Two-dimensional microcolumn separation platform for proteomics consisting of on-line coupled capillary isoelectric focusing and capillary electrochromatography. 1. Evaluation of the capillary-based two-dimensional platform with proteins, peptides, and human serum

In this report, an on-line coupling of capillary isoelectric focusing (CIEF) to capillary electrochromatography (CEC) is developed via a nanoinjector valve for performing two-dimensional (2D) proteomics separation. CIEF constitutes the first separation dimension, while CEC operates as the second separation dimension. Besides the orthogonal migration mechanisms of the two capillary-based separation modes, which lead to a 2D system whose overall peak capacity is the product of the peak capacity of the individual modes, the solvent of the CIEF mode is a weak eluent for the reversed-phase CEC (RP-CEC) mode, thus, allowing the transferring of focused fractions from CIEF to CEC without inducing band broadening, and instead zone sharpening would result. In fact, the transferred focused protein fraction from the CIEF column to the CEC column will stay tightly adsorbed to the inlet top of the CEC column until it will be eluted and separated into its protein components with a hydro-organic mobile phase. The theoretical peak capacity of the CIEF-CEC 2D platform is estimated at n(CIEF) (= 560) x n(CEC) (= 97) = 54 320. This peak capacity is more than needed for proteomics profiling. Also, only a fraction of this peak capacity is needed when looking at heart cuts for performing subproteomics. The 2D platform described here offers the convenience to generate the needed peak capacity to solve a given proteomic separation problem. This is facilitated by the RP-CEC dimension, which ensures rapid isocratic separation of proteins and peptides and rapid solvent change and column equilibration and avoids lengthy gradient elution. The RP-CEC column is based on neutral C17 monolith, which offers high separation efficiency and relatively high column permeability. To the best of our knowledge, the proposed 2D platform combining CIEF and CEC is reported for the first time for proteins and proteomics.     

Adsorption kinetics of low-density lipoproteins with Langmuir monolayer

The present work utilizes the Langmuir monolayer technique to detect the adsorption kinetics of native low-density lipoproteins and their oxidized form with the lipid monolayer. We found that low-density lipoproteins and oxidized low-density lipoproteins are able to penetrate the LM up to pressure π?=?9.9 and 11.6 mN/m. Also, the adsorption constants of both particles were found to depend strongly on the monolayer initial pressure. It is found that less compressed lipid monolayers could accommodate more native low-density lipoproteins than the oxidized ones due their higher binding affinity toward monolayers. The probable α-helical regions along the apoproteinB-100 secondary structure and average hydrophobicity could explain partially their adsorption kinetics into lipid monolayers. This simplified ‘in vitro’ study of low-density lipoprotein–monolayer interaction may serve as a step further to understand the mechanism and bioactivity of the atherosclerotic process. Also, it may shed light on the oxidized low-density lipoprotein’s role in plaque formation in the innermost arterial wall in blood vessels.     

Spectral signatures of heterogeneous protein ensembles revealed by MD Simulations of 2DIR spectra

A model for the calculation of amide I FTIR and 2DIR spectra taking into account fluctuations in hydrogen bonding and structure from molecular dynamics (MD) simulations is tested on three systems. It is found that although the homogeneous lineshape approximation yields satisfactory FTIR spectra, 2DIR spectra are sensitive to the inhomogeneity naturally present in any solvated protein and the common approximations of a static structure and averaged-effect solvent are invalid. By building on the local amide Hamiltonian and incorporating site energy variation with electrostatic-based models and disorder from MD trajectories, good agreement is obtained between calculated and measured 2DIR spectra. The largest contribution to the observed inhomogeneity is found to be the fluctuating site energies, which in turn are most sensitive to the water solvent. With the ability to accurately predict 2DIR spectra from atomistic simulations, new opportunities to test force fields and mechanistic predictions from MD are revealed.     

Fifty years of violent war deaths from Vietnam to Bosnia: analysis of data from the world health survey programme

Objective To provide an accurate estimate of violent war deaths. Design Analysis of survey data on mortality, adjusted for sampling bias and censoring, from nationally representative surveys designed to measure population health. Estimated deaths compared with estimates in database of passive reports.Setting 2002-3 World health surveys, in which information was collected from one respondent per household about sibling deaths, including whether such deaths resulted from war injuries.Main outcome measure Estimated deaths from war injuries in 13 countries over 50 years.Results From 1955 to 2002, data from the surveys indicated an estimated 5.4 million violent war deaths (95% confidence interval 3.0 to 8.7 million) in 13 countries, ranging from 7000 in the Democratic Republic of Congo to 3.8 million in Vietnam. From 1995 to 2002 survey data indicate 36 000 war deaths annually (16 000 to 71 000) in the 13 countries studied. Data from passive surveillance, however, indicated a figure of only a third of this. On the basis of the relation between world health survey data and passive reports, we estimate 378 000 globalwar deaths annually from 1985-94, the last years for which complete passive surveillance data were available.Conclusions The use of data on sibling history from peacetime population surveys can retrospectively estimate mortality from war. War causes more deaths than previously estimated, and there is no evidence to support a recent decline in war deaths.     

Isolation and characterization of an {alpha}1,2-mannosidase cDNA from the lepidopteran insect cell line Sf9

As part of our ongoing efforts to characterize the N-glycosylationpathway of lepidopteran insect cells, we have isolated an     

oriX: A new replication origin in E. coli

Replication of the chromosome of E. coli at 42°C in an integratively suppressed dnaA mutant (dnaA46 Sin Hfr) occurs predominantly from the origin of replication of the integrated plasmid (oriV). We have carried out a detailed marker frequency analysis on such Hfrs. This analysis indicates that replication at 42°C occurs not only from oriV, but also from an origin, oriX, located in the terminal region of the chromosome close to, but distinct from, the prophage rac (oriJ). In an oxal mutant of one of these Hfrs, we have shown that replication proceeds at 42°C from all three origins: oriV, oriX, and oriC. Loss of the integrated plasmid results in a temperature- and rich-medium-sensitive strain that replicates the chromosome from oriC and oriX. Replication from oriX proceeds slowly and bidirectionally. We suggest that oriX may be involved in the coupling between replication and cell division.