the cost for production of enzymes that degrade cellulose to fermentable sugars. The aim of this study was to evaluate the behavior of the species of cactus pear Opuntia ficus indica and Nopalea cochenillifera, commonly found in northeastern Brazil, as raw materials for the production of: 1) cellulosic ethanol by simultaneous saccharification and fermentation (SSF) process, using two different strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11), and 2) cellulolytic enzymes by semi-solid state fermentation (SSSF) using the filamentous fungus Penicillium chrysogenum. Before alcoholic fermentation process, the material was conditioned and pretreated by three different strategies: alkaline hydrogen peroxide, alkaline using NaOH and acid using H2SO4 followed by alkaline delignification with NaOH. Analysis of composition, crystallinity and enzymatic digestibility were carried out with the material before and after pretreatment. In addition, scanning electron microscopy images were used to compare qualitatively the material and observe the effects of pretreatments. An experimental design 2² with triplicate at the central point was used to evaluate the influence of temperature (30, 40 and 45 °C) and the initial charge of substrate (3, 4 and 5% cellulose) in the SSF process using the material obtained through the best condition and testing both strains of S. cerevisiae, one of them flocculent (LNF CA-11). For cellulase production, the filamentous fungus P. chrysogenum was tested with N. cochenillifera in the raw condition (without pretreatment) and pretrated hydrothermically, varying the pH of the fermentative medium (3, 5 and 7). The characterization of cactus pear resulted in 31.55% cellulose, 17.12% hemicellulose and 10.25% lignin for N. cochenillifera and 34.86% cellulose, 19.97% hemicellulose and 15.72% lignin for O. ficus indica. It has also been determined, to N. cochenillifera and O. ficus indica, the content of pectin (5.44% and 5.55% of calcium pectate, respectively), extractives (26.90% and 9.69%, respectively) and ashes (5.40% and 5.95%). Pretreatment using alkaline hydrogen peroxide resulted in the best cellulose recovery results (86.16% for N. cochenillifera and 93.59% for O. ficus indica) and delignification (48.79% and 23.84% for N. cochenillifera and O. ficus indica, respectively). This pretreatment was also the only one which did not increase the crystallinity index of the samples, in the case of O. ficus indica. However, when analyzing the enzymatic digestibility of cellulose, alkali pretreatment was the one which showed the best yields and therefore it was chosen for the tests in SSF. The experiments showed higher yield of conversion of cellulose to ethanol by PE-2 strain using the pretreated N. cochenillifera (93.81%) at 40 °C using 4% initial charge of cellulose. N. cochenillifera gave better yields than O. ficus indica and PE-2 strain showed better performance than CA-11. N. cochenillifera proved to be a substrate that can be used in the SSSF for enzymes production, reaching values of 1.00 U/g of CMCase and 0.85 FPU/g. The pretreatment was not effective to increase the enzymatic activity values

Properties of a new fungal b-galactosidase with potential application in the dairy industry

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

Screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by Bacillus sp

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

Síntese e processamento de cerâmicas em forno de microondas doméstico

O uso de microondas no processamento e obtenção de materiais tem adquirido nos últimos anos crescente interesse por parte de diversas áreas do conhecimento como a química e a engenharia de materiais. Neste sentido, aparatos especialmente projetados têm começado a ser descritos na literatura como reatores e câmaras de processamento a microondas visando aplicação na pesquisa e na indústria. em particular o uso de fornos de microondas domésticos em atividades científicas tem se mostrado de interesse dado uma série de novas aplicações, simplicidade e baixo custo. O presente trabalho reporta um dispositivo desenvolvido a partir de um forno de microondas doméstico, capaz de efetuar sínteses e processamentos de sólidos mesmo quando os materiais envolvidos não possuem susceptibilidade as microondas. O novo dispositivo tem sido utilizado com sucesso na síntese de óxidos metálicos e, em especial na sinterização e ordenamento de redes cristalinas de filmes finos.

Nutritional-requirements of Cryptococcus-laurentii in sugar-cane vinasse medium

The growth of the yeast Cryptococcus laurentii in complete broth for yeast and in liquid and semi-solid sugar cane vinasse media supplemented with several nitrogen and phosphorus sources was analysed in order to evaluate its potential utilization as biomass producer from vinasse. The trials were performed in a 1-liter fermentor, at 30-degrees-C, under magnetic agitation for 48 hours, and initial pH 5.0 for the liquid media. The parameters analysed were biomass, protein, and final pH. For the semi-solid media, agitated flasks under rotational agitation for 24 hours, at 30-degrees-C, and initial pH 5.0 were used. The results obtained showed that the yeast did not grow well in liquid sugar cane vinasse media, both supplemented or not, in comparison to the complete broth for yeast. The protein content was also lower in liquid sugar cane vinasse media. The medium pH did not alter markedly during the cultivation. The best results were obtained in semi-solid sugar cane vinasse media with supplementation, probably due to the known polysaccharide production by this species, which allows a better survival to solid substrates.

Purification and characterization of two beta-glucosidases from the thermophilic fungus Thermoascus aurantiacus

beta-Glucosidase from the fungus Thermoascus aurantiacus grown oil semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps - ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, beta-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80 degreesC. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl beta-D-glucopyranoside as substrate, K-m, values of 1.17 +/- 0.35 and 1.38 +/- 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

Avaliação de diferentes sistemas de cultivo in vitro para a micropropagação de Psychotria ipecacuanha (Brot.) Stokes

This work was carried out with Psychotria ipecacuanha, a Brazilian medicinal plant the roots of which contain emetine. The main objective was to develop a protocol for the micro-propagation of these species, by testing different culture techniques, the temporary immersion system, and the semi-solid and liquid media systems. In the semi-solid system, experiments were developed in flasks of two different sizes containing MS, B5, and WP media to which were added different growth regulators. Innoculum density was also evaluated. The liquid medium system consisted of MS medium supplemented with different growth regulators. For the temporary immersion system, the MS medium received an addition of 1.5mg/L BAP and 0.5mg/L GA3, and a reverse digital apparatus and vacuum pump were used. The liquid medium system with MS medium supplemented with 1.5mg/L BAP and 0.5mg/L GA3 presented the best results for shoot proliferation in a period of 30 days in culture (2.37 ± 0.32 shoots/explant). Cultures carried out for 90 days in the semi-solid system, using 8.5 × 5.5cm flasks and 3 explants per flask, developed 1.80 ± 0.20 shoots/explant, achieving 3.06 ± 0.51 cm of height adn presented superior survival ratio (96%). Explants cultured in temporary immersion system for 90 days showed 2.30 ± 1.10 shoots/explant achieving a growth of 2.08 ± 0.12 cm and 52% survival.

Purification and characterization of two β-glucosidases from the thermophilic fungus Thermoascus aurantiacus

β-Glucosidase from the fungus Thermoascus aurantiacus grown on semi-solid fermentation medium (using ground corncob as substrate) was partially purified in 5 steps-ultrafiltration, ethanol precipitation, gel filtration and 2 anion exchange chromatography runs, and characterized. After the first anion exchange chromatography, β-glucosidase activity was eluted in 3 peaks (Gl-1, Gl-2, Gl-3). Only the Gl-2 and Gl-3 fractions were adsorbed on the gel matrix. Gl-2 and Gl-3 exhibited optimum pH at 4.5 and 4.0, respectively. The temperature optimum of both glucosidases was at 75-80°C. The pH stability of Gl-2 (4.0-9.0) was higher than Gl-3 (5.5-8.5); both enzyme activities showed similar patterns of thermostability. Under conditions of denaturing gel chromatography the molar mass of Gl-2 and Gl-3 was 175 and 157 kDa, respectively. Using 4-nitrophenyl β-D-glucopyranoside as substrate, Km values of 1.17 ± 0.35 and 1.38 ± 0.86 mmol/L were determined for Gl-2 and Gl-3, respectively. Both enzymes were inhibited by Ag+ and stimulated by Ca2+.

In vitro propagation of Maytenus ilicifolia (Celastraceae) as potential source for antitumoral and antioxidant quinomethide triterpenes production. A rapid quantitative method for their analysis by reverse-phase high-performance liquid chromatography

Cell culture of Maytenus ilicifolia were established in order to produce and to quantify the antitumoral and antioxidant quinonemethide triterpenes. In vitro calli were induced from leaf explants of native plants and cultured in semi-solid medium under controlled conditions of humidity, temperature and photoperiod. The quinonemethide triterpenes showed maximum accumulation in the logarithmic phase growth of the cell culture. A rapid, sensitive and reliable reverse-phase HPLC method was used for quantitative determination of the antitumoral and antioxidant quinonemethide triterpenes, 22β-hydroxymaytenin and maytenin in callus of Maytenus ilicifolia. Well resolved peaks with good detection response and linearity in the range 1.0 - 100 μg/mL were obtained. This quantitative work was performed by an external standard method.

Efeitos da interação e toxicidade das proteínas Cry1 e Vip3Aa de Bacillus thuringiensis, Berliner em Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae)

Pós-graduação em Microbiologia Agropecuária - FCAV

Prevalência do lado preferencial mastigatório e sua relação com a alimentação, a saúde periodontal, o lado preferencial no primeiro ciclo mastigatório e a dominância lateral nas dentições decídua, mista e permanente

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudos de pré-formulação e formulação de Heliotropium indicum (L.) DC (Boraginaceae)

Heliotropium indicum L. (Boraginaceae) é um subarbusto que atinge até 70 cm de altura, é amplamente distribuído geograficamente. A espécie é conhecida como fedegoso na região norte e nordeste. Diante do potencial biológico desta espécie este trabalho objetiva determinar parâmetros de qualidade da droga vegetal até o produto final e avaliar a atividade antimicrobiana de suas folhas, com finalidade de se obter uma formulação fitoterápica semi-sólida. Para isso utilizou-se parâmetros de controle de qualidade físico, químico e físico-químico descritos na Farmacopéia Brasileira e literatura pertinente. A droga vegetal foi classificada como pó grosso, apresentou valores médios de perda por dessecação e cinzas totais de 12,88% e 17,14%, respectivamente. A análise termogravimétrica do pó e do extrato liofilizado mostrou que ambos apresentaram boa estabilidade térmica até 180°C. Os espectros na região do IV mostraram um aumento na intensidade das bandas de absorção do extrato liofilizado, que pode está relacionado à extração dos constituintes químicos da matriz celular. A prospecção química do extrato confirmou a presença de classes de metabólitos secundários já relatados em literatura. A fração clorofórmica sugere a presença de alcalóides pelo teste de precipitação com reagente de Dragendorff. A CCD e a CLAE mostraram uma possível presença de uma mesma substância nas frações alcaloídicas e hexânicas. O extrato bruto de H. indicum L. inibiu o crescimento de Staphylococcus aureus apresentando halos de 12,5 mm±0,707 e 10,5 mm±0,707 para as concentrações de 500 mg/mL e 250 mg/mL, respectivamente. As misturas físicas do extrato com os adjuvantes farmacêuticos, utilizados no desenvolvimento da formulação fitoterápica, não apresentaram incompatibilidade física e não houve modificações significativas no perfil de absorção entre os compostos analisados. A formulação fitoterápica semi-sólida manteve-se estável após sua preparação, após a submissão do gel a força centrípeta e após ação do estresse térmico.