Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1

The ATP-binding cassette transporter ABCA1 is essential for high density lipoprotein (HDL) formation and considered rate-controlling for reverse cholesterol transport. Expression of the Abca1 gene is under control of the liver X receptor (LXR). We have evaluated effects of LXR activation by the synthetic agonist T0901317 on hepatic and intestinal cholesterol metabolism in C57BL/6J and DBA/1 wild-type mice and in ABCA1-deficient DBA/1 mice. In wild-type mice, T0901317 increased expression of Abca1 in liver and intestine, which was associated with a similar to60% rise in HDL. Biliary cholesterol excretion rose 2.7-fold upon treatment, and fecal neutral sterol output was increased by 150-300%. Plasma cholesterol levels also increased in treated Abca1(-/-) mice (+120%), but exclusively in very low density lipoprotein-sized fractions. Despite the absence of HDL, hepatobiliary cholesterol output was stimulated upon LXR activation in Abca1(-/-) mice, leading to a 250% increase in the biliary cholesterol/phospholipid ratio. Most importantly, fecal neutral sterol loss was induced to a similar extent (+300%) by the LXR agonist in DBA/1 wild-type and Abca1(-/-) mice. Expression of Abcg5 and Abcg8, recently implicated in biliary excretion of cholesterol and its intestinal absorption, was induced in T0901317-treated mice. Thus, activation of LXR in mice leads to enhanced hepatobiliary cholesterol secretion and fecal neutral sterol loss independent of (ABCA1-mediated) elevation of HDL and the presence of ABCA1 in liver and intestine

Endocytosis and intracellular processing of platelet microparticles by brain endothelial cells

Platelet-derived microparticles (PMP) bind and modify the phenotype of many cell types including endothelial cells. Recently, we showed that PMP were internalized by human brain endothelial cells (HBEC). Here we intend to better characterize the internalization mechanisms of PMP and their intracellular fate. Confocal microscopy analysis of PKH67-labelled PMP distribution in HBEC showed PMP in early endosome antigen 1 positive endosomes and in LysoTracker-labelled lysosomes, confirming a role for endocytosis in PMP internalization. No fusion of calcein-loaded PMP with HBEC membranes was observed. Quantification of PMP endocytosis using flow cytometry revealed that it was partially inhibited by trypsin digestion of PMP surface proteins and by extracellular Ca2+ chelation by EDTA, suggesting a partial role for receptor-mediated endocytosis in PMP uptake. This endocytosis was independent of endothelial receptors such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and was not increased by tumour necrosis factor stimulation of HBEC. Platelet-derived microparticle internalization was dramatically increased in the presence of decomplemented serum, suggesting a role for PMP opsonin-dependent phagocytosis. Platelet-derived microparticle uptake was greatly diminished by treatment of HBEC with cytochalasin D, an inhibitor of microfilament formation required for both phagocytosis and macropinocytosis, with methyl-β-cyclodextrin that depletes membrane cholesterol needed for macropinocytosis and with amiloride that inhibits the Na+/H+ exchanger involved in macropinocytosis. In conclusion, PMP are taken up by active endocytosis in HBEC, involving mechanisms consistent with both phagocytosis and macropinocytosis. These findings identify new processes by which PMP could modify endothelial cell phenotype and functions. © 2011 The Authors. Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd

Efficacy and tolerability of a specific blend of amino acids in patients with anorexia nervosa treated in a hospital setting: study protocol for a randomized controlled trial

BackgroundSpecific blends of essential amino acids (EAA) containing a high percentage of branched-chain amino acids preserves mitochondrial metabolism and higher physical resistance in elderly mice, increasing their survival and improving physical performance and cognitive functions in malnourished elderly patients. However, no study has been yet done on patients with anorexia nervosa (AN) who regain weight with specialized intensive treatment. The present study aims to evaluate the efficacy of supplementation with EAA on the change in lean body mass (LBM) and other physical and psychological outcomes in patients with AN who are undergoing specialist treatment for eating disorders.MethodsThis is a 13-week randomized, double-blind, placebo-controlled study. Patients will be randomized to either a mixture of a complex blend of EAA and intermediates of the tricarboxylic acid (TCA) cycle (citrate, malate, succinate) supplementation (or placebo) upon admission at the intensive residential and day-hospital treatment for eating disorders. Ninety-two participants with AN aged 16-50 years will be recruited from a specialized intensive treatment of eating disorders. Double-blind assessment will be conducted at baseline (T0) and the end of the 13 weeks of treatment (T1). The study's primary aim is to evaluate the efficacy of supplementation with EAA and TCA intermediates on the change in lean body mass (LBM) with weight restoration in patients with AN who are undergoing specialist treatment for eating disorders. The secondary aims of the study are to assess the effect of dietary supplementation on physical fitness, weight restoration, modification of AN and general psychopathology, and psychosocial impairment.DiscussionThe study's results will inform researchers and clinicians on whether supplementing a mixture of EAA and TCA cycle intermediates will improve the increase of LBM and other important physical and psychological outcomes in patients with AN who regain weight with specialized intensive treatment

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide

Chemical, Physical And Oxidative Characteristics Of Broilers Meat Supplemented With Passion Fruit Seed Oil

Residues that could be discarded and transformed in high biological value protein is a measure of environmental preservation combined with the sustainability of animal production. This study evaluated the effects of the addition of passion fruit seed oil (PFSO) on broiler diet under the resistance of skin, physical-chemical characteristic, fatty acid profile and lipid oxidation (under storage) of breast meat obtained from these animals. There was improvement in the condition of skin rupture and meat tenderness; apart from that, oxidative reactions decreased, as PFSO was added to the diet (P<0.05). On the other hand, no effect on colour, pH, water retention capacity and weight loss on cooking (P>0.05) was observed. There was a linear effect increasing only for the margaric (C17:0) and cis10-Heptadecanoic (C17:1; P<0,05) fatty acids. However, due to the low amount of these fatty acids in PFSO, the found content was still low in all evaluated treatments. However, the evaluated levels did not contribute to the increase of the polyunsaturated fatty acid profile (P>0.05). Thus, it can be concluded that PFSO improves the resistance of broiler skin, which becomes softer and less susceptible to oxidative effects due to the oil’s antioxidant action, suggesting that it may be an ingredient that brings substantial benefits in the poultry industry

Composted sewage sludge and different irrigation layers on Acacia polyphylla development

É cada vez mais comum a adição de componentes orgânicos aos substratos em viveiros florestais, com o objetivo de potencializar seus atributos biológicos, químicos e físicos. Entre esses componentes destaca-se o lodo de esgoto que, após passar por processo de estabilização, torna-se um componente rico em matéria orgânica, podendo ser utilizado na agricultura, e em viveiros florestais. O trabalho teve como objetivo analisar o comportamento da espécie em diferentes composições de substratos e lâminas de irrigação. Utilizaram-se 5 formulações distintas de substrato compostas por substrato comercial (SC) e lodo e esgoto compostado (LEC). Avaliaram-se simultaneamente 3 lâminas de água, perfazendo um total de 300 mudas no estudo. Os parâmetros analisados foram: altura de planta, diâmetro do coleto, número de folhas, massa seca de parte aérea e raiz. Observou-se que os melhores tratamentos consistiram nas seguintes formulações: S1 (100% LEC), S2 (25% SC + 75% LEC), e S3 (50% SC + 50% SC). Mostrou-se que, a partir do LEC puro, houve melhor desenvolvimento das mudas, expressando um aporte com o aumento no diâmetro do coleto, altura da planta, maior crescimento inicial e desenvolvimento radicular, ou seja, além do potencial como fertilizante, o LEC atua como condicionador de atribuições físicas, química e biológicas necessárias às mudas. As diferentes lâminas de irrigação não apresentaram influência significativa em nenhum dos parâmetros avaliados.The selection of organic components to substrates in forest nurseries is increasingly common, with the aim of enhancing biological, chemical and physical data. The main components are the sewage process that goes through a stabilization process, becoming a rich component in industry that can be used in agriculture, and becomes a rich component in the composition of substrates. The objective of this work is to analyze the behavior of the species in different substrate compositions and irrigation depths. Five distinct formulations of composite substrate and commercial substrate (SC) and composted sludge and sewage (LEC) were used. The slide and irrigation evaluation was divided into three different irrigations, a total of 300 samples were analyzed. The parameters used were: plant height, stem diameter, leaf number, dry mass and root dry mass. Based on the results, the best alternatives for producing seedlings of wine in nurseries are observed, consisting of the following formulations: S1 (100% LEC), S2 (25% SC + 75% LEC), and S3 (50% SC + 50% LEC). The combination of 100% LEC resulted in the best development of seedlings, expressing a contribution with the increase in stem diameter, plant height, greater initial growth and root development, that is, in addition to the potential as fertilizer, it acts as a conditioner for physical assignments, chemical and biological necessary for seedlings. However, the different irrigation depths do not minimize the influences in any of the upper stages

Detection of human disease conditions by single-cell morpho-rheological phenotyping of blood

Blood is arguably the most important bodily fluid and its analysis provides crucial health status information. A first routine measure to narrow down diagnosis in clinical practice is the differential blood count, determining the frequency of all major blood cells. What is lacking to advance initial blood diagnostics is an unbiased and quick functional assessment of blood that can narrow down the diagnosis and generate specific hypotheses. To address this need, we introduce the continuous, cell-by-cell morpho-rheological (MORE) analysis of diluted whole blood, without labeling, enrichment or separation, at rates of 1000 cells/sec. In a drop of blood we can identify all major blood cells and characterize their pathological changes in several disease conditions in vitro and in patient samples. This approach takes previous results of mechanical studies on specifically isolated blood cells to the level of application directly in blood and adds a functional dimension to conventional blood analysis

Cooperation between Engulfment Receptors: The Case of ABCA1 and MEGF10

The engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway