A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells

Mouse embiyonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with ß-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohor-monal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation. Copyright © 2008 Cognizant Comm. Corp.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

In vitro bioactivity of titanium-doped bioglass

Previous studies have suggested that incorporating relatively small quantities of titanium dioxide into bioactive glasses may result in an increase in bioactivity and hydroxyapatite formation. The present work therefore investigated the in vitro bioactivity of a titanium doped bioglass and compared the results with 45S5 bioglass. Apatite formation was evaluated for bioglass and Ti-bioglass in the presence and absence of foetal calf serum. Scanning electron microscopy (SEM) images were used to evaluate the surface development and energy dispersive X-ray measurements provided information on the elemental ratios. X-ray diffraction spectra confirmed the presence of apatite formation. Cell viability was assessed for bone marrow stromal cells under direct and indirect contact conditions and cell adhesion was assessed using SEM. © 2014 Springer Science+Business Media.

Differential effects of chlorinated and oxidized phospholipids in vascular tissue:implications for neointima formation

The presence of inflammatory cells and MPO (myeloperoxidase) in the arterial wall after vascular injury could increase neointima formation by modification of phospholipids. The present study investigates how these phospholipids, in particular oxidized and chlorinated species, are altered within injured vessels and how they affect VSMC (vascular smooth muscle cell) remodelling processes. Vascular injury was induced in C57BL/6 mice and high fat-fed ApoE-/- (apolipoprotein E) mice by wire denudation and ligation of the left carotid artery (LCA). Neointimal and medial composition was assessed using immunohistochemistry and ESI-MS. Primary rabbit aortic SMCs (smooth muscle cells) were utilized to examine the effects of modified lipids on VSMC proliferation, viability and migration at a cellular level. Neointimal area, measured as intima-to-media ratio, was significantly larger in wire-injured ApoE-/- mice (3.62±0.49 compared with 0.83±0.25 in C57BL/6 mice, n=3) and there was increased oxidized low-density lipoprotein (oxLDL) infiltration and elevated plasma MPO levels. Relative increases in lysophosphatidylcholines and unsaturated phosphatidylcholines (PCs) were also observed in wire-injured ApoE-/- carotid arteries. Chlorinated lipids had no effect on VSMC proliferation, viability or migration whereas chronic incubation with oxidized phospholipids stimulated proliferation in the presence of fetal calf serum [154.8±14.2% of viable cells at 1 μM PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) compared with control, n=6]. In conclusion, ApoE-/- mice with an inflammatory phenotype develop more neointima in wire-injured arteries and accumulation of oxidized lipids in the vessel wall may propagate this effect.

A comparison of individual cow versus group concentrate allocation strategies on dry matter intake, milk production, tissue changes, and fertility of Holstein-Friesian cows offered a grass silage diet

A diverse range of concentrate allocation strategies are adopted on dairy farms. The objectives of this study were to examine the effects on cow performance [dry matter (DM) intake (DMI), milk yield and composition, body tissue changes, and fertility] of adopting 2 contrasting concentrate allocation strategies over the first 140 d of lactation. Seventy-seven Holstein-Friesian dairy cows were allocated to 1 of 2 concentrate allocation strategies at calving, namely group or individual cow. Cows on the group strategy were offered a mixed ration comprising grass silage and concentrates in a 50:50 ratio on a DM basis. Cows on the individual cow strategy were offered a basal mixed ration comprising grass silage and concentrates (the latter included in the mix to achieve a mean intake of 6 kg/cow per day), which was formulated to meet the cow’s energy requirements for maintenance plus 24 kg of milk/cow per day. Additional concentrates were offered via an out-of-parlor feeding system, with the amount offered adjusted weekly based on each individual cow’s milk yield during the previous week. In addition, all cows received a small quantity of straw in the mixed ration part of the diet (approximately 0.3 kg/cow per day), plus 0.5 kg of concentrate twice daily in the milking parlor. Mean concentrate intakes over the study period were similar with each of the 2 allocation strategies (11.5 and 11.7 kg of DM/cow per day for group and individual cow, respectively), although the pattern of intake with each treatment differed over time. Concentrate allocation strategy had no effect on either milk yield (39.3 and 38.0 kg/d for group and individual cow, respectively), milk composition, or milk constituent yield. The milk yield response curves with each treatment were largely aligned with the concentrate DMI curves. Cows on the individual cow treatment had a greater range of concentrate DMI and milk yields than those on the group treatment. With the exception of a tendency for cows on the individual cow treatment to lose more body weight to nadir than cows on the group treatment, concentrate allocation strategy had little effect on either body weight or body condition score over the experimental period. Cows on the individual cow treatment had a higher pregnancy rate to first and second service and tended to have a higher 100-d in calf rate than cows on the group treatment. This study demonstrates that concentrate allocation strategy had little effect on overall production performance.

Qual a contribuição das características antropométricas na velocidade de corrida de curta distância ?

v. 19, n. 2, abr./jun. 2016.

Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

Desenvolvimento ponderal de bubalinos da raça Murrah criados em pastagem de Brachiaria brizantha no Centro-Oeste do Estado de São Paulo, Brasil

Foram analisados os dados de desempenho ponderal de bubalinos Murrah do Sistema de Produção de Leite da Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia, campus de Botucatu. Os pesos foram corrigidos às diversas idades-padrão e o modelo incluiu os efeitos de sexo (S), mês (M) e ano (A) de nascimento, classe de idade da búfala ao parto (C) e as interações S x M, S x A, S x C e M x A. As médias ajustadas e respectivos erros-padrão estimados para as características estudadas foram: Peso ao Nascer (PN): 37,71 ± 8,25kg; Peso aos 120 dias (P120): 102,08 ± 16,27kg; Peso aos 240 dias (P240): 169,84 ± 22,83kg; Peso aos 365 dias (P365): 250,59 ± 25,12kg; Peso aos 550 dias (P550): 326,13 ± 39,27kg e Peso aos 730 dias (P730): 389,80 ± 31,26kg. O efeito de sexo (S) foi significativo somente para PN e P365, sendo que machos tenderam a nascer mais pesados que fêmeas. O mês de nascimento (M) exerceu efeito sobre o PN, P120 e P730 sendo que animais nascidos em maio foram os mais pesados ao nascer, enquanto os nascidos em janeiro e maio, foram os mais pesados aos 120 e 730 dias, respectivamente. O efeito de ano de foi significativo sobre o PN, P120, P240 e P730. Os filhos de búfalas das classes de idade 1 (3 anos ou menos) e da classe 6 (10,11 e 12 anos) foram os mais leves e mais pesados ao nascer, respectivamente. O fato de a classe de idade da búfala não exercer efeito sobre os P365, P550 e P730 sugere que, em rebanhos comerciais possa ser feita a substituição de búfalas não gestantes por novilhas prenhes, apesar de esta prática reduzir a média de idade do rebanho de cria. Bubalinos da raça Murrah oriundos de rebanhos leiteiros podem ser utilizados para a produção de carne.

Modelagem univariada e regressão múltipla na análise da oferta e do preço do boi gordo no mercado brasileiro

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronegócios, 2016.

3D Porous Architecture of Stacks of β-TCP Granules Compared with That of Trabecular Bone: A microCT, Vector Analysis, and Compression Study

International audience

Impacto del uso de los sistemas de alimentación a corral como estrategia para el engorde de bovinos para carne

El confinamiento estratégico de los bovinos Argentina es una herramienta muy útil para lograr la terminación de animales destinados al consumo interno y a la exportación. La utilidad de estos encierres consiste permitir un mejor aprovechamiento del forraje y reduciendo la edad a la faena. Para su aplicación es necesario considerar los factores que afectan su resultado económico tales como la característica de las dietas utilizadas, los niveles de consumo y la eficiencia de conversión. Esto interactúa a su vez con el tipo de animal a encerrar. Son dos los sistemas de confinamiento estratégico que van a ser considerados en esta presentación: los corrales de engorde o terminación y los de recría o engorde de los terneros posdete. La actividad del engorde a corral durante los últimos años, ha estado ligada a variaciones operadas en el precio del grano y del tipo de producto logrado. Hay variaciones atribuidas al cambio en las preferencias del consumidor y también al efecto de la concentración de las ventas de carne en determinadas bocas de expendio que exigen uniformidad en la entrega de animales y del tamaño de los cortes. Se comentan entonces algunos aspectos relevantes del engorde a corral americano y de los engordes a corral en Argentina, además de algunos factores que pueden afectar el resultado económico de la actividad. En cuanto al corral de recría, tiene como objetivo la mejora de la ganancia de peso durante esta etapa frente a condiciones de recría a pasto donde pueden existir limitantes en la cantidad y calidad del forraje producido. En esta presentación se hace el comentario y el análisis de los factores a tener en cuenta en los dos tipos de manejo de los animales a corral: los de terminación o engorde final y los de recría o engorde de terneros después del destete. En el primer caso, los efectos del sexo, la edad, la eficiencia de conversión, la raza, el estado nutricional previo, así como factores de la dieta en si mismo. En el segundo caso se analizan sobre todo los distintos tipos de terneros que pueden incorporar al sistema, así como el uso de diferentes dietas. El desarrollo de los encierres de terneros permitió diseñar nuevos sistemas de producción basados en un aumento de la carga, de la ganancia de peso, la modificación del peso final y los posibles cambios en el momento de terminación. En el caso de los corrales de terminación, han sido una herramienta muy útil para lograr la terminación de animales destinados al consumo interno como exportación.

Interaction of Multiwalled Carbon Nanotube Produces Structural Alteration of DNA

Multiwalled carbon nanotube (MWCNT) has been found to produce structural changes in Calf Thymus-DNA (CT-DNA). The interaction or binding of the multi-walled carbon nanotubes (MWCNT) was investigated in order to discover if it brings about any significant changes of the DNA double helix using CD spectra of the CT-DNA at two concentration levels of MWCNT representing an increasing MWCNT/DNA molar ratio. In addition, spectrophotometric titrations between MWCNT and CT-DNA were carried out in order to utilize spectral changes as a means of detecting specific binding modes of either intercalation or degradation of DNA. Interactions of MWCNT induced significant changes in the CD spectra of the B-form of natural DNA. The intensities of the positive CD band at 280 nm decreased significantly. This decrease was found to be concentration-dependent. Following spectrophotometric titrations; specific subtle conformational changes were observed with a molar ratio combination of 2:1 between MWCNT and CT-DNA and these were characterized by a formation constant of the order of 103 M-1 and a negative Gibbs free energy suggesting that MWCNT avidly binds to DNA. Thermodynamic considerations revealed that electrostatic interactions between the DNA base pairs and the MWCNT are taking place accounting for the negative free energy change, positive enthalpy change with a small entropy change. The results obtained in the study of the binding interactions of MWCNT with DNA confirm that a cytogenetic effect of MWCNT with DNA is a possibility in vivo.

Neural Modulation of Leg Stiffness in Response to Neuromuscular Fatigue

The spring-mass model is able to accurately represent hopping spring-like behavior (leg and joint stiffness), and leg and joint stiffness changes can reveal overall motor control responses to neural and muscular contributors of neuromuscular fatigue. By understanding leg stiffness modulation, we can determine which variables the nervous system targets to maintain motor performance and stability. The purpose of this study was to determine how neuromuscular fatigue affects hopping behavior by examining leg and joint stiffness before and after a single-leg calf raise fatiguing protocol. Post-fatigue, leg stiffness decreased for the exercised leg, but not for the non-exercised leg. Ankle and knee joint stiffness did not significantly change for either leg. This indicates that leg stiffness decreases primarily from muscular fatigue, but was not explained by ankle and knee joint stiffness. The decrease in leg stiffness may be an attempt to soften landing impact, while at the same time maintaining performance.

Estudo da transferência de imunidade passiva, em vitelos, no concelho de Guimarães

Dissertação de Mestrado Integrado em Medicina Veterinária

Tratamento de diarreias neonatais em vitelos

Dissertação de Mestrado Integrado em Medicina Veterinária