A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

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Highlights

• •Liver Mallory-Denk-Body inducers elicited an IκBα-loss and NF-κB-activation.
• •IκBα-loss was due to its sequestration into insoluble cytoplasmic aggregates.
• •Four proteomic approaches identified 10 IκBα-interacting/aggregating proteins.
• •Nup153/RanBP2-aggregation prevented IκBα nuclear entry for ending NF-κB-activation.

     

iTRAQ‐Based Proteomics Reveals that the Tomato ms1035 Gene Causes Male Sterility through Compromising Fat Acid Metabolism

So far, over 50 spontaneous male sterile mutants of tomato have been described and most of them are categorized as genetic male sterility. To date, the mechanism of tomato genetic male sterility remained unclear. In this study, differential proteomic analysis is performed between genetic male sterile line (2‐517), which carries the male sterility (ms10³⁵) gene, and its wild‐type (VF‐11) using isobaric tags for relative and absolute quantification‐based strategy. A total of 8272 proteins are quantified in the 2–517 and VF‐11 lines at the floral bud and florescence stages. These proteins are involved in different cellular and metabolic processes, which express obvious functional tendencies toward the hydroxylation of the ω‐carbon in fatty acids, the tricarboxylic acid cycle, the glycolytic, and pentose phosphate pathways. Based on the results, a protein network explaining the mechanisms of tomato genetic male sterility is proposed, finding the compromising fat acid metabolism may cause the male sterility. These results are confirmed by parallel reaction monitoring, quantitative Real‐time PCR (qRT‐PCR), and physiological assays. Taken together, these results provide new insights into the metabolic pathway of anther abortion induced by ms10³⁵ and offer useful clues to identify the crucial proteins involved in genetic male sterility in tomato.     

Source apportionment and health risk quantification for heavy metal sources in soils near aluminum-plastic manufacturing facilities in northeast China

Abstract

Identifying the source effect on heavy metals to human health risk is essential for devising and implementing restoration policies for polluted soils. For this purpose, eight heavy metals (As, Cd, Hg, Cr, Cu, Ni, Pb, and Zn) in soil profile samples (0–10, 10–20, 20–30, and 30–40?cm) collected in the area around aluminum-plastic manufacturing facilities (APMF) were determined. An absolute principal component score multiple linear regression (APCS-MLR) model supported by a health risk assessment (HRA) model was developed to determine the source apportionment of soil heavy metals and contribution rate of pollution sources to human health risk. Results showed significant accumulations of eight metals in the topsoil (0–20?cm), parent material, transportation, APMF, and agricultural practices were the four major contributing sources for heavy metals in soils, with average contribution percentages of 21.69%, 24.99%, 29.60%, and 14.25%, respectively. Carcinogenic risk factors for adults (1.23E-04) and children (1.32E-04) were found to be above the acceptable level (1E-06 to 1E-04). The health risk quantification results indicated that parent material, APMF, transportation, agricultural practices, and unidentified factors accounted for 55.76%, 14.48%, 12.09%, 10.13%, and 7.54% of the carcinogenic risk for children and adults. The adverse impacts of Cd, Zn, and Pb accumulations in soil coming from APMF activities were significant and need to receive more attention.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.     

3D strain map of axially loaded mouse tibia: a numerical analysis validated by experimental measurements

A combined experimental/numerical study was performed to calculate the 3D octahedral shear strain map in a mouse tibia loaded axially. This study is motivated by the fact that the bone remodelling analysis, in this in vivo mouse model should be performed at the zone of highest mechanical stimulus to maximise the measured effects. Accordingly, it is proposed that quantification of bone remodelling should be performed at the tibial crest and at the distal diaphysis. The numerical model could also be used to furnish a more subtle analysis as a precise correlation between local strain and local biological response can be obtained with the experimentally validated numerical model.     

Isobaric protein and peptide quantification: perspectives and issues

An important challenge for proteomics is the ability to compare protein levels across biological samples. Since their introduction, isotopic and isobaric peptide labeling have played an important role in relative quantitative comparisons of proteomes. One important drawback of most of the isotopic-labeling techniques is an increase in sample complexity. This problem was successfully addressed with the construction of isobaric labeling strategies, such as isobaric tag for relative and absolute quantification (iTRAQ), tandem mass tagging, the cleavable isobaric affinity tag, dimethylated leucines and isobaric peptide termini labeling. Furthermore, numerous applications for multiplexing using iTRAQ and tandem mass tagging have been reported.     

The Plasma Proteome: High Abundance versus Low Abundance

The field of proteomics is rapidly turning towards targeted mass spectrometry (MS) methods to quantify putative markers or known proteins of biological interest. Historically, the enzyme-linked immunosorbent assay (ELISA) has been used for targeted protein analysis, but, unfortunately, it is limited by the excessive time required for antibody preparation, as well as concerns over selectivity. Despite the ability of proteomics to deliver increasingly quantitative measurements, owing to limited sensitivity, the leads generated are in the microgram per milliliter range. This stands in stark contrast to ELISA, which is capable of quantifying proteins at low picogram per milliliter levels. To bridge this gap, targeted liquid chromatography (LC) tandem MS (MS/MS) analysis of tryptic peptide surrogates using selected reaction monitoring detection has emerged as a viable option for rapid quantification of target proteins. The precision of this approach has been enhanced by the use of stable isotope-labeled peptide internal standards to compensate for variation in recovery and the influence of differential matrix effects. Unfortunately, the complexity of proteinaceous matrices, such as plasma, limits the usefulness of this approach to quantification in the mid-nanogram per milliliter range (medium-abundance proteins). This article reviews the current status of LC/MS/MS using selected reaction monitoring for protein quantification, and specifically considers the use of a single antibody to achieve superior enrichment of either the protein target or the released tryptic peptide. Examples of immunoaffinity-assisted LC/MS/MS are reviewed that demonstrate quantitative analysis of low-abundance proteins (subnanogram per milliliter range). A strategy based on this technology is proposed for the expedited evaluation of novel protein biomarkers, which relies on the synergy created from the complementary nature of MS and ELISA.     

Microplate-based,label-free detection of biomolecular interactions: applications in proteomics

This review describes a new type of label-free optical biosensor that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard format microplates to enable highly sensitive, high-throughput detection of small molecules, proteins and cells. The biosensor and associated detection instrumentation are applied to review two fundamental limiting issues for assays in proteomics research and drug discovery: requirement for quantitative measurement of protein concentration and specific activity, and measurements made with complex systems in highly parallel measurements. SRU BIosystems, Inc.’s BIND? label-free detection will address these issues using data examples for hybridoma screening, epitope binning and mapping, small-molecule screening, and cell-based functional assays. The review describes several additional applications that are under development for the system, and the key issues that will drive adoption of the technology over the next 5 years.     

Application of two SH-based methods for metallothionein determination in mussels and intercalibration of the spectrophotometric method: laboratory and field studies in the Mediterranean Sea

Metallothionein (MT) induction is widely used as a biomarker of exposure to metals in mussels. The aims of the present work were first to compare the suitability of spectrophotometry and differential pulse polarography (DPP) for MT detection in mussels exposed to 200 ppb cadmium for 9 days in a laboratory experiment and in mussels sampled in different seasons from expected pollution gradients along the Mediterranean Sea; second, to intercalibrate the widely used spectrophotometric method using mussels from Saronikos Gulf. In the intercalibration of the spectrophotometric method, similar results (p>0.05) were obtained by two different research teams indicating a good reproducibility of the technique. However, polarographic and spectrophotometric methods gave significantly (p<0.05) different results in laboratory and field studies. In the laboratory experiment, MT values detected with DPP were nine times higher than with spectrophotometry. The results obtained by the two methods were significantly correlated. Both methods could discriminate between control and exposed mussels. In field studies, MT values obtained by DPP were 34–38-fold higher than with spectrophotometry, and MT concentrations measured by both methods were not correlated. This discrepancy could be due to several factors, including the low levels of bioavailable metals in the studied areas and the possibility that the different methods can measure MT isoforms differentially. Further work is needed to decipher the functions of MT isoforms in mussels. This information is relevant for the application of MT as a biomarker in biomonitoring programmes.     

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.