Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns

<p>Abstract</p> <p>Background</p> <p>In cerebrospinal fluid (CSF), which is a rich source of biomarkers for neurological diseases, identification of biomarkers requires methods that allow reproducible detection of low abundance proteins. It is therefore crucial to decrease dynamic range and improve assessment of protein abundance.</p> <p>Results</p> <p>We applied LC-MS/MS to compare the performance of two CSF enrichment techniques that immunodeplete either albumin alone (IgYHSA) or 14 high-abundance proteins (IgY14). In order to estimate dynamic range of proteins identified, we measured protein abundance with APEX spectral counting method.</p> <p>Both immunodepletion methods improved the number of low-abundance proteins detected (3-fold for IgYHSA, 4-fold for IgY14). The 10 most abundant proteins following immunodepletion accounted for 41% (IgY14) and 46% (IgYHSA) of CSF protein content, whereas they accounted for 64% in non-depleted samples, thus demonstrating significant enrichment of low-abundance proteins. Defined proteomics experiment metrics showed overall good reproducibility of the two immunodepletion methods and MS analysis. Moreover, offline peptide fractionation in IgYHSA sample allowed a 4-fold increase of proteins identified (520 vs. 131 without fractionation), without hindering reproducibility.</p> <p>Conclusions</p> <p>The novelty of this study was to show the advantages and drawbacks of these methods side-to-side. Taking into account the improved detection and potential loss of non-target proteins following extensive immunodepletion, it is concluded that both depletion methods combined with spectral counting may be of interest before further fractionation, when searching for CSF biomarkers. According to the reliable identification and quantitation obtained with APEX algorithm, it may be considered as a cheap and quick alternative to study sample proteomic content.</p

Review of epidemiologic data on the debate over smokeless tobacco's role in harm reduction

Some tobacco researchers have argued that the European Union should remove its ban on a form of low-nitrosamine smokeless tobacco referred to as Swedish 'snus'. This argument has developed in to an international debate over the use of smokeless tobacco as a measure of harm reduction for smokers. Leading authorities in the USA have firmly stated that there is no safe tobacco - a message which does not allow for any discussion of comparative tobacco risks. This commentary is intended to review the origin of the controversy over Swedish 'snus', to examine briefly the meta-analysis on cancer risks by Peter Lee and Jan Hamling (published in July in BMC Medicine) and to discuss the anticipated direction of the debate on tobacco-harm reduction in the USA. We anticipate that much of the debate will shift from the discussion of epidemiologic data to the discussion of the marketing, health communication and economics of smokeless tobacco. While the Food and Drug Administration's newly approved authority over tobacco will undoubtedly affect the smokeless products, it may not be the sole determinant of harm reduction's fate in the USA

What potential has tobacco control for reducing health inequalities? The New Zealand situation

In this Commentary, we aim to synthesize recent epidemiological data on tobacco and health inequalities for New Zealand and present it in new ways. We also aim to describe both existing and potential tobacco control responses for addressing these inequalities. In New Zealand smoking prevalence is higher amongst Māori and Pacific peoples (compared to those of "New Zealand European" ethnicity) and amongst those with low socioeconomic position (SEP). Consequently the smoking-related mortality burden is higher among these populations. Regarding the gap in mortality between low and high socioeconomic groups, 21% and 11% of this gap for men and women was estimated to be due to smoking in 1996–99. Regarding the gap in mortality between Māori and non-Māori/non-Pacific, 5% and 8% of this gap for men and women was estimated to be due to smoking. The estimates from both these studies are probably moderate underestimates due to misclassification bias of smoking status. Despite the modest relative contribution of smoking to these gaps, the absolute number of smoking-attributable deaths is sizable and amenable to policy and health sector responses. There is some evidence, from New Zealand and elsewhere, for interventions that reduce smoking by low-income populations and indigenous peoples. These include tobacco taxation, thematically appropriate mass media campaigns, and appropriate smoking cessation support services. But there are as yet untried interventions with major potential. A key one is for a tighter regulatory framework that could rapidly shift the nicotine market towards pharmaceutical-grade nicotine (or smokeless tobacco products) and away from smoked tobacco

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Feedback from the Field: Feasibility of the Perinatal Food Group Recall Form

Background: Maintaining a healthy weight can reduce risks of excessive weight gain during pregnancy and help prevent maternal and child obesity. Low-income women are high-risk for excessive weight gain during pregnancy. Nutrition counseling can help improve dietary habits, however, it is often provided by paraprofessionals who lack formal training in dietetics. There is a need for a brief dietary assessment form for paraprofessionals serving low-income perinatal women. Purpose: The purpose of this study was to provide feedback on the feasibility of the Perinatal Food Group Recall (PFGR) form among Comprehensive Perinatal Health Workers (CPHWs) serving low-income pregnant and post-partum mothers. Methods: Nine CPHWs at four clinics were asked to complete their regular dietary assessment form and then administer the PFGR. Key informant interviews with the nine paraprofessionals were conducted to obtain their feedback on the form. Results: The findings suggest that the form provides a feasible and easyto-administer dietary assessment tool for use by practitioners without formal dietetics training who serve clinics in low-income communities with heavy client flow. Conclusion: The form can facilitate the provision of nutrition counseling among low-income mothers and build capacity among community health workers

European Union policy on smokeless tobacco: a statement in favour of evidence based regulation for public health

Rationale: This statement is an updated version of one released by the same authors in February 2003. The statement was produced to follow up the Royal College of Physicians (RCP) Tobacco Advisory Group report "Protecting smokers, saving lives: the case for a tobacco and nicotine regulatory authority",(1) which argued for an evidence based regulatory approach to smokeless tobacco and harm reduction and posed a series of questions that regulators must address in relation to smokeless tobacco. The purpose of this statement is to provide arguments of fact and principle to follow the RCP's report and to outline the public health case for changing existing European Union (EU) regulation in this area. A review of regulation in relation to harm reduction and regulation of tobacco products other than cigarettes is required in Article 11 of EU directive 2001/37/EC,(2) and this is a contribution towards forming a consensus in the European public health community about what policy the EU should adopt in the light of this review, or following ongoing legal action that may potentially strike out the existing regulation altogether. Public health case: We believe that the partial ban applied to some forms of smokeless tobacco in the EU should be replaced by regulation of the toxicity of all smokeless tobacco. We hold this view for public health reasons: smokeless tobacco is substantially less harmful than smoking and evidence from Sweden suggests it is used as a substitute for smoking and for smoking cessation. To the extent there is a "gateway" it appears not to lead to smoking, but away from it and is an important reason why Sweden has the lowest rates of tobacco related disease in Europe. We think it is wrong to deny other Europeans this option for risk reduction and that the current ban violates rights of smokers to control their own risks. For smokers that are addicted to nicotine and cannot or will not stop, it is important that they can take advantage of much less hazardous forms of nicotine and tobacco—the alternative being to "quit or die"... and many die. While nicotine replacement therapies (NRT) may have a role in harm reduction, tobacco based harm reduction options may reach more smokers and in a different, market based, way. Chewing tobacco is not banned or regulated in the EU but is often highly toxic, and our proposal could remove more products from the market than it permitted. Regulatory options: We believe that the EU policy on smokeless tobacco should adapt to new scientific knowledge and that the European Commission should bring forward proposals to amend or replace Article 8 of directive 2001/37/EC with a new regulatory framework. Canada has developed testing regimens for tobacco constituents and these could be readily adapted to the European situation. A review of EU policy in this area is required no later than December 2004, and we believe the Commission should expedite the part of its review that deals with harm reduction and regulation of tobacco products other than cigarettes so as to reconsider its policy on smokeless tobacco. We held this view before Swedish Match brought its legal proceedings to challenge EU legislation and we will continue to hold these views if its action fails

Reproducibility of tryptic digestion investigated by quantitative fourier transform ion cyclotron resonance mass spectrometry

In this study, the reproducibility of tryptic digestion of complex solutions was investigated using liquid chromatography Fourier transform ion cyclotron resonance (LC FT-ICR) mass spectrometry. Tryptic peptides, from human cerebrospinal fluid, (CSF) were labeled with Quantification-Using-Enhanced-Signal-Tags (QUEST)-markers, or 1-([H-4]nicotinoyloxy)- and 1-([D-4]nicotinoyloxy)-succinimide ester markers. The analysis was performed on abundant proteins with respect-to-intensity ratios and sequence coverage and obtained by comparing differently labeled components from one or different pools. To interpret the dynamics in the proteome, one must be able to estimate the error introduced in each experimental steps. The intra sample variation due to derivatization was approximately 10%. The inter sample variation depending on derivatization and tryptic digestion was not more than approximately 30%. These experimental observations provide a range for the up- and clown-regulations that are possible to study with electrospray ionization LC FT-ICR mass spectrometry