Niagara, County of and Niagara County White Collar Employee Unit, CSEA Local 1000, AFSCME, AFL-CIO, Local 832 (2012) (MOA)

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) and multiple reaction monitoring mass spectrometry (MRM-MS) proteomics analyses were performed on eccrine sweat of healthy controls, and the results were compared with those from individuals diagnosed with schizophrenia (SZ). This is the first large scale study of the sweat proteome. First, we performed LC–MS/MS on pooled SZ samples and pooled control samples for global proteomics analysis. Results revealed a high abundance of diverse proteins and peptides in eccrine sweat. Most of the proteins identified from sweat samples were found to be different than the most abundant proteins from serum, which indicates that eccrine sweat is not simply a plasma transudate and may thereby be a source of unique disease-associated biomolecules. A second independent set of patient and control sweat samples were analyzed by LC–MS/MS and spectral counting to determine qualitative protein differential abundances between the control and disease groups. Differential abundances of selected proteins, initially determined by spectral counting, were verified by MRM-MS analyses. Seventeen proteins showed a differential abundance of approximately 2-fold or greater between the SZ pooled sample and the control pooled sample. This study demonstrates the utility of LC–MS/MS and MRM-MS as a viable strategy for the discovery and verification of potential sweat protein disease biomarkers

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste’s proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste’s predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes’ complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://[email protected])

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste’s proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste’s predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes’ complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://[email protected])

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste’s proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste’s predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes’ complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://[email protected])

Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of <i>Bacillus anthracis</i> Spore-Challenged Mice

<div><p>Anthrax is a frequently fatal infection of many animal species and men. The causative agent <i>Bacillus anthracis</i> propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissesction. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host.</p></div

Immunohistochemical staining of formalin-fixed sections of popliteal LNs for Puma (brown) in control (left panel) and <i>B</i>. <i>anthracis</i> Sterne-challenged (right panel) mice.

<p><b>Hematoxylin counterstain (blue) was used.</b> The dSterne-challenged mice demonstrated to Puma stain (not shown).</p

Immunohistochemical staining of formalin-fixed sections of popliteal LNs for phosphorylated ERK1/2 (brown) in naïve (A) and <i>B</i>. <i>anthracis</i> Sterne-challenged mice (B) with hematoxylin counterstain (blue) at different magnifications.

<p>The morphology of a typical CD11b staining is shown for comparison with the ERK1/2 one (right panels).</p

Mortality curves (A) and bacterial load in LNs and the spleen of spore-challenged DBA/2J mice (B-E).

<p>Animals were challenged with the toxinogenic, non-encapsulated <i>B</i>. <i>anthracis</i> Sterne 34F2 or non-toxinogenic, non-encapsulated delta-Sterne (dSterne) spores (4x10⁶ spores in 20 μL of PBS, intradermally into both hind footpads, 3 animals per challenge group. In (B) at the indicated times the animals were anesthetized with isoflurane and 20 μl of a mixture containing 1% tracer dye Evans Blue in PBS were injected into foot pads. The LNs were surgically removed and homogenized for the PFU determination on agar plates before and after heat inactivation of the vegetative bacteria.</p

Immunohistochemical staining of formalin-fixed sections of popliteal LNs for survivin (brown) in naïve (A) and <i>B</i>. <i>anthracis</i> Sterne-challenged mice (B-D) at different magnifications.

<p>Hematoxylin counterstain (blue) was used. The majority of survinin+ cells (immunostained brown) localize to the cortical zones morphologically similar to B-cell lymphoid follicles (dotted lines in A and C) overlapping with bacteria in infected mice (bottom panels C, D). The boxed region in the left bottom panel C is magnified on the right.</p

Immunohistochemical staining of formalin-fixed sections of popliteal LNs for macrosialin/CD68 (brown) in naïve (A) and <i>B</i>. <i>anthracis</i> Sterne-challenged mice (B) with hematoxylin counterstain (blue) at different magnifications.

<p>CD68 expression is induced in the Sterne-infected LNs (B,C,E). The staining demonstrates a punctate pattern mainly in subcapsular (B) and medullary (C) regions overlapping with bacteria (B, E). Control uninfected LNs and dSterne-infected LNs stain negative for CD68 (A, D, respectively). Phagocytic cells contain ingested bacteria with CD68 “caps” (F,G). The phagocytic uptake seems to be required for the staining of bacteria because the un-phagocytosed cells do not demonstrate the staining (F, arrows).</p