Stingless bee propolis, metformin, and their combination alleviate diabetic cardiomyopathy

Abstract Background and aim: Stingless bee propolis, a resinous compound processed by mandibular secretion of stingless bees, is used for maintenance of hygiene and stability of beehives. Research on stingless bee propolis shows therapeutic properties attributed to polyphenols exhibiting antioxidative, antihyperglycemic and antiischemic effect. However, the cardioprotective effect of stingless bee propolis on diabetic cardiomyopathy is unknown. Methods: Adult male Sprague Dawley rats were randomised to five groups: normal group, diabetic group, diabetic given metformin (DM+M), diabetic given propolis (DM+P) and diabetic given combination therapy (DM+M+P) and treated for four weeks. Body weight, fasting blood glucose, food and water intake were taken weekly. At the end of experiment, biomarkers of oxidative damage were measured in serum and heart tissue. Antioxidants in heart tissue were quantified. Part of left ventricle of heart was processed for histological staining including Haematoxylin and Eosin (H&E) stain for myocyte size and Masson's Trichrome (MT) stain for heart fibrosis and perivascular fibrosis. Results: Propolis alleviated features of diabetic cardiomyopathy such as myocyte hypertrophy, heart fibrosis and perivascular fibrosis associated with improvement in antioxidative status. Conclusion: This study reports beneficial effect of propolis and combination with metformin in alleviating histopathological feature of diabetic cardiomyopathy by modulating antioxidants, making propolis an emerging complementary therapy.

Produção de biscoitos recheados com formação de organogel na base gordurosa / Production of sandwich cookies with the formation of organogel in the fatty base

A fim de atender à demanda do público que atualmente busca por alimentos mais saudáveis, as indústrias têm procurado alternativas que possibilitem a aplicação de ingredientes que agreguem valor nutricional aos produtos. A redução de gorduras saturadas e trans em produtos alimentícios, bem como a inserção de cereais ou farinhas nutricionais, vem sendo aplicadas em produtos de panificação. Biscoitos recheados possuem como bases geralmente biscoitos à base de farinha de trigo. O objetivo foi desenvolver formulação de biscoitos recheados com substituição de gordura vegetal por organogel no recheio e de farinha de trigo por farinha de sorgo no biscoito, a fim de agregar valor nutricional ao produto. Foram desenvolvidos biscoitos recheados: 1) recheio controle e com substituição da gordura vegetal dos recheios por organogel elaborado com sistema emulsionado (colágeno + óleo vegetal + água), a fim de diminuir concentrações de gorduras saturadas e trans. 2) para a base elaborouse biscoitos controle (farinha de trigo) e com substituição parcial e total de farinha de trigo por farinha de sorgo em 50% (50FS) e 100% (100FS). Foram conduzidas nos recheios e das bases dos biscoitos análises físicas e físico-químicas (textura, atividade de água, cor, composição centesimal e reologia) para avaliação e para análise de estabilidade de 6 semanas. Os resultados apresentaram que o biscoito 50FS obteve melhor valor de textura (Controle: 16,09 ± 1,28 N; 50FS: 19,63 ± 5,68 N e 100FS: 10,09 ± 0,65 N) e menor teor de atividade de água (Semana 01: 0,327±0,01 e Semana 06: 0,389 ± 0,00) do que o biscoito controle, durante análise de estabilidade. O biscoito 100FS apresentou coloração mais avermelhada. Os biscoitos 50FS e 100FS apresentaram maior teor proteico do que o controle (Controle: 5,37 ± 0,23 %; 50FS: 5,64 ± 0,49 % e 100FS: 5,75 ± 0,49 %). O recheio com organogel apresentou maior dureza (N) durante análise de estabilidade do que o recheio controle (Semana 6 Organogel: 6,81±1,48; Controle: 4,29±0,38). Os parâmetros de adesividade, coesividade e gomosidade do recheio com organogel não apresentaram diferenças significativas (p > 0,05). Os valores de atividade de água da formulação com organogel foram mais altos do que o recheio controle (Semana 6 Organogel: 0,730±0,00; Controle: 0,555±0,01). O valor de L* foi maior para o recheio controle, apresentando coloração mais amarelada do que a formulação com organogel. O recheio com organogel apresentou redução de 65 % do teor lipídico e aumento do teor proteico. Os recheios controle, com organogel e de mercado apresentaram comportamento tixotrópico durante a avaliação reológica, sendo que o produto de mercado teve comportamento próximo à formulação controle, com recuperação quase total da estrutura. Foram desenvolvidos cinco produtos, sendo três inovadores com valor nutricional agregado, atendendo às legislações vigentes, vida útil mínima de 6 semanas e ao apelo do mercado atual, podendo ser comercializados como biscoito recheado

In order to satisfy the demand of the public that is currently looking for healthier foods industries have been looking for alternatives that allow the application of ingredients that add nutritional value to the products. The reduction of saturated and trans fats in food products, as well as the insertion of cereals or nutritional flours, has been applied in bakery products. Filled cookies are usually based on wheat flour. The objective was to develop a formulation of filled cookies with replacement of vegetable fat for organogel in the filling and wheat flour for sorghum flour in the biscuit, in order to add nutritional value to the product. In this study, cookies filled with vegetable fat and wheat flour were used as a control where: 1) filling was replaced by organogel elaborated with an emulsified system (collagen + vegetable oil + water); and 2) base was prepared with partial and total replacer of wheat flour for sorghum flour in 50% (50FS) and 100% (100FS). Physical and physicochemical analyzes (texture, water activity, color, proximate composition and rheology) were carried out on the fillings and bases of the biscuits for evaluation and for the stability analysis of 6 weeks. The results showed that the 50FS cookies had a better texture value (Control: 16,09±1,28 N; 50FS: 19,63±5,68N and 10,09±0,65 N) and lower content of water activity (Week 1: 0,327±0,01 and Week 6: 0,389±0,00) than the control cookie during stability analysis. The 100FS had a more reddish color. The 50FS and 100FS cookies had a higher protein content than the control (Control: 5,37±0,23 %; 50FS 5,64±0,49 %). The fillings with organogel showed a higher hardness (N) than the control during stability analysis (Week 6 Organogel: 6,81±1,48; Control: 4,29±0,38). The parameters of adhesiveness, cohesiveness and guminess of the filling with organogel showed no significant differences (p> 0.05). The water activity values of the organogel formulation were higher than the control filling (Week 6 Organogel: 0,730±0,00; Control: 0,555±0,01). The value of L * was higher for the control filling, showing a more yellowish color than the formulation with organogel. The filling with organogel showed a 65% reduction in lipid content and an increase in protein content. The control, organogel and market fillings showed a thixotropic behavior in the rheological evaluation, and the market product had a behavior close to the control formulation, with almost total recovery of the structure. Five products were developed, three of which were innovative with added nutritional value, in compliance with current legislation, a minimum shelf life of 6 weeks, which can be sold as a stuffed cookies.

The Calvarial Injection Assay.

This chapter describes the calvarial injection method, whereby the effect of a substance on bone is tested by subcutaneous injection over the calvarium of a mouse. This assay allows testing of the effect of substances on both bone resorption and bone formation in a relatively simple in vivo model. The analysis is carried out by histological means, usually in glycolmethacrylate-embedded tissue, allowing for histochemical analysis and for a variety of different histological staining methods which are also described in detail.

High Content, Phenotypic Assays and Screens for Compounds Modulating Cellular Processes in Primary Neurons.

High content, phenotypic screens offer a powerful approach to systems biology at the cellular level. The approach employs cells carrying fluorescently labeled molecules or organelles in 384- or 1536-well microplates, and an automated confocal screening microscope for capturing images from each well. Although some specifics vary according to the assay type, each will apply some degree of image processing and feature extraction followed by a data analysis pipeline to identify the perturbations (small molecules, etc.) of interest. We describe and discuss the advantages and limitations of high content assays and screens using the specific example of assaying mitochondrial dynamics in primary neurons. We provide a detailed description of our culturing methods, imaging and data analysis techniques and provide an open source, ready to use CellProfiler pipeline for high-throughput image segmentation and quantification tool for mitochondrial parameters.

Protocol for Detecting the Primo Vascular System in the Lymph Ducts of Mice.

The primo vascular system (PVS), which is the proposed conduit for the acupuncture Qi, is a complex network distributed throughout an animal's body. However, even with a microscope, it is not easily detectable because of its transparency. Thus, its existence is largely unknown in current anatomy. A convincing demonstration of its existence is needed. The lymph-primo vessel (PV), which is a subsystem of the PVS, is a very effective visual demonstration of the PVS. The lymph-PVS is a mobile threadlike structure floating in lymph ducts that has been observed in rabbits, rats, and mice by several independent teams. The involved techniques are novel and rather complicated; therefore, we have already provided detailed protocols for the surgery; for the injection of the staining dye; and for the detection, extraction, and identification of the PVS in rabbits and rats. However, the mouse is one of the most important laboratory animals used for various biomedical research purposes. For the convenience of researchers who wish to initiate the PVS experiments in mice, we provide a shortened version of the protocol, despite many similarities with previously published protocols. Thus, researcher can easily obtain the samples of the lymph-PVS of mice.

Improved quality control of [18F]FDG by HPLC with UV detection.

A conventional high-performance liquid chromatographic (HPLC) method for the analysis of 2-fluoro-2-deoxy-d-glucose (FDG) and 2-deoxy-2-chloro-d-glucose (ClDG) in [18F]FDG preparations is described. This method was based on a postcolumn derivatization with 2-cyanoacetamide (2-CA) and UV detection. FDG and ClDG were separated on a normal-phase column using acetonitrile/water as the mobile phase. The eluate was mixed with 2-CA in sodium borate buffer solution at the outlet of a PTFE coil (10 m x 0.5 mm id) from the column, and the reaction was carried out at 100 degrees C during the passage through the coil. The UV absorbance of the resultant product was monitored at 276 nm. Under optimum conditions, the detection limits [signal-to-noise (S/N) ratio=3] for FDG and ClDG were 0.31 and 0.17 microg/ml for a 20-microl injection volume, respectively, and the linearity ranges were 0.5-100 microg/ml for both compounds. The intra- and interday reproducibilities were better than 2.2% [relative standard deviation (R.S.D.)]. This HPLC separation procedure is also useful for determining the radiochemical purity of [18F]FDG preparations since it allows the analysis of 2-[18F]fluoro-1,3,4,6-tetra-O-acetyl-d-glucose ([18F]TAG), partially hydrolyzed [18F]TAG and [18F]F-. This method can be used at many positron emission tomography (PET) facilities since it does not require an expensive, sophisticated electrochemical detector.

Fully automated synthesis of [18F]fluoromisonidazole using a conventional [18F]FDG module.

We developed a new fully automated method for the synthesis of [18F]fluoromisonidazole ([18F]FMISO) by modifying a commercial FDG synthesizer and its disposable fluid pathway. A three-step procedure was used to prepare the tosylate precursor, 1-(2'-nitro-1'-imidazolyl)-2-O-tetrahydrofuranyl-3-O-toluenesulfonylpropanediol. Using glycerol as the starting material, the precursor was synthesized with a yield of 21%. The optimal labeling conditions for the automated synthesis of [18F]FMISO was 10 mg of precursor in acetonitrile (2 ml heated at 105 degrees C for 360 s, followed by heating at 75 degrees C for 280 s and hydrolysis with 1 N HCl at 105 degrees C for 300 s. Using 3.7 GBq of [18F]F- as a starting activity, [18F]FMISO was obtained with high end-of-synthesis (EOS) radiochemical yields of 58.5+/-3.5% for 60.0+/-5.2 min with high-performance liquid chromatography (HPLC) purification. When solid-phase purification steps were added, the EOS radiochemical yields were 54.5+/-2.8% (337+/-25 GBq/micromol) for 70.0+/-3.8 min (n=10 for each group, decay-corrected). With a high starting radioactivity of 37.0 GBq, we obtained radiochemical yields of 54.4+/-2.9% and 52.8+/-4.2%, respectively (n=3). The solid-phase purification removed unreacted [18F]fluoride and polar impurities before the HPLC procedure. Long-term tests showed a good stability of 98.2+/-1.5%. This new automated synthesis procedure combines high and reproducible yields with the advantage of using a disposable cassette system.

Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions. Dozens of astrocytes can be labeled in single slice cultures by gene gun-mediated ballistic delivery of gold or tungsten particles carrying cDNAs (Biolistics), lipophilic dyes (DiOlistics), or fluorescent intracellular calcium indicators (Calistics). Expression of a membrane-targeted form of eGFP (Lck-GFP) is superior to soluble eGFP for resolving fine astrocytic processes. Time-lapse confocal imaging of Lck-GFP transfected astrocytes or "calistically" labeled astrocytes show structural remodeling and calcium transients, respectively. This approach provides an in vitro system for investigating the functional architecture, development and dynamic remodeling of astrocytes and their relationships to neurons and glia in live mammalian brain tissues.

A new principle in polychrome staining: a system of automated staining, complementary to hematoxylin and eosin, and usable as a research tool.

A staining system is described in which each stage forms a separate module or unit. All reagents, concentrations of dye, ratios of phosphotungstic acid to dye, pH values, temperature and staining times are standardized and only aqueous solutions used. The technic uses equal strength solutions of orange G, acid fuchsin and methyl (or aniline) blue, in ascending order of molecular size, at pH 2.5 (range: 2.3 to 2.7). Phosphotungstic acid is incorporated in the dyebaths, not used separately, and the combination of this with ferric alum hematoxylin (Lillie's by preference) and either naphthol yellow S or picric acid as a primer, enables fibrin and cytoplasmic components to be demonstrated vividly, with other tissues shown in clear contrasting colors. Erythrocytes are yellow, fibrin red and collagen blue. The system permits substitution of dyes, lending itself to both manual and computer recording and analysis, helped by a notation system for identifying variants. Many of the factors are variable at will. The system aids research into the mechanism of polychrome staining, and, by extrapolation, into the mechanism of action of other stains. Two manually or machine usable progressive polychrome technics intended for routine use are described. They identify tissue components consistently, complementing the standard hematoxylin and eosin stain, and deserve equal attention during reporting. Variants may be used for one-minute one-stage staining of frozen sections, or to give strong colors with 2 millimicrons acrylic sections.

Lead and uranium stain artefacts in electron microscopy: a technique for minimizing their occurrence.

A new technique is presented which significantly reduced the occurrence of artefacts by eliminating the air-stain interface when staining thin sections of biological material. It is designed for simultaneous treatment of large numbers of samples. The experiment described deals mainly with the specific staining problems encountered when using grids coated with Formvar.