Advances in Low Volume Sample Analysis Using Microfluidic Separation Techniques

During the last decades, a great interest has been shown for miniaturised separation techniques. The use of microfluidic techniques fulfills the constant needs for increasing sample throughput and analysis sensitivity, while reducing costs and sample volume consumption. In this chapter, three microfluidic separation techniques will be addressed: capillary electrophoresis, gas chromatography and liquid chromatography. A special attention will be paid to miniaturised liquid chromatography, with a deep investigation of its advantages compared with classical liquid chromatography. Sample preparation adapted to low volumes (a few µl) will also be discussed

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore.

This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple

Two biomarkers for the screening of cardiac risk among runners ?

Background Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight protein involved in the intracellular uptake and buffering of long chain fatty in the myocardium. Troponin T is a component of the contractile apparatus of the striated musculature. Both are early markers for acute coronary syndrome. Objective The aim of our study was to compare the results obtained with the H-FABP and the highly sensitive cardiac troponins (hsTnT) and to test their cardiospecificity in healthy runners. Design Prospective, cohort study. Setting Amateur marathon runners. Patients 23 runners (marathon) were enrolled. Interventions We drowned blood samples at three times: just before (T0), just after (T1), and three hours after the end of the race (T3). Main outcome measurements H-FABP and hs-TnT were performed according to the manufacturer's instructions. A linear regression was calculated to observe if there is any correlation between the two biomarkers. Values above the 95th percentile for H-FABP (2.5 ng/mL) and the 99th percentile for hsTnT (14 ng/L) were considered as positive. Results At T0, none of the subjects were positive for hsTnT but 35% were positive for H-FABP; at T1, 83% for hsTnT and 100% for H-FABP; at T3, 83% for hsTnT and 96% for H-FABP. At T0, the regression equation was H-FABP T0=3.9454–0.1001×hsTnT T0; at T1: H-FABP T1=51.838–1.7026×hsTnT T1; at T3: H-FABP T3=47.977–1.6193×hsTnT T3. No correlation was observed between the 2 biomarkers. Conclusion We observed a significant increase of H-FABP and hsTnT in runners. These markers are independent to each other. These values could biologically correspond to a heart ischemia. These biomarkers could be helpful for the screening of cardiac risk among runners

Separation of aminoglutethimide enantiomers using single and dual cyclodextrin system in capillary electrophoresis

peer reviewedaudience: researcher, professional, student, popularizatio

Erythroferrone and hepcidin as mediators between erythropoiesis and iron metabolism during allogeneic hematopoietic stem cell transplant.

Hematopoietic cell transplantation (HCT) brings important alterations in erythropoiesis and iron metabolism. Hepcidin, which regulates iron metabolism, increases in iron overload or inflammation and decreases with iron deficiency or activated erythropoiesis. Erythroferrone (ERFE) is the erythroid regulator of hepcidin. We investigated erythropoiesis and iron metabolism after allogeneic HCT in 70 patients randomized between erythropoietin (EPO) treatment or no EPO, by serially measuring hepcidin, ERFE, CRP (inflammation), soluble transferrin receptor (sTfR, erythropoiesis), serum iron and transferrin saturation (Tsat; iron for erythropoiesis) and ferritin (iron stores). We identified biological and clinical factors associated with serum hepcidin and ERFE levels. Serum ERFE correlated overall with sTfR and reticulocytes and inversely with hepcidin. Erythroferrone paralleled sTfR levels, dropping during conditioning and recovering with engraftment. Inversely, hepcidin peaked after conditioning and decreased during engraftment. Erythroferrone and hepcidin were not significantly different with or without EPO. Multivariate analyses showed that the major determinant of ERFE was erythropoiesis (sTfR, reticulocytes or serum Epo). Pretransplant hepcidin was associated with previous RBC transfusions and ferritin. After transplantation, the major determinants of hepcidin were iron status (ferritin at all time points and Tsat at day 56) and erythropoiesis (sTfR or reticulocytes or ERFE), while the impact of inflammation was less clear and clinical parameters had no detectable influence. Hepcidin remained significantly higher in patients with high compared to low pretransplant ferritin. After allogeneic HCT with or without EPO therapy, significant alterations of hepcidin occur between pretransplant and day 180, in correlation with iron status and inversely with erythroid ERFE

O uso das novas tecnologias nas aulas de Geografia para a melhoria do ensino e aprendizagem em escolas de ensino básico

Este trabalho tem como objetivo apresentar a importância do uso das novas tecnologias como instrumento para a melhoria do ensino e aprendizagem em Geografia. A atividade foi realizada em duas escolas do ensino básico na cidade de Fortaleza no ano 2014, onde se desenvolveram práticas com o uso de recursos tecnológicos, tais como a plataforma Moodle, criação e reprodução de slides, além de vídeos, havendo uma maior socialização e produção de conhecimentos e uma nova forma de conduzir as aulas de Geografia. Podemos perceber a importância e os desafios quanto ao uso das novas tecnologias

Challenges for Biomarker Discovery in Body Fluids Using SELDI-TOF-MS

Protein profiling using SELDI-TOF-MS has gained over the past few years an increasing interest in the field of biomarker discovery. The technology presents great potential if some parameters, such as sample handling, SELDI settings, and data analysis, are strictly controlled. Practical considerations to set up a robust and sensitive strategy for biomarker discovery are presented. This paper also reviews biological fluids generally available including a description of their peculiar properties and the preanalytical challenges inherent to sample collection and storage. Finally, some new insights for biomarker identification and validation challenges are provided