A leukocyte cryopreservation technique for cytogenetic studies

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

MINERAL COMPOSITION of RAW MATERIAL, SUBSTRATE and FRUITING BODIES of Pleurotus ostreatus IN CULTURE

In a culture of a Pleurotus ostreatus (oyster mushroom) strain, macro and micronutrients of the raw material and the initial and spent substrates were evaluated. Substrates were formulated with sawdust from Simarouba amara Aubl. and Ochroma piramidale Cav. ex. Lam., crushed Bactris gasipaes Kunth and crushed Saccharum officinarum (sugar cane). Samples were solubilized by means of acid digestion (nitric-peridrol). Ca, Mg, Fe, Cu, Zn and Mn were determined by atomic absorption spectrophotometry, Na and K by atomic emission, and P by colorimetry. The mineral composition of the fruiting body varied with the substrates, which made possible the production of a fruiting body rich in K, P Mg and Fe. Potassium was the mineral with the highest content in the fruiting body in all substrates tested (36.83-42.18g.kg(-1)). There was an increase of protein and mineral content in the spent substrate in relation to the initial one.

Isolation, Culture and Differentiation of Buffaloes Bone Marrow Mesenchymal Stem Cells Obtained from the Coxal Tuberosity

Currently, much attention has been devoted to the renewal of knowledge about Stem Cells and Cell Therapy in domestic species. In this sense, the present work aimed to develop a methodology for collecting, processing and cultivation of mesenchymal stem cells obtained from bone marrow of coxal tuberosity in buffaloes. The collection was performed using a Komiyashiki needle, which was introduced in the coxal tuberosity and the bone marrow aspirated into a heparinized syringe with the aid of negative pressure. Directly after collection samples were processed at the laboratory at FMVZ - UNESP. The samples took approximately 32 days to reach 80% confluence, when the first passage and differentiation was performed. To confirm the mesenchymal origin, cells were induced to differentiate into adipogenic and osteogenic lineages. Samples showed morphological changes during differentiation protocol, but not all presented production of extracellular deposits of calcium or intracellular fat droplets, observed after staining with Alizarin Red and Oil Red respectively. Compared with the material obtained from other species and processed in the same laboratory, the primary culture was longer. Therefore, more studies are needed to standardize the age of animals used and to test other inducers of cell differentiation.

Mediação pedagógica com apoio de material didático para Educação de Jovens e Adultos

Pós-graduação em Docência para a Educação Básica - FC

Chiral HPLC analysis of donepezil, 5-O-desmethyl donepezil and 6-O-desmethyl donepezil in culture medium: application to fungal biotransformation studies

An high performance liquid chromatography (HPLC) method for the enantioselective determination of donepezil (DPZ), 5-O-desmethyl donepezil (5-ODD), and 6-O-desmethyl donepezil (6-ODD) in Czapek culture medium to be applied to biotransformation studies with fungi is described for the first time. The HPLC analysis was carried out using a Chiralpak AD-H column with hexane/ethanol/methanol (75:20:5, v/v/v) plus 0.3 % triethylamine as mobile phase and UV detection at 270 nm. Sample preparation was carried out by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 100-10,000 ng mL(-1) for each enantiomer of DPZ (r a parts per thousand yenaEuro parts per thousand 0.9985) and of 100-5,000 ng mL(-1) for each enantiomer of 5-ODD (r a parts per thousand yenaEuro parts per thousand 0.9977) and 6-ODD (r a parts per thousand yenaEuro parts per thousand 0.9951). Within-day and between-day precision and accuracy evaluated by relative standard deviations and relative errors, respectively, were lower than 15 % for all analytes. The validated method was used to assess DPZ biotransformation by the fungi Beauveria bassiana American Type Culture Collection (ATCC) 7159 and Cunninghamella elegans ATCC 10028B. Using the fungus B. bassiana ATCC 7159, a predominant formation of (R)-5-ODD was observed while for the fungus C. elegans ATCC 10028B, DPZ was biotransformed to (R)-6-ODD with an enantiomeric excess of 100 %.

Evaluation of 3D cell culture systems for host-pathogen interaction studies

Traditional cell culture models have limitations in extrapolating functional mechanisms that underlie strategies of microbial virulence. Indeed during the infection the pathogens adapt to different tissue-specific environmental factors. The development of in vitro models resembling human tissue physiology might allow the replacement of inaccurate or aberrant animal models. Three-dimensional (3D) cell culture systems are more reliable and more predictive models that can be used for the meaningful dissection of host–pathogen interactions. The lung and gut mucosae often represent the first site of exposure to pathogens and provide a physical barrier against their entry. Within this context, the tracheobronchial and small intestine tract were modelled by tissue engineering approach. The main work was focused on the development and the extensive characterization of a human organotypic airway model, based on a mechanically supported co-culture of normal primary cells. The regained morphological features, the retrieved environmental factors and the presence of specific epithelial subsets resembled the native tissue organization. In addition, the respiratory model enabled the modular insertion of interesting cell types, such as innate immune cells or multipotent stromal cells, showing a functional ability to release pertinent cytokines differentially. Furthermore this model responded imitating known events occurring during the infection by Non-typeable H. influenzae. Epithelial organoid models, mimicking the small intestine tract, were used for a different explorative analysis of tissue-toxicity. Further experiments led to detection of a cell population targeted by C. difficile Toxin A and suggested a role in the impairment of the epithelial homeostasis by the bacterial virulence machinery. The described cell-centered strategy can afford critical insights in the evaluation of the host defence and pathogenic mechanisms. The application of these two models may provide an informing step that more coherently defines relevant molecular interactions happening during the infection.

Human bone chips release of sclerostin and FGF-23 into the culture medium: an in vitro pilot study

Abstract OBJECTIVE: Signaling molecules derived from osteocytes have been proposed as a mechanism by which autografts contribute to bone regeneration. However, there have been no studies that determined the role of osteocytes in bone grafts. MATERIAL AND METHOD: Herein, it was examined whether bone chips and demineralized bone matrix release sclerostin and FGF-23, both of which are highly expressed by osteocytes. RESULTS: Bone grafts from seven donors were placed in culture medium. Immunoassay showed that bone chips released sclerostin (median 1.0 ng/ml) and FGF-23 (median 9.8 relative units/ml) within the first day, with declining levels overtime. Demineralized bone matrix also released detectable amounts of sclerostin into culture medium, while FGF-23 remained close to the detection limit. In vitro expanded isolated bone cells failed to release detectable amounts of sclerostin and FGF-23. CONCLUSION: These results suggest that autografts but also demineralized bone matrix can release signaling molecules that are characteristically produced by osteocytes. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. KEYWORDS: FGF-23; autologous bone; bone grafts; demineralized bone matrix; osteocytes; sclerostin

Integrated studies for the evaluation of conservation treatments on stone material from archaeological sites. Application to the case of Mérida (Spain)

In recent decades archaeological sites have been subject of many interventions. The application of conservation treatments, such us consolidation and protection ones by means of using, for instance, synthetic resins or organosilicic compounds, has been demonstrated inadequate in many cases, and even harmful for the heritage materials [1]. Evaluation studies should be a mandatory task, ideally before and after the intervention, but both tasks are complex and unusual in the case of archaeological heritage. Moreover, there is a general lack of knowledge in the mid and long term effects of these treatments, and how to act when these have resulted in deterioration of the original material. Remains of Roman Augusta Emerita, located in Merida (Spain), have gone through many interventions since the first archaeological campaign, in 1910. Some of them have demonstrated already to be harmful [2], others, more recent, must be evaluated in order to determine its effectiveness and durability, considering that many of these treatments are currently still applied. For this purpose a range of parameters has been measured such as color, surface hardness and roughness, mechanical or hydric properties, porosity, etc. on the original material (granite, marble and mortars mainly), and then the transformations of those same parameters analyzed after treatment, both in situ, in places where a intervention is documented, and in the laboratory, in samples. The study is being conducted both in the laboratory (Petrophysics Laboratory within IGEO) and in situ, on selected archaeological sites of Mérida (Theater and House of Mitreo). The comparison of results in untreated and treated areas of the site, and in treated-untreated samples, allows the distinction of variables that affect the interaction between products and stone material, issues such us effectiveness and durability of treatment and its validation or dismissal.

Integrated Studies for the Evaluation of Conservation Treatments on Stone Material from Archaeological Sites. Application to the Case of Merida (Spain)

The application of conservation treatments, such as consolidation and protection ones, has been demonstrated ineffective in many cases, and even harmful. Evaluation studies should be a mandatory task, ideally before and after the intervention, but both tasks are complex and unusual in the case of archaeological heritage. This study is mainly focused on analyzing changes in petrophysical properties of stone material from archaeological sites of Merida (Spain), evaluating, both on site and in laboratory, effects derived from different conservation treatments applied in past interventions, throughout the integration of different non-destructive techniques (NDT) and portable devices of analysis available at the Institute of Geosciences (CSIC,UCM). These techniques allow, not only assessment of effectiveness and alteration processes, but also monitoring durability of treatments, focused mainly on 1996 intervention in the case of Roman Theater, as well as different punctual interventions from the 90’s until date in the House of Mitreo. Studies carried out on archaeological sites of Merida permit us to compare outcomes and also check limitations in the use of those equipments. In this paper we discuss about the use of some techniques, their integration and limits, for the assessment of conservation treatments, showing some examples of Merida’s case study.