Unveiling the Role of Endoplasmic Reticulum Stress Pathways in Canine Demodicosis.

Canine demodicosis is a prevalent skin disease caused by overpopulation of a commensal species of Demodex mite, yet its precise cause remains unknown. Research suggests that T-cell exhaustion, increased immunosuppressive cytokines, induction of regulatory T cells and increased expression of immune checkpoint inhibitors may contribute to its pathogenesis. This study aimed to gain a deeper understanding of the molecular changes occurring in canine demodicosis using mass spectrometry and pathway enrichment analysis. The results indicate that endoplasmic reticulum stress promotes canine demodicosis through regulation of three linked signalling pathways: eIF2, mTOR, and eIF4 and p70S6K. These pathways are involved in the modulation of Toll-like receptors, most notably TLR2, and have been shown to play a role in the pathogenesis of skin diseases in both dogs and humans. Moreover, these pathways are also implicated in the promotion of immunosuppressive M2 phenotype macrophages. Immunohistochemical analysis, utilising common markers of dendritic cells and macrophages, verified the presence of M2 macrophages in canine demodicosis. The proteomic analysis also identified immunological disease, organismal injury and abnormalities and inflammatory response as the most significant underlying diseases and disorders associated with canine demodicosis. This study demonstrates that Demodex mites, through ER stress, unfolded protein response and M2 macrophages contribute to an immunosuppressive microenvironment, thereby assisting in their proliferation.

A proteomic profile of the healthy human placenta.

BACKGROUND: The placenta remains one of the least studied organs within the human body. Yet, placental dysfunction has been associated with various pregnancy complications leading to both maternal and fetal death and long-term health consequences. The aim of this study was to characterise the protein networks of healthy term placental sub-anatomical regions using label free quantification mass spectrometry. METHODS: Three healthy placentae were sampled at five sample sites and each biopsy was dissected into maternal-, middle-, and fetal- sub-anatomical regions. Quadrupole-orbitrap mass spectrometer was used in data dependant analysis mode to identify 1859 unique proteins before detailed differential expression between regions. RESULTS: Protein profiling identified 1081, 1086, and 1101 proteins in maternal, middle, and fetal sub-anatomical regions respectively. Differentially expressed proteins were identified considering the effect between sample site location and sub-anatomical region on protein expression. Of these, 374 differentially expressed proteins (Two-way ANOVA adjusted p-value < 0.05, HSD Tukey adjusted p-value 0.05) were identified between sample site locations and sub-anatomical regions. The placenta specific disease map NaviCenta ( https://www.sbi.uni-rostock.de/minerva/index.xhtml?id=NaviCenta ) was used to focus functional analysis results to the placenta specific context. Subsequently, functional analysis with a focus on senescence, and mitochondrial function were performed. Significant differences were observed between sub-anatomical regions in protein intensity and composition. A decrease in anti-senescent proteins within the maternal sub-anatomical region, and an increase in proteins associated with a switch from ATP to fatty acid consumption as a source of energy between middle and fetal sub-anatomical regions were observed. CONCLUSION: These results suggest that normal proteomic variations exist within the anatomical structure of the placenta, thus recommending serial sectioning methodology for consistent placental research.

Complement pathway changes at age 12 are associated with psychotic experiences at age 18 in a longitudinal population-based study: evidence for a role of stress.

The complement cascade is a major component of the immune defence against infection, and there is increasing evidence for a role of dysregulated complement in major psychiatric disorders. We undertook a directed proteomic analysis of the complement signalling pathway (n = 29 proteins) using data-independent acquisition. Participants were recruited from the UK avon longitudinal study of parents and children (ALSPAC) cohort who participated in psychiatric assessment interviews at ages 12 and 18. Protein expression levels at age 12 among individuals who reported psychotic experiences (PEs) at age 18 (n = 64) were compared with age-matched controls (n = 67). Six out of the 29 targeted complement proteins or protein subcomponents were significantly upregulated following correction for multiple comparisons (VTN↑, C1RL↑, C8B↑, C8A↑, CFH↑, and C5↑). We then undertook an unbiased plasma proteomic analysis of mice exposed to chronic social stress and observed dysregulation of 11 complement proteins, including three that were altered in the same direction in individuals with PE (C1R↑, CFH↑, and C5↑). Our findings indicate that dysregulation of the complement protein pathway in blood is associated with incidence of psychotic experiences and that these changes may reflect exposure to stress.

Predicting response to vascular endothelial growth factor inhibitor and chemotherapy in metastatic colorectal cancer.

BACKGROUND: Bevacizumab improves progression free survival (PFS) and overall survival (OS) in metastatic colorectal cancer patients however currently there are no biomarkers that predict response to this treatment. The aim of this study was to assess if differential protein expression can differentiate patients who respond to chemotherapy and bevacizumab, and to assess if select proteins correlate with patient survival. METHODS: Pre-treatment serum from patients with metastatic colorectal cancer (mCRC) treated with chemotherapy and bevacizumab were divided into responders and nonresponders based on their progression free survival (PFS). Serum samples underwent immunoaffinity depletion and protein expression was analysed using two-dimensional difference gel electrophoresis (2D-DIGE), followed by LC-MS/MS for protein identification. Validation on selected proteins was performed on serum and tissue samples from a larger cohort of patients using ELISA and immunohistochemistry, respectively (n = 68 and n = 95, respectively). RESULTS: 68 proteins were identified following LC-MS/MS analysis to be differentially expressed between the groups. Three proteins (apolipoprotein E (APOE), angiotensinogen (AGT) and vitamin D binding protein (DBP)) were selected for validation studies. Increasing APOE expression in the stroma was associated with shorter progression free survival (PFS) (p = 0.0001) and overall survival (OS) (p = 0.01), DBP expression (stroma) was associated with shorter OS (p = 0.037). Increasing APOE expression in the epithelium was associated with a longer PFS and OS, and AGT epithelial expression was associated with a longer PFS (all p < .05). Increasing serum AGT concentration was associated with shorter OS (p = 0.009). CONCLUSIONS: APOE, DBP and AGT identified were associated with survival outcomes in mCRC patients treated with chemotherapy and bevacizumab.

An Electrophysiological and Proteomic Analysis of the Effects of the Superoxide Dismutase Mimetic, MnTMPyP, on Synaptic Signalling Post-Ischemia in Isolated Rat Hippocampal Slices.

Metabolic stress and the increased production of reactive oxygen species (ROS) are two main contributors to neuronal damage and synaptic plasticity in acute ischemic stroke. The superoxide scavenger MnTMPyP has been previously reported to have a neuroprotective effect in organotypic hippocampal slices and to modulate synaptic transmission after in vitro hypoxia and oxygen-glucose deprivation (OGD). However, the mechanisms involved in the effect of this scavenger remain elusive. In this study, two concentrations of MnTMPyP were evaluated on synaptic transmission during ischemia and post-ischemic synaptic potentiation. The complex molecular changes supporting cellular adaptation to metabolic stress, and how these are modulated by MnTMPyP, were also investigated. Electrophysiological data showed that MnTMPyP causes a decrease in baseline synaptic transmission and impairment of synaptic potentiation. Proteomic analysis performed on MnTMPyP and hypoxia-treated tissue indicated an impairment in vesicular trafficking mechanisms, including reduced expression of Hsp90 and actin signalling. Alterations of vesicular trafficking may lead to reduced probability of neurotransmitter release and AMPA receptor activity, resulting in the observed modulatory effect of MnTMPyP. In OGD, protein enrichment analysis highlighted impairments in cell proliferation and differentiation, such as TGFß1 and CDKN1B signalling, in addition to downregulation of mitochondrial dysfunction and an increased expression of CAMKII. Taken together, our results may indicate modulation of neuronal sensitivity to the ischemic insult, and a complex role for MnTMPyP in synaptic transmission and plasticity, potentially providing molecular insights into the mechanisms mediating the effects of MnTMPyP during ischemia.

Butyrate limits human natural killer cell effector function.

The gut microbiota regulates chronic inflammation and has been implicated in the pathogenesis of a broad spectrum of disease including autoimmunity and cancer. Microbial short-chain fatty acids (SCFAs) e.g., butyrate have demonstrated immunomodulatory effects and are thought to be key mediators of the host-microbiome interaction. Here, we investigated the effect of butyrate on effector functions of blood derived human NK cells stimulated for 18 h with a combination of IL-12/IL-15, a potent mix of cytokines that drive NK cell activation. We show that butyrate has a strong anti-inflammatory effect on NK cells. NK cells cultured in the presence of butyrate expressed lower levels of activating receptors (TRAIL, NKp30, NKp44) and produced lower levels of cytokines (IFNγ, TNF-α, IL-22, granzyme B, granzyme A, perforin) in response to IL-12/IL-15. Butyrate restricted NK cell function by downregulation of mTORC1 activity, c-Myc mRNA expression and metabolism. Using a shotgun proteomic approach, we confirmed the effect of butyrate on NK cell cytokine signaling and metabolism and identified BRD2, MAT2A and EHD1 as downstream mediators of these effects. This insight into the immunomodulatory activity of butyrate on human NK cell function might help to develop new ways to limit NK cell function during chronic inflammation.

Platelet proteo-transcriptomic profiling validates mediators of thrombosis and proteostasis in patients with myeloproliferative neoplasms.

Patients with chronic Myeloproliferative Neoplasms (MPN) including polycythemia vera (PV) and essential thrombocythemia (ET) exhibit unique clinical features, such as a tendency toward thrombosis and hemorrhage, and risk of disease progression to secondary bone marrow fibrosis and/or acute leukemia. Although an increase in blood cell lineage counts (quantitative features) contribute to these morbid sequelae, the significant qualitative abnormalities of myeloid cells that contribute to vascular risk are not well understood. Here, we address this critical knowledge gap via a comprehensive and untargeted profiling of the platelet proteome in a large (n= 140) cohort of patients (from two independent sites) with an established diagnosis of PV and ET (and complement prior work on the MPN platelet transcriptome from a third site). We discover distinct MPN platelet protein expression and confirm key molecular impairments associated with proteostasis and thrombosis mechanisms of potential relevance to MPN pathology. Specifically, we validate expression of high-priority candidate markers from the platelet transcriptome at the platelet proteome (e.g., calreticulin (CALR), Fc gamma receptor (FcγRIIA) and galectin-1 (LGALS1) pointing to their likely significance in the proinflammatory, prothrombotic and profibrotic phenotypes in patients with MPN. Together, our proteo-transcriptomic study identifies the peripherally-derived platelet molecular profile as a potential window into MPN pathophysiology and demonstrates the value of integrative multi-omic approaches in gaining a better understanding of the complex molecular dynamics of disease.

Benefits of heat treatment to the protease packed neutrophil for proteome analysis: halting protein degradation.

Neutrophils, cells of the innate immune system, contain an array of proteases and reactive oxygen species-generating enzymes that assist in controlling the invasion of bacteria and pathogens. The high content of intracellular proteolytic enzymes makes them difficult cells to work with as they can degrade proteins of potential interest. Here, we describe the benefits of heat treatment of neutrophils in reducing protein degradation for subsequent proteome analysis. Neutrophils isolated from four healthy volunteers were each divided into three aliquots and subjected to different preparation methods for 2-DE: (i) Heat treatment, (ii) resuspension in NP40 lysis buffer and (iii) resuspension in standard 2-DE lysis buffer. Representative spots found to be statistically significant between groups (p<0.01) were excised and identified by LC-MS/MS, three of which were validated by immunoblotting. Heat-treated samples contained proteins in the high-molecular-weight range that were absent from NP40-treated samples. Moreover, NP40-treated samples showed an increase in spot number and volume at lower molecular weights suggestive of protein degradation. Incorporating heat treatment into sample preparation resulted in the identification of proteins that may not have previously been detected due to sample degradation, thus leading to a more comprehensive 2-DE map of the human neutrophil proteome.

Glycomic and glycoproteomic analysis of serum from patients with stomach cancer reveals potential markers arising from host defense response mechanisms.

Despite the reduced incidence of gastric cancer in the developed world, a diagnosis of stomach carcinoma still carries a poor prognosis due to the asymptomatic nature of the disease in the early stages, subsequent advanced stage diagnosis, and a low 5 year survival rate. Endoscopy remains the primary standard for diagnosis of stomach carcinoma and the current marker, carbohydrate antigen 19-9 (CA19-9) lacks the levels of sensitivity and specificity required in order to make it clinically useful for diagnostic monitoring. Therefore, there is a current need for additional markers to improve the diagnostic accuracy for the early stages of stomach cancer. Together, glycomic, proteomic, and glycoproteomic analyses of serum have the potential to identify such probable markers. A discovery study is reported here using preoperative serum from 80 stomach cancer patients, 10 patients bearing benign stomach disease, and 20 matched controls. Glycomic analysis of the total and immunoaffinity depleted serum revealed statistically significant increases in the levels of sialyl Lewis X epitopes (SLe(X)) present on triantennary glycans accompanied by increased levels of core fucosylated agalactosyl biantennary glycans present on IgG (referred to as the IgG G0 glycoform) which are associated with increasing disease pathogenesis. Protein expression analysis using 2D-DiGE returned a number of differentially expressed protein candidates in the depleted serum, many of which were shown to carry triantennary SLe(X) during subsequent glycomic investigations. Biological pathway analysis of the experimental data returned complement activation and acute phase response signaling as the most significantly altered pathways in the stomach cancer patient serum. Upon the basis of these findings, it is suggested that increased expression of IgG G0 and complement activation are a host response to the presence of the stomach tumor while the increased expression of SLe(X) and acute phase response proteins is a result of pro-inflammatory cytokine signaling, including IL-6, during carcinogenesis. The approach presented herein provides an insight into the underlying mechanisms of disease and the resulting changes in the glycome and glycoproteome offer promise as potential markers for diagnosis and prognostic monitoring in stomach cancer.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts.

Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 µM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

2-D DIGE analysis of the budding yeast pH 6-11 proteome in meiosis.

Meiosis is the cell division that generates haploid gametes from diploid precursors. To provide insight into the functional proteome of budding yeast during meiosis, a 2-D DIGE kinetic approach was used to study proteins in the pH 6-11 range. Nearly 600 protein spots were visualised and 79 spots exhibited statistically significant changes in abundance as cells progressed through meiosis. Expression changes of up to 41-fold were detected and protein sequence information was obtained for 48 spots. Single protein identifications were obtained for 21 spots including different gel mobility forms of 5 proteins. A large number of post-translational events are suggested for these proteins, including processing, modification and import. The data are incorporated into an online 2-DE map of meiotic proteins in budding yeast, which extends our initial DIGE investigation of proteins in the pH 4-7 range. Together, the analyses provide peptide sequence data for 84 protein spots, including 50 single-protein identifications and gel mobility isoforms of 8 proteins. The largest classes of identified proteins include carbon metabolism, protein catabolism, protein folding, protein synthesis and the oxidative stress response. A number of the corresponding genes are required for yeast meiosis and recent studies have identified similar classes of proteins expressed during mammalian meiosis. This proteomic investigation and the resulting protein reference map make an important contribution towards a more detailed molecular view of yeast meiosis.

Analysis of the budding yeast pH 4-7 proteome in meiosis.

Meiosis, the developmental programme generating haploid gametes from diploid precursors, requires two cell divisions and many innovations. In budding yeast, a large number of genes are expressed exclusively during meiosis while others are repressed compared to vegetative growth. Microarray analysis has shown that gene expression during meiosis is highly regulated, and has been used to classify yeast genes according to meiotic temporal expression pattern. In this study, we have begun to investigate the kinetics of meiotic protein expression using a proteomics approach. 2-D DIGE was used to characterise the temporal protein expression patterns of the budding yeast pH 4-7 proteome in meiosis. More than 1400 meiotic protein spots were visualised and at least 63 spots were temporally regulated during meiosis in a statistically significant manner. Gel spots with significant expression changes were excised and 26 unique proteins were identified using LC-MS/MS. The identified proteins could be classified into functional categories and the genes encoding a number of these were previously shown to be involved in yeast sporulation and meiosis. This data set was used to assemble the first differential 2-D PAGE map of budding yeast meiosis, which can be accessed through a web server. This work represents one of the first quantitative proteomic analyses of meiosis in yeast and will provide a valuable resource for future investigations.

Stratification and monitoring of juvenile idiopathic arthritis patients by synovial proteome analysis.

Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish patients in whom inflammation extends to affect a large number of joints, early in the disease process. SF samples from 22 JIA patients were analyzed: 10 with oligoarticular arthritis, 5 extended oligoarticular and 7 polyarticular disease. SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression further verified by Western immunoblotting and immunohistochemistry. Hierarchical clustering based on the expression levels of a set of 40 proteins segregated the extended oligoarticular from the oligoarticular patients (p < 0.05). Expression patterns of the isolated protein panel have also been observed over time, as disease spreads to multiple joints. The data indicates that synovial fluid proteome profiles could be used to stratify patients based on risk of disease extension. These protein profiles may also assist in monitoring therapeutic responses over time and help predict joint damage.

ApoE elevation is associated with the persistence of psychotic experiences from age 12 to age 18: Evidence from the ALSPAC birth cohort.

Apolipoproteins, which play important roles in lipid metabolism, innate immunity and synaptic signalling, have been implicated in first episode psychosis and schizophrenia. This is the first study to investigate plasma apolipoprotein expression in children with psychotic experiences that persist into adulthood. Here, using semi-targeted proteomic analysis we compared plasma apolipoprotein expression levels in age 12 subjects who reported psychotic experiences at both age 12 and age 18 (n = 37) with age-matched subjects who only experienced psychotic experiences (PEs) at age 12 (n = 38). Participants were recruited from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) cohort who participated in psychiatric assessment interviews at ages 12 and 18. We identified apoE, a protein with significant regulatory activity on cholesterol metabolism in the brain, to be significantly up regulated (p < 0.003) in those with persistent psychotic experiences. We confirmed this finding in these samples using ELISA. Our findings indicate elevated plasma apoE in age 12 children who experience PEs is associated with persistence psychotic experiences.

Integrated Lipidomics and Proteomics Point to Early Blood-Based Changes in Childhood Preceding Later Development of Psychotic Experiences: Evidence From the Avon Longitudinal Study of Parents and Children.

BACKGROUND: The identification of early biomarkers of psychotic experiences (PEs) is of interest because early diagnosis and treatment of those at risk of future disorder is associated with improved outcomes. The current study investigated early lipidomic and coagulation pathway protein signatures of later PEs in subjects from the Avon Longitudinal Study of Parents and Children cohort. METHODS: Plasma of 115 children (12 years of age) who were first identified as experiencing PEs at 18 years of age (48 cases and 67 controls) were assessed through integrated and targeted lipidomics and semitargeted proteomics approaches. We assessed the lipids, lysophosphatidylcholines (n = 11) and phosphatidylcholines (n = 61), and the protein members of the coagulation pathway (n = 22) and integrated these data with complement pathway protein data already available on these subjects. RESULTS: Twelve phosphatidylcholines, four lysophosphatidylcholines, and the coagulation protein plasminogen were altered between the control and PEs groups after correction for multiple comparisons. Lipidomic and proteomic datasets were integrated into a multivariate network displaying a strong relationship between most lipids that were significantly associated with PEs and plasminogen. Finally, an unsupervised clustering approach identified four different clusters, with one of the clusters presenting the highest case-control ratio (p < .01) and associated with a higher concentration of smaller low-density lipoprotein cholesterol particles. CONCLUSIONS: Our findings indicate that the lipidome and proteome of subjects who report PEs at 18 years of age are already altered at 12 years of age, indicating that metabolic dysregulation may contribute to an early vulnerability to PEs and suggesting crosstalk between these lysophosphatidylcholines, phosphatidylcholines, and coagulation and complement proteins.

Blood-Based Protein Changes in Childhood Are Associated With Increased Risk for Later Psychotic Disorder: Evidence From a Nested Case-Control Study of the ALSPAC Longitudinal Birth Cohort.

The identification of early biological changes associated with the psychotic disorder (PD) is important as it may provide clues to the underlying pathophysiological mechanisms. We undertook the first proteomic profiling of blood plasma samples of children who later develop a PD. Participants were recruited from the UK Avon Longitudinal Study of Parents and Children (ALSPAC) cohort who also participated in psychiatric assessment interviews at age 18. Protein expression levels at age 11 were compared between individuals who developed PD at age 18 (n = 37) with population-based age-matched controls (n = 38). Sixty out of 181 plasma proteins profiled were found to be differentially expressed (P < .05) in children with an outcome of the PD. Thirty-four of these proteins were found to be differentially expressed following correction for multiple comparisons. Pathway analysis implicated the complement and coagulation cascade. A second, targeted proteomic approach was used to verify these findings in age 11 plasma from subjects who reported psychotic experiences at age 18 (n = 40) in comparison to age-matched controls (n = 66). Our findings indicate that the complement and coagulation system is dysregulated in the blood during childhood before the development of the PD.

Dataset of mouse hippocampus profiled by LC-MS/MS for label-free quantitation.

This dataset reports on the analysis of mouse hippocampus by LC-MS/MS, from mice fed a diet that was either deficient in n-3 FA (n-3 Def) or sufficient in n-3 FA (n-3 Adq). Label free quantitative (LFQ) analysis of the mass spectrometry data identified 1008 quantifiable proteins, 115 of which were found to be differentially expressed between the two dietary groups (n=8 per group). This data article refers to the research article "Omega-3 fatty acid deficiency disrupts endocytosis, neuritogenesis, and mitochondrial protein pathways in the mouse hippocampus" (English et al., 2013 [1]), in which a more comprehensive interpretation and analysis of the data is given.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

A 2-D guinea pig lung proteome map.

Guinea pigs represent an important model for a number of infectious and non-infectious pulmonary diseases. The guinea pig genome has recently been sequenced to full coverage, opening up new research avenues using genomics, transcriptomics and proteomics techniques in this species. In order to further annotate the guinea pig genome and to facilitate future pulmonary proteomics in this species we constructed a 2-D guinea pig proteome map including 486 protein identifications and post translational modifications (PTMs). The map has been up-loaded to the UCD 2D-PAGE open access database (http://proteomics-portal.ucd.ie/). Transit peptides, N-terminal acetylations and other PTMs are available via Peptideatlas (ftp://PASS00619:NM455hi@ftp.peptideatlas.org/). This dataset is associated with a research article published in the Journal of Proteomics [1].