Influence of gold nanoparticles on collagen fibril morphology quantified using transmission electron microscopy and image analysis

BACKGROUND: Development of implantable biosensors for disease detection is challenging because of poor biocompatibility of synthetic materials. A possible solution involves engineering interface materials that promote selfassembly and adhesion of autologous cells on sensor surfaces. Crosslinked type-I collagen is an acceptable material for developing engineered basement membranes. In this study, we used functionalized gold nanoparticles as the crosslinking agent. Functionalized nanoparticles provide sites for crosslinking collagen as well as sites to deliver signaling compounds that direct selfassembly and reduce inflammation. The goal of this study was to obtain a quantitative parameter to objectively determine the presence of crosslinks. METHODS: We analyzed TEM images of collagen fibrils by two methods: Run length analysis and topology analysis after medial axis transform. RESULTS: Run length analysis showed a significant reduction of the interfibril spaces in the presence of nanoparticles (change of 40%, P < 0.05), whereas the fibril thickness remained unchanged. In the topological network, the number of elements, number of branches and number of sides increased significantly in the presence of nanoparticles (P < 0.05). Other parameters, especially the number of loops showed only a minimal and nonsignificant change. We chose a ratiometric parameter of the number of branches normalized by the number of loops to achieve independence from gross fibril density. This parameter is lower by a factor of 2.8 in the presence of nanoparticles (P < 0.05). CONCLUSION: The numerical parameters presented herein allow not only to quantify fibril mesh complexity and crosslinking, but also to help quantitatively compare cell growth and adhesion on collagen matrices of different degree of crosslinking in further studies

Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apo)A-I from murine RAW 264.7 macrophages

<p>Abstract</p> <p>Background</p> <p>Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function.</p> <p>Methods</p> <p>Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [³H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, <it>Gapdh</it>, and combined with studies of this molecule on cholesterol esterification, <it>de novo</it> lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post <it>t</it>-tests, as appropriate.</p> <p>Results</p> <p>The positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; <it>p</it><0.01) and oligomycin (55%; <it>p</it><0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in <it>Abca1</it> mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (<it>Abca1</it>, <it>Abcg4</it>, <it>Stard1</it>) and cholesterol biosynthesis (<it>Hmgr</it>, <it>Mvk</it>, <it>Scap</it>, <it>Srebf2</it>), indicating profound dysregulation of cholesterol homeostasis.</p> <p>Conclusions</p> <p>Acute loss of mitochondrial function, and in particular Δψ_m, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.</p

Serotonin transporter (SERT) and translocator protein (TSPO) expression in the obese ob/ob mouse

Background: An ever growing body of evidences is emerging concerning metabolism hormones, neurotransmitters or stress-related biomarkers as effective modulators of eating behavior and body weight in mammals. The present study sought at examining the density and affinity of two proteins related to neurotransmission and cell metabolism, the serotonin transporter SERT and the cholesterol import-benzodiazepine site TSPO (translocator protein), in a rodent leptin-lacking mutant, the obese ob/ob mouse. Binding studies were thus carried out in brain or peripheral tissues, blood platelets (SERT) and kidneys (TSPO), of ob/ob and WT mice supplied with a standard diet, using the selective radiochemical ligands [(3)H]-paroxetine and [(3)H]-PK11195. Results: We observed comparable SERT number or affinity in brain and platelets of ob/ob and WT mice, whilst a significantly higher [(3)H]-PK11195 density was reported in the brain of ob/ob animals. TSPO binding parameters were similar in the kidneys of all tested mice. By [(3)H]-PK11195 autoradiography of coronal hypothalamic-hippocampal sections, an increased TSPO signal was detected in the dentate gyrus (hippocampus) and choroids plexus of ob/ob mice, without appreciable changes in the cortex or hypothalamic-thalamic regions. Conclusions: These findings show that TSPO expression is up-regulated in cerebral regions of ob/ob leptin-deficient mice, suggesting a role of the translocator protein in leptin-dependent CNS trophism and metabolism. Unchanged SERT in mutant mice is discussed herein in the context of previous literature as the forerunner to a deeper biochemical investigation

Genomic and neural analysis of the estradiol-synthetic pathway in the zebra finch

<p>Abstract</p> <p>Background</p> <p>Steroids are small molecule hormones derived from cholesterol. Steroids affect many tissues, including the brain. In the zebra finch, estrogenic steroids are particularly interesting because they masculinize the neural circuit that controls singing and their synthesis in the brain is modulated by experience. Here, we analyzed the zebra finch genome assembly to assess the content, conservation, and organization of genes that code for components of the estrogen-synthetic pathway and steroid nuclear receptors. Based on these analyses, we also investigated neural expression of a cholesterol transport protein gene in the context of song neurobiology.</p> <p>Results</p> <p>We present sequence-based analysis of twenty steroid-related genes using the genome assembly and other resources. Generally, zebra finch genes showed high homology to genes in other species. The diversity of steroidogenic enzymes and receptors may be lower in songbirds than in mammals; we were unable to identify all known mammalian isoforms of the 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase families in the zebra finch genome assembly, and not all splice sites described in mammals were identified in the corresponding zebra finch genes. We did identify two factors, Nobox and NR1H2-RXR, that may be important for coordinated transcription of multiple steroid-related genes. We found very little qualitative overlap in predicted transcription factor binding sites in the genes for two cholesterol transport proteins, the 18 kDa cholesterol transport protein (TSPO) and steroidogenic acute regulatory protein (StAR). We therefore performed in situ hybridization for TSPO and found that its mRNA was not always detected in brain regions where StAR and steroidogenic enzymes were previously shown to be expressed. Also, transcription of TSPO, but not StAR, may be regulated by the experience of hearing song.</p> <p>Conclusions</p> <p>The genes required for estradiol synthesis and action are represented in the zebra finch genome assembly, though the complement of steroidogenic genes may be smaller in birds than in mammals. Coordinated transcription of multiple steroidogenic genes is possible, but results were inconsistent with the hypothesis that StAR and TSPO mRNAs are co-regulated. Integration of genomic and neuroanatomical analyses will continue to provide insights into the evolution and function of steroidogenesis in the songbird brain.</p

Deriva de equipamentos costais na aplicação de glyphosate

RESUMOObjetivou-se, neste trabalho, comparar o risco potencial de deriva provocada por três equipamentos que utilizam diferentes taxas de aplicação do herbicida glyphosate. Os equipamentos avaliados foram: pulverizador costal de acionamento manual, pulverizador costal pressurizado e um pulverizador de micronização centrífuga acionado eletricamente. Os equipamentos foram testados com as pontas de pulverização AXI 110015 e AXI 11003. O risco potencial de deriva foi avaliado em túnel de vento com coletas nas distâncias de 5, 10 e 15 m em relação ao local de pulverização e nas alturas de 0,2; 0,4; 0,6; 0,8 e 1,0 m em referência ao túnel de vento. O pulverizador de micronização centrífuga acionado eletricamente apresentou risco de deriva inferior aos demais equipamentos; a mesma ponta de pulverização, AXI 110015, avaliada em túnel de vento, pode oferecer risco de deriva diferente quando utilizada em pulverizadores costais de acionamento manual e pressurizado