Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

A number of commercial software packages are currently available to perform digital two-dimensional electrophoresis (2D-GE) gel analysis. However, both the high cost of the commercial packages and the unavailability of a standard data analysis workflow, have prompted several groups to develop freeware systems to perform certain steps of gel analysis. Unfortunately, to the best of our knowledge none of them offer a package that performs all the steps envisaged in a 2D-GE gel analysis. Here we describe an ImageJ-based procedure, able to manage all the steps of a 2D-GE gel analysis. ImageJ is a free available image processing and analysis application developed by National Institutes of Health (NIH) and widely used in different life sciences fields as medical imaging, microscopy, western blotting and PAGE. Nevertheless no one has yet developed a procedure enabled to compare spots on 2D-GE gels. We collected all used ImageJ tools in a plug-in that allows us to perform the whole 2D-GE analysis. To test it, we performed a set of 2D-GE experiments on plasma samples from 9 patients victims of acute myocardial infarction and 8 controls, and we compared the results obtained by our procedure to those obtained using a widely diffuse commercial package, finding similar performance

Image Analysis Workflow for 2-D Electrophoresis Gels Based on ImageJ

A number of commercial software packages are currently available to perform digital two-dimensional electrophoresis (2D-GE) gel analysis. However, both the high cost of the commercial packages and the unavailability of a standard data analysis workflow, have prompted several groups to develop freeware systems to perform certain steps of gel analysis. Unfortunately, to the best of our knowledge none of them offer a package that performs all the steps envisaged in a 2D-GE gel analysis. Here we describe an ImageJ-based procedure, able to manage all the steps of a 2D-GE gel analysis. ImageJ is a free available image processing and analysis application developed by National Institutes of Health (NIH) and widely used in different life sciences fields as medical imaging, microscopy, western blotting and PAGE. Nevertheless no one has yet developed a procedure enabled to compare spots on 2D-GE gels. We collected all used ImageJ tools in a plug-in that allows us to perform the whole 2D-GE analysis. To test it, we performed a set of 2D-GE experiments on plasma samples from 9 patients victims of acute myocardial infarction and 8 controls, and we compared the results obtained by our procedure to those obtained using a widely diffuse commercial package, finding similar performances

LCAT cholesterol esterification is associated with the increase of ApoE/ApoA-I ratio during atherosclerosis progression in rabbit

Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E, and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analyzed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8 weeks with 2 % cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters/cholesterol molar ratio, and in vitro. Apolipoproteins levels were analyzed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7- and 2.5-fold, respectively, compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4 weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle. © 2012 University of Navarra

LCAT cholesterol esterification is associated with the increase of ApoE/ApoA-I ratio during atherosclerosis progression in rabbit

Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E, and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analyzed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8 weeks with 2 % cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters/cholesterol molar ratio, and in vitro. Apolipoproteins levels were analyzed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7- and 2.5-fold, respectively, compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4 weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle. © 2012 University of Navarra

Glucose-6-phosphate dehydrogenase plays a crucial role in the protection from redox-stress induced apoptosis.

Glucose-6-phosphate dehydrogenase-deleted embryonic stem (ES) cells (G6pdD) proliferate in vitro without special requirements, but when challenged with oxidants fail to sustain glutathione disulphide reconversion to reduced glutathione (GSH), entering a condition of oxidative stress. Here, we investigate the signalling events downstream of GSH oxidation in G6pdD and wild-type (wt) ES cells. We found that G6pdD ES cells are very sensitive to oxidants, activating an apoptotic pathway at oxidant concentrations otherwise sublethal for wt ES cells. We show that the apoptotic pathway activated by low oxidant concentrations is accompanied by mitochondria dysfunction, and it is therefore blocked by the overexpression of Bcl-XL. Bcl-XL does not inhibit the decrease in cellular GSH and reactive oxygen species formation following oxidant treatment. We also found that oxidant treatment in ES cells is followed by the activation of the MEK/ extracellular signal-regulated kinase (ERK) pathway. Interest¬ingly, ERK activation has opposite outcomes in G6pdD ES cells compared to wt, which has a proapoptotic function in the first and a prosurvival function in the latter. We show that this phenomenon can be regulated by the cellular GSH level

Haptoglobin from Psoriatic Patients Exhibits Decreased Activity in Binding Hemoglobin and Inhibiting LCAT Activity.

Objective: The aim of this work was to assess whether psoriasis is associated with phenotype prevalence and altered activity of Haptoglobin (Hpt). Background: Hpt is a plasma acute phase glycoprotein, displaying in humans three phenotypes. Phenotype prevalence or structure modification of Hpt was associated with several diseases. The Hpt main function is to bind and carry to the liver free hemoglobin for degradation and iron recycling. Hpt was recently found able to bind the apolipoprotein A-I (ApoA-I), thus impairing its stimulation on the activity of the enzyme lecithin-cholesterol acyl-transferase (LCAT) Study design: Hpt was isolated from patients with psoriasis vulgaris, and its activity in hemoglobin or ApoA-I binding and LCAT inhibition was compared with that of normal protein. Methods: Two affinity chromatography steps, the first using resin-coupled hemoglobin and the second anti-Hpt antibodies, were used to purify Hpt. The protein phenotype was assessed by electrophoresis. Binding experiments were performed by ELISA with stationary hemoglobin or ApoA-I, Hpt in solution, and anti-Hpt antibodies for detection of bound Hpt. Standard LCAT assays were carried out in the presence of Hpt purified from patients or healthy subjects. Results: Phenotype prevalence of Hpt in psoriasis was not found. After affinity chromatography by hemoglobin, albumin and ApoA-I were routinely found heavily contaminating only Hpt from normal subjects. Isolated Hpt from patients had lower activity than normal protein in both hemoglobin binding and LCAT inhibition. Conclusions: In psoriasis, Hpt displays some structure modification(s) which might be associated with the protein function in the disease

Epidemiology of Strongyloides stercoralis in northern Italy: Results of a multicentre case-control study, February 2013 to July 2014

Strongyloides stercoralis is a soil-transmitted helminth widely diffused in tropical and subtropical regions of the world. Autochthonous cases have been also diagnosed sporadically in areas of temperate climate. We aimed at defining the epidemiology of strongyloidiasis in immigrants and Italians living in three northern Italian Regions. Screening for S. stercoralis infection was done with serology, confirmation tests were a second serological method or stool agar culture. A case-control approach was adopted and patients with a peripheral eosinophil count 65 500/mcL were classified as cases. Of 2,701 individuals enrolled here 1,351 were cases and 1,350 controls; 86% were Italians, 48% women. Italians testing positive were in 8% (97/1,137) cases and 1% (13/1,178) controls (adjusted odds ratio (aOR) 8.2; 95% confidence interval (CI): 4.5-14.8), while positive immigrants were in 17% (36/214) cases and in 2% (3/172) controls (aOR 9.6; 95% CI: 2.9-32.4). Factors associated with a higher risk of infection for all study participants were eosinophilia (p < 0.001) and immigration (p = 0.001). Overall, strongyloidiasis was nine-times more frequent in individuals with eosinophilia than in those with normal eosinophil count

Liver Enzyme Abnormalities and Associated Risk Factors in HIV Patients on Efavirenz-Based HAART with or without Tuberculosis Co-Infection in Tanzania.

To investigate the timing, incidence, clinical presentation, pharmacokinetics and pharmacogenetic predictors for antiretroviral and anti-tuberculosis drug induced liver injury (DILI) in HIV patients with or without TB co-infection. A total of 473 treatment naïve HIV patients (253 HIV only and 220 with HIV-TB co-infection) were enrolled prospectively. Plasma efavirenz concentration and CYP2B6*6, CYP3A5*3, *6 and *7, ABCB1 3435C/T and SLCO1B1 genotypes were determined. Demographic, clinical and laboratory data were collected at baseline and up to 48 weeks of antiretroviral therapy. DILI case definition was according to Council for International Organizations of Medical Sciences (CIOMS). Incidence of DILI and identification of predictors was evaluated using Cox Proportional Hazards Model. The overall incidence of DILI was 7.8% (8.3 per 1000 person-week), being non-significantly higher among patients receiving concomitant anti-TB and HAART (10.0%, 10.7 per 1000 person-week) than those receiving HAART alone (5.9%, 6.3 per 1000 person-week). Frequency of CYP2B6*6 allele (p = 0.03) and CYP2B6*6/*6 genotype (p = 0.06) was significantly higher in patients with DILI than those without. Multivariate cox regression model indicated that CYP2B6*6/*6 genotype and anti-HCV IgG antibody positive as significant predictors of DILI. Median time to DILI was 2 weeks after HAART initiation and no DILI onset was observed after 12 weeks. No severe DILI was seen and the gain in CD4 was similar in patients with or without DILI. Antiretroviral and anti-tuberculosis DILI does occur in our setting, presenting early following HAART initiation. DILI seen is mild, transient and may not require treatment interruption. There is good tolerance to HAART and anti-TB with similar immunological outcomes. Genetic make-up mainly CYP2B6 genotype influences the development of efavirenz based HAART liver injury in Tanzanians

Four lectures on secant varieties

This paper is based on the first author's lectures at the 2012 University of Regina Workshop "Connections Between Algebra and Geometry". Its aim is to provide an introduction to the theory of higher secant varieties and their applications. Several references and solved exercises are also included.Comment: Lectures notes to appear in PROMS (Springer Proceedings in Mathematics & Statistics), Springer/Birkhause

Impact of social determinants on antiretroviral therapy access and outcomes entering the era of universal treatment for people living with HIV in Italy

Background: Social determinants are known to be a driving force of health inequalities, even in high income countries. Aim of our study was to determine if these factors can limit antiretroviral therapy (ART) access, outcome and retention in care of people living with HIV (PLHIV) in Italy. Methods: All ART naïve HIV+ patients (pts) of Italian nationality enrolled in the ICONA Cohort from 2002 to 2016 were included. The association of socio-demographic characteristics (age, sex, risk factor for HIV infection, educational level, occupational status and residency area) with time to: ART initiation (from the first positive anti-HIV test), ART regimen discontinuation, and first HIV-RNA < 50 cp/mL, were evaluated by Cox regression analysis, Kaplan Meier method and log-rank test. Results: A total of 8023 HIV+ pts (82% males, median age at first pos anti-HIV test 36 years, IQR: 29-44) were included: 6214 (77.5%) started ART during the study period. Women, people who inject drugs (PWID) and residents in Southern Italy presented the lowest levels of education and the highest rate of unemployment compared to other groups. Females, pts aged > 50 yrs., unemployed vs employed, and people with lower educational levels presented the lowest CD4 count at ART initiation compared to other groups. The overall median time to ART initiation was 0.6 years (yrs) (IQR 0.1-3.7), with a significant decrease over time [2002-2006 = 3.3 yrs. (0.2-9.4); 2007-2011 = 1.0 yrs. (0.1-3.9); 2012-2016 = 0.2 yrs. (0.1-2.1), p < 0.001]. By multivariate analysis, females (p < 0.01) and PWID (p < 0.001), presented a longer time to ART initiation, while older people (p < 0.001), people with higher educational levels (p < 0.001), unemployed (p = 0.02) and students (p < 0.001) were more likely to initiate ART. Moreover, PWID, unemployed vs stable employed, and pts. with lower educational levels showed a lower 1-year probability of achieving HIV-RNA suppression, while females, older patients, men who have sex with men (MSM), unemployed had higher 1-year risk of first-line ART discontinuation. Conclusions: Despite median time to ART start decreased from 2002 to 2016, socio-demographic factors still contribute to disparities in ART initiation, outcome and durability