bims-prodis Biomed News
on Proteomics in disease
Issue of 2018–11–18
sixty-one papers selected by
Nancy Gough, Bioserendipity
  1. Biomarker and Drug Target Discovery Using Quantitative Proteomics Post-Intracerebral Hemorrhage Stroke in the Rat Brain.
  2. Proteome characterization in various biological fluids of Trypanosoma brucei gambiense-infected subjects.
  3. Levels of human proteins in plasma associated with acute paediatric malaria.
  4. Integrative proteomic analysis reveals potential high-frequency alternative open reading frame-encoded peptides in human colorectal cancer.
  5. Transcriptomics and proteomics analyses of anti-cancer mechanisms of TR35-An active fraction from Xinjiang Bactrian camel milk in esophageal carcinoma cell.
  6. Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid.
  7. Identification of TEX101-associated proteins through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974.
  8. Deep Profiling of the Aggregated Proteome in Alzheimer's Disease: From Pathology to Disease Mechanisms.
  9. High sensitivity proteomics of prostate cancer tissue microarrays to discriminate between healthy and cancerous tissue.
  10. Tear Proteins Calcium binding protein A4 (S100A4) and Prolactin Induced Protein (PIP) are Potential Biomarkers for Thyroid Eye Disease.
  11. Comparative proteomic analysis identifies exosomal Eps8 protein as a potential metastatic biomarker for pancreatic cancer.
  12. Novel protein signatures suggest progression to muscular invasiveness in bladder cancer.
  13. Identification and validation of DOCK4 as a potential biomarker for risk of bone metastasis development in patients with early breast cancer.
  14. High Throughput Identification of Plasma Proteomic Signature of Inflammatory Bowel Disease.
  15. Computational analysis reveals histotype-dependent molecular profile and actionable mutation effects across cancers.
  16. Accelerated Barocycler Lysis and Extraction (ABLE) sample preparation for clinical proteomics by mass spectrometry.
  17. Proteomic Signatures of Clostridium difficile Stressed with Metronidazole, Vancomycin, or Fidaxomicin.
  18. Targeting the CALR interactome in myeloproliferative neoplasms.
  19. Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress.
  20. Targeted Quantitative Profiling of GTP-binding Proteins in Cancer Cells Using Isotope-Coded GTP Probes.
  21. Proteomic Identification of Biomarkers Associated with Eating Control and Bariatric Surgery Outcomes in Patients with Morbid Obesity.
  22. Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer.
  23. Integrative proteomics and immunochemistry analysis of the factors in the necrosis and repair in acetaminophen-induced acute liver injury in mice.
  24. A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex.
  25. Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.
  26. O-GlcNAc-induced nuclear translocation of hnRNP-K is associated with progression and metastasis of cholangiocarcinoma.
  27. MALDI-TOF mass spectrometry-based serotyping of V. parahaemolyticus isolated from the Zhejiang province of China.
  28. Integrated Proteomics and Lipidomics Investigation of the Mechanism Underlying the Neuroprotective Effect of N-benzylhexadecanamide.
  29. No Plasmatic Proteomic Signature at Clinical Disease Onset Associated With 11 Year Clinical, Cognitive and MRI Outcomes in Relapsing-Remitting Multiple Sclerosis Patients.
  30. Effect of IAPP on the proteome of cultured Rin-5F cells.
  31. Proteomics reveals ablation of PlGF increases antioxidant and neuroprotective proteins in the diabetic mouse retina.
  32. Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors.
  33. A Targeted Quantitative Proteomic Analysis Revealed Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells.
  34. Genetic determinants of steatosis and fibrosis progression in pediatric non-alcoholic fatty liver disease.
  35. Computational and Experimental Approaches to Predict Host-Parasite Protein-Protein Interactions.
  36. Tumor-Derived Autoantibodies Identify Malignant Pulmonary Nodules.
  37. C/VDdb: A multi-omics expression profiling database for a knowledge-driven approach in cardiovascular disease (CVD).
  38. KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism.
  39. Epitope-based immunoinformatics approach on RNA-dependent RNA polymerase (RdRp) protein complex of Nipah virus (NiV).
  40. S100a14 is Increased in Activated Nk Cells and Plasma of HIV-Exposed Seronegative People Who Inject Drugs and Promotes Monocyte-Nk crosstalk.
  41. Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression.
  42. HiPLA: High-throughput imaging Proximity Ligation Assay.
  43. Molecular biomarkers of Graves' ophthalmopathy.
  44. Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting.
  45. Calcium-activated chloride channel regulator 1 as a prognostic biomarker in pancreatic ductal adenocarcinoma.
  46. Opisthorchis viverrini Proteome and Host-Parasite Interactions.
  47. Kaurenoic acid and its sodium salt derivative: antibacterial activity against Porphyromonas gingivalis and their mechanism of action.
  48. Rapid detection of colistin-resistant Klebsiella pneumoniae using MALDI-TOF MS peak-based assay.
  49. dbPTM in 2019: exploring disease association and cross-talk of post-translational modifications.
  50. Identification of DKK-1 as a novel mediator of statin effects in human endothelial cells.
  51. Paraquat-Mediated Oxidative Stress in Anopheles gambiae Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response.
  52. Image-guided phenotyping of ovariectomized mice: altered functional connectivity, cognition, myelination, and dopaminergic functionality.
  53. Prognostic models in primary biliary cholangitis.
  54. MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis.
  55. Proteome-wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of FASN.
  56. Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.
  57. Autophagy maintains tumour growth through circulating arginine.
  58. ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasis.
  59. Urinary proteomics: fancy gadgetry or a clinically useful diagnostic instrument? The end-user's perspective.
  60. Cell-Membranes Immunotherapy Based on Natural Killer Cell-Membranes-Coated Nanoparticles for Effective Inhibition of Primary and Abscopal Tumor Growth.
  61. MALDI-TOF MS Applications to the Detection of Antifungal Resistance: State of the Art and Future Perspectives.
  1. J Mol Neurosci. 2018 Nov 14.
    Biomarker and Drug Target Discovery Using Quantitative Proteomics Post-Intracerebral Hemorrhage Stroke in the Rat Brain.
    Shuixiang Deng, Shengjie Feng, Wei Wang, Feng Zhao, Ye Gong.
      The pathological mechanisms of acute intracerebral hemorrhage (ICH) remain unknown and unverified. In the present study, we used quantitative proteomics to elucidate the pathological mechanisms and to identify novel biomarker and therapeutic target candidates via tissue proteome in a rat model of acute ICH. Rats were experimentally induced with ICH (n = 6) or Sham (n = 6), and their brain tissue was obtained by 24 h. The TMT-LC-MS/MS-based proteomics approach was used to quantify the differential proteomes across brain tissue, and the results were further analyzed by ingenuity pathway analysis to explore canonical pathways and the relationship involved in the uploaded data. Upon quantification, we found that 96 secreted proteins that were identified in the ICH 24-h group were significantly different those in the control group (P < 0.05); among these proteins, 57 increased and 39 decreased in abundance. Bioinformatic analyses of differentially expressed proteins demonstrated that the protein localization and ERK1 and ERK2 cascade were the top two biological processes with the highest concentrations of differentially proteins. The top protein-protein action network with high confidence levels of protein was the albumin and ERK signaling pathways. Albumin, ERK, and p-ERK were assessed in brain tissue by western blot analysis, and higher expression levels of albumin and p-ERK were observed in the ICH group. Our proteomic results highlight important change in the biological processes of ERK1 and ERK2 cascade, which are possible targets for future interventions of ICH. To our knowledge, this study provides in-depth analysis of ICH in brain tissue, and we propose 96 new biomarker candidates for ICH, including albumin and ERK.
    Keywords:  Acute intracerebral hemorrhage; Albumin; Biomarkers; ERK; Proteomics
    DOI:  https://doi.org/10.1007/s12031-018-1206-z
  2. J Proteomics. 2018 Nov 07. pii: S1874-3919(18)30395-6. [Epub ahead of print]
    Proteome characterization in various biological fluids of Trypanosoma brucei gambiense-infected subjects.
    Julien Bonnet, Camille Garcia, Thibaut Leger, Marie-Pauline Couquet, Philippe Vignoles, Vatunga Gedeao, Joseph Ndung'u, Clotilde Boudot, Sylvie Bisser, Bertrand Courtioux.
      Human African trypanosomiasis (HAT) is a neglected tropical disease that is endemic in sub-Saharan Africa. Control of the disease has been recently improved by better screening and treatment strategies, and the disease is on the WHO list of possible elimination. However, some physiopathological aspects of the disease transmission and progression remain unclear. We propose a new proteomic approach to identify new targets and thus possible new biomarkers of the disease. We also focused our attention on fluids classically associated with HAT (serum and cerebrospinal fluid (CSF)) and on the more easily accessible biological fluids urine and saliva. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) established the proteomic profile of patients with early and late stage disease. The serum, CSF, urine and saliva of 3 uninfected controls, 3 early stage patients and 4 late stage patients were analyzed. Among proteins identified, in CSF, urine and saliva, respectively, 37, 8 and 24 proteins were differentially expressed and showed particular interest with regards to their function. The most promising proteins (Neogenin, Neuroserpin, secretogranin 2 in CSF; moesin in urine and intelectin 2 in saliva) were quantified by enzyme-linked immunosorbent assay in a confirmatory cohort of 14 uninfected controls, 23 patients with early stage disease and 43 patients with late stage disease. The potential of two proteins, neuroserpin and moesin, with the latter present in urine, were further characterized. Our results showed the potential of proteomic analysis to discover new biomarkers and provide the basis of the establishment of a new proteomic catalogue applied to HAT-infected subjects and controls. SIGNIFICANCE: Sleeping sickness, also called Human African Trypanosomiasis (HAT), is a parasitic infection caused by a parasitic protozoan, Trypanosoma brucei gambiense or T. b. rhodesiense which are transmitted via an infected tsetse fly: Glossina. For both, the haemolymphatic stage (or first stage) signs and symptoms are intermittent fever, lymphadenopathy, hepatosplenomegaly, headaches, pruritus, and for T. b. rhodesiense infection a chancre is often formed at the bite site. Meningoencephalitic stage (or second stage) occurs when parasites invade the CNS, it is characterised by neurological signs and symptoms such as altered gait, tremors, neuropathy, somnolence which can lead to coma and death if untreated. first stage of the disease is characterizing by fevers, headaches, itchiness, and joint pains and progressive lethargy corresponding to the second stage with confusion, poor coordination, numbness and trouble sleeping. Actually, diagnosing HAT requires specialized expertise and significant resources such as well-equipped health centers and qualified staff. Such resources are lacking in many endemic areas that are often in rural locales, so many individuals with HAT die before the diagnosis is established. In this study, we analysed by mass spectrometry the entire proteome of serum, CSF, urine and saliva samples from infected and non-infected Angolan individuals to define new biomarkers of the disease. This work of proteomics analysis is a preliminary stage to the characterization of the whole proteome, of these 4 biological fluids, of HAT patients. We have identified 69 new biomarkers. Five of them have been thoroughly investigated by ELISA quantification. Neuroserpine and Moesin are respectively promising new biomarkers in CSF and urine's patient for a better diagnosis.
    Keywords:  Biological fluids; Proteomic; Sleeping sickness
    DOI:  https://doi.org/10.1016/j.jprot.2018.11.005
  3. Malar J. 2018 Nov 15. 17(1): 426
    Levels of human proteins in plasma associated with acute paediatric malaria.
    Philippa Reuterswärd, Sofia Bergström, Judy Orikiiriza, Elisabeth Lindquist, Sven Bergström, Helene Andersson Svahn, Burcu Ayoglu, Mathias Uhlén, Mats Wahlgren, Johan Normark, Ulf Ribacke, Peter Nilsson.
       BACKGROUND: The intimate interaction between the pathophysiology of the human host and the biology of the Plasmodium falciparum parasite results in a wide spectrum of disease outcomes in malaria. Development of severe disease is associated with a progressively augmented imbalance in pro- and anti-inflammatory responses to high parasite loads and sequestration of parasitized erythrocytes. Although these phenomena collectively constitute common denominators for the wide variety of discrete severe malaria manifestations, the mechanistic rationales behind discrepancies in outcome are poorly understood. Exploration of the human pathophysiological response by variations in protein profiles in plasma presents an excellent opportunity to increase the understanding. This is ultimately required for better prediction, prevention and treatment of malaria, which is essential for ongoing elimination and eradication efforts.
    RESULTS: An affinity proteomics approach was used to analyse 541 paediatric plasma samples collected from community controls and patients with mild or severe malaria in Rwanda. Protein profiles were generated with an antibody-based suspension bead array containing 255 antibodies targetting 115 human proteins. Here, 57 proteins were identified with significantly altered levels (adjusted p-values < 0.001) in patients with malaria compared to controls. From these, the 27 most significant proteins (adjusted p-values < 10-14) were selected for a stringent analysis approach. Here, 24 proteins showed elevated levels in malaria patients and included proteins involved in acute inflammatory response as well as cell adhesion. The remaining three proteins, also implicated in immune regulation and cellular adhesivity, displayed lower abundance in malaria patients. In addition, 37 proteins (adjusted p-values < 0.05) were identified with increased levels in patients with severe compared to mild malaria. This set includes, proteins involved in tissue remodelling and erythrocyte membrane proteins. Collectively, this approach has been successfully used to identify proteins both with known and unknown association with different stages of malaria.
    CONCLUSION: In this study, a high-throughput affinity proteomics approach was used to find protein profiles in plasma linked to P. falciparum infection and malaria disease progression. The proteins presented herein are mainly involved in inflammatory response, cellular adhesion and as constituents of erythrocyte membrane. These findings have a great potential to provide increased conceptual understanding of host-parasite interaction and malaria pathogenesis.
    Keywords:  Affinity proteomics; Cytoadhesion; Human plasma profiling; Malaria; Plasmodium falciparum, suspension bead arrays; Sequestration
    DOI:  https://doi.org/10.1186/s12936-018-2576-y
  4. Life Sci. 2018 Nov 09. pii: S0024-3205(18)30723-9. [Epub ahead of print]
    Integrative proteomic analysis reveals potential high-frequency alternative open reading frame-encoded peptides in human colorectal cancer.
    Tianfang Wang, Yining Liu, Qi Liu, Scott Cummins, Min Zhao.
      Identification of alternative open reading frame-encoded peptides (AEPs) for the diagnosis of colorectal cancer at the proteome level is largely unexplored because of a lack of comprehensive proteomics data. Here, we performed a comprehensive integrative analysis of mass spectral data published by Clinical Proteomic Tumor Analysis Consortium and characterized 93 high-confident AEPs encoded within 75 genes. There are four cancer-related genes appeared to have AEPs identified frequently in >20 out of 95 colorectal cancer samples, including ABCF2, AR, RBM10 and NRG1. Further network analysis of the identified AEPs found the enrichment of novel AEPs within hormone androgen receptor and a highly-modularised network with 42 genes associated with patient survival. Our results not only suggested a mechanistic view of how AEPs work in cancer progression, but also shed light on somatic amino acid mutations in AEPs, which might be overlooked previously because of their low frequencies. In particular, potential high-frequency mutations in 77 samples associated with EDARADD may contribute to the discovery of new biomarkers and the development of innovative therapeutic approaches.
    Keywords:  Biological network; Cancer genetics; Proteomics; Systems biology
    DOI:  https://doi.org/10.1016/j.lfs.2018.11.018
  5. Clin Nutr. 2018 Oct 27. pii: S0261-5614(18)32484-1. [Epub ahead of print]
    Transcriptomics and proteomics analyses of anti-cancer mechanisms of TR35-An active fraction from Xinjiang Bactrian camel milk in esophageal carcinoma cell.
    Jie Yang, Zhihua Dou, Xi Peng, Hongjuan Wang, Tong Shen, Jun Liu, Guan Li, Yang Gao.
       BACKGROUND & AIMS: The aim of the paper is to investigate the effect of the active fraction extracted from the Xinjiang Bactrian camel whey on the human cancer cells using an in vitro and in vivo model of human carcinoma of the esophagus.
    METHODS AND RESULTS: Our results demonstrated that an antitumor active fraction, TR35, isolated from Xinjiang Bactrian camel milk could significantly inhibit Eca109 cell proliferation and induce its apoptosis (indicated by MTT assay, Annexin V-FITC Apoptosis Detection, and caspase-3 activity). Moreover, we found that TR35 could inhibit the growth of xenografted tumor in nude mice without loss in body weight. Furthermore, we used RNA-Seq and 2-DE combined Mass Spectrometry analysis to identify differentially expressed RNA and protein markers of apoptosis and necrosis. Compared with untreated Eca109 cells, a total of 405 differentially expressed genes and 55 differentially expressed proteins were identified in TR35 treated Eca109 cells. KEGG analysis uncovered signaling pathways closely associated with cancer inhibition that were enriched in the TR35-treated cells.
    CONCLUSIONS: These results might implicate that downregulation of specific proteins identified in this study may be the cause of this tumor growth inhibition. This study sheds light on the potential therapeutic advantages based on the historical anti-cancer activities of camel milk.
    Keywords:  Anti-cancer; Camel milk; Esophageal carcinoma; Proteomics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.clnu.2018.10.013
  6. Proteomics Clin Appl. 2018 Nov 15. e1800085
    Novel Application of Aptamer Proteomic Analysis in Cystic Fibrosis Bronchoalveolar Lavage Fluid.
    Emily M DeBoer, Brandie D Wagner, Jonathan Popler, J Kirk Harris, Edith T Zemanick, Frank J Accurso, Scott D Sagel, Robin R Deterding.
       PURPOSE: Biomarkers are needed in cystic fibrosis (CF) to understand disease progression, assess response to therapy, and enrich enrollment for clinical trials. Aptamer-based proteomics have proven useful in blood samples. We aimed to evaluate proteins in bronchoalveolar lavage fluid (BALF) in CF children compared to controls and identify endotypes during CF exacerbations.
    EXPERIMENTAL DESIGN: BALF was collected clinically from 50 patients with CF and 9 disease controls, processed, and stored per protocol. BALF supernatants were analyzed for 1129 proteins by aptamer approach (SOMAscan proteomics platform). Proteins were compared across groups and used for pathway analysis. Endotypes were identified within the CF group.
    RESULTS: CF BALF had increased concentrations of neutrophil elastase, myeloperoxidase and decreased concentration of protein folding and host defense proteins. Pathways that distinguished CF subjects included interferon gamma signaling, membrane trafficking, and phospholipid metabolism. In the CF group, unbiased analysis of proteins identified two distinct endotypes that differed based on BALF white blood cell and neutrophil counts and detection of CF pathogens.
    CONCLUSIONS AND CLINICAL RELEVANCE: Proteomic analysis of the CF airway demonstrates a complex environment of proteins and pathways. This work provides evidence that aptamer-based proteomics can differentiate between groups and can determine endotypes within CF. This article is protected by copyright. All rights reserved.
    Keywords:  inflammation; pediatrics; pseudomonas aeruginosa
    DOI:  https://doi.org/10.1002/prca.201800085
  7. Mol Cell Proteomics. 2018 Nov 14. pii: mcp.RA118.001170. [Epub ahead of print]
    Identification of TEX101-associated proteins through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974.
    Christina Schiza, Dimitrios Korbakis, Keith Jarvi, Eleftherios P Diamandis, Andrei P Drabovich.
      TEX101 is a germ cell-specific protein and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility, and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 men for a common missense variant rs35033974 of TEX101, and identified 52 heterozygous and 4 homozygous men. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with the near-complete degradation (>97%) of the corresponding G99V TEX101 form, and suggested that spermatozoa of homozygous men could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified 8 cell-surface and 9 secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte interaction (including LY6K and ADAM29) were confirmed by targeted proteomics and western blotting assays. Since recent population-scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a monogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found 5-fold lower (P=0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction.
    Keywords:  Clinical proteomics; Immunoaffinity; Label-free quantification; Parallel reaction monitoring; Protein Identification*; Proteogenomics; Selected reaction monitoring; TEX101; rs35033974; single nucleotide variant; spermatozoa; testis-specific proteins
    DOI:  https://doi.org/10.1074/mcp.RA118.001170
  8. Proteomes. 2018 Nov 12. pii: E46. [Epub ahead of print]6(4):
    Deep Profiling of the Aggregated Proteome in Alzheimer's Disease: From Pathology to Disease Mechanisms.
    Brianna M Lutz, Junmin Peng.
      Hallmarks of Alzheimer's disease (AD), a progressive neurodegenerative disease causing dementia, include protein aggregates such as amyloid beta plaques and tau neurofibrillary tangles in a patient's brain. Understanding the complete composition and structure of protein aggregates in AD can shed light on the as-yet unidentified underlying mechanisms of AD development and progression. Biochemical isolation of aggregates coupled with mass spectrometry (MS) provides a comprehensive proteomic analysis of aggregates in AD. Dissection of these AD-specific aggregate components, such as U1 small nuclear ribonucleoprotein complex (U1 snRNP), provides novel insights into the deregulation of RNA splicing in the disease. In this review, we summarize the methodologies of laser capture microdissection (LCM) and differential extraction to analyze the aggregated proteomes in AD samples, and discuss the derived novel insights that may contribute to AD pathogenesis.
    Keywords:  Alzheimer’s disease; U1 snRNP; laser capture microdissection; mass spectrometry; protein aggregation; proteome; proteomics; splicing
    DOI:  https://doi.org/10.3390/proteomes6040046
  9. J Proteomics. 2018 Nov 12. pii: S1874-3919(18)30399-3. [Epub ahead of print]
    High sensitivity proteomics of prostate cancer tissue microarrays to discriminate between healthy and cancerous tissue.
    Lilla Turiák, Oliver Ozohanics, Gábor Tóth, András Ács, Ágnes Révész, Károly Vékey, András Telekes, László Drahos.
      Biopsies, in the form of tissue microarrays (TMAs) were studied to identify anomalies indicative of prostate cancer at the proteome level. TMAs offer a valuable source of well-characterized biological material. However, because of the small tissue sample size method development was essential to provide the sensitivity and reliability necessary for the analysis. Surface digestion of TMA cores was followed by peptide extraction and shotgun proteomics analysis. About 5 times better sensitivity was achieved by the optimized surface digestion compared to bulk digestion of the same TMA spot and it allowed the identification of over 500 proteins from individual prostate TMA cores. Label-free quantitation showed that biological variability among all samples was about 3 times larger than the technical reproducibility. We have identified 189 proteins which showed statistically significant changes (t-test p-value <.05) in abundance between healthy and cancerous tissue samples. The proteomic profile changed according to cancer grade, but did not show a correlation with cancer stage. Results of this pilot study were further evaluated using bioinformatics tools, identifying various protein pathways affected by prostate cancer progression indicating the usefulness of studying TMA cores to identify quantitative changes in tissue proteomics. SIGNIFICANCE: Detailed proteomics analysis of TMAs presents a good alternative for tissue analysis. Here we present a novel method, based on tissue surface digestion and nano-LC-MS measurements, which is capable of identifying and quantifying over 500 proteins from a 1.5 mm diameter tissue section. We compared healthy and cancerous prostate tissues samples, and tissues with various grades and stages of cancer. Tissue proteomics clearly distinguished healthy and cancerous samples, furthermore the results correlated well with cancer grade, but not with cancer stage. Over 100 proteins showed statistically significant abundance changes (t-test p-value <.05) between various groups. This was sufficient for a meaningful bioinformatics evaluation; showing e.g. increased abundance of proteins in cancer in the KEGG ribosome pathway, GO mRNA splicing via spliceosome, and chromatin assembly biological processes. The results highlight the feasibility of the developed method for future large-scale tissue proteomics studies using commercially available TMAs.
    Keywords:  Label-free quantitation; Mass spectrometry; Prostate cancer; Proteomics; Surface digestion; Tissue microarray
    DOI:  https://doi.org/10.1016/j.jprot.2018.11.009
  10. Sci Rep. 2018 Nov 16. 8(1): 16936
    Tear Proteins Calcium binding protein A4 (S100A4) and Prolactin Induced Protein (PIP) are Potential Biomarkers for Thyroid Eye Disease.
    Chiaw-Ling Chng, Lay Leng Seah, Morgan Yang, Sunny Yu Shen, Siew Kwan Koh, Yan Gao, Lu Deng, Louis Tong, Roger Wilmer Beuerman, Lei Zhou.
      There are no reliable biomarkers to predict thyroid eye disease (TED) in patients with autoimmune thyroid disease (AITD) currently. Several evidences support the involvement of the lacrimal gland in TED. The aim of our study was to quantitatively correlate the changes in tear protein profile with increasing severity of TED. Tear samples were collected from four groups of patients; AITD without TED (AITD), AITD with mild TED (mild TED), AITD with severe TED (severe TED) and normal controls. A total of 72 patients were recruited for the study. In discovery phase, isobaric tags for relative and absolute quantification (iTRAQ) 4-plex was used for quantitative proteomics analysis. For verification of results from discovery phase, sequential window acquisition of all theoretical fragment ion spectra (SWATH) was used to analyze an independent cohort from normal controls, AITD, mild TED and severe TED. Two proteins, S100A4 and PIP showed consistent dysregulation trends in the discovery and validation phase experiments. Our study demonstrated the differences in tear proteome across the spectrum of different severity and activity of TED in patients with AITD. Two tear proteins, S100A4 and PIP may serve as potential biomarkers to predict progression to severe TED in patients with AITD.
    DOI:  https://doi.org/10.1038/s41598-018-35096-x
  11. Oncol Rep. 2018 Nov 15.
    Comparative proteomic analysis identifies exosomal Eps8 protein as a potential metastatic biomarker for pancreatic cancer.
    Keiichi Ohshima, Keiichi Hatakeyama, Kaori Kanto, Tomomi Ide, Yuko Watanabe, Sachi Moromizato, Kanako Wakabayashi-Nakao, Naoki Sakura, Ken Yamaguchi, Tohru Mochizuki.
      Exosomes are small vesicles found in extracellular environments including blood, urine, and cell culture medium. Their contents are cell‑type specific, and molecules embedded in exosomes can be useful fluid‑based clinical biomarkers. To identify proteins with metastatic marker potential, we conducted a comparative exosomal proteome analysis using human pancreatic cancer cell lines derived from metastasis, ascites, and primary tumors. Metastatic potential of cell lines was assessed by migratory and invasive activities. A pancreatic cancer cell line from metastasis (SU.86.86) revealed 23‑fold and 20‑fold increases in cell migratory and invasive activities, respectively, compared to the MIA PaCa‑2 cell line derived from primary tumor cells. Liquid chromatography‑mass spectrometry‑based proteome analysis and subsequent validation by immunoblot analysis revealed that epidermal growth factor receptor pathway substrate 8 (Eps8) was highly abundant in exosomes from metastasis‑derived SU.86.86 cells. Comparison of 12 pancreatic cancer cell lines derived from different stages of malignancy revealed a strong relationship between exosomal Eps8 protein levels and cell motile activities (migration: r=0.85, P=4.2x10‑4; invasion: r=0.60, P=3.2x10‑2). Conversely, relationships between intracellular Eps8 protein levels and cell motile activities were moderate (migration: r=0.65, P=2.0x10‑2; invasion: r=0.51, P=9.2x10‑2). It was therefore concluded that exosomal Eps8 protein levels were correlated with the migratory cell potential of human pancreatic cancer cells, indicating that exosomal Eps8 has the potential to be a metastatic biomarker for human pancreatic cancer.
    DOI:  https://doi.org/10.3892/or.2018.6869
  12. PLoS One. 2018 ;13(11): e0206475
    Novel protein signatures suggest progression to muscular invasiveness in bladder cancer.
    Magnus Berle, Luiza Ghila, Heidrun Vethe, Adeel Chaudhry, Hilde Garberg, Christian Beisland, Øystein Ariansen Haaland, Eystein Oveland, Ole Johan Halvorsen, Thomas Davidsson, Simona Chera.
      Patients with bladder cancer need frequent controls over long follow-up time due to high recurrence rate and risk of conversion to muscle invasive cancer with poor prognosis. We identified cancer-related molecular signatures in apparently healthy bladder in patients with subsequent muscular invasiveness during follow-up. Global proteomics of the normal tissue biopsies revealed specific proteome fingerprints in these patients prior to subsequent muscular invasiveness. In these presumed normal samples, we detected modulations of proteins previously associated with different cancer types. This study indicates that analyzing apparently healthy tissue of a cancer-invaded organ may suggest disease progression.
    DOI:  https://doi.org/10.1371/journal.pone.0206475
  13. J Pathol. 2018 Nov 14.
    Identification and validation of DOCK4 as a potential biomarker for risk of bone metastasis development in patients with early breast cancer.
    Jules A Westbrook, Steven L Wood, David A Cairns, Kathryn McMahon, Renu Gahlaut, Helene Thygesen, Mike Shires, Stephanie Roberts, Helen Marshall, Maria R Oliva, Mark J Dunning, Andrew M Hanby, Peter J Selby, Valerie Speirs, Georgia Mavria, Robert E Coleman, Janet E Brown.
      Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid. We used quantitative proteomics (SILAC-MS), to compare protein expression in a bone-homed variant (BM1) of the human breast cancer cell line MDA-MB-231 with parental non-bone-homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC-MS showed that Dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone-homing BM1 cells, confirmed by Western blotting. BM1 cells also had enhanced invasive ability compared with parental cells which could be reduced by DOCK4-shRNA. In a training Tissue Microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p=0.004) but not oestrogen receptor status (p=0.19) or lymph node involvement (p=0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n=689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronic acid) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06-4.30, p=0.034). No corresponding association was found in patients who received zoledronic acid (HR 0.812, 95%CI 0.176-3.76, p=0.790), suggesting that treatment with zoledronic acid may counteract the higher risk for bone relapse from high DOCK4-expressing tumours. High DOCK4 expression was not associated with metastasis to non-skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. This article is protected by copyright. All rights reserved.
    DOI:  https://doi.org/10.1002/path.5197
  14. J Crohns Colitis. 2018 Nov 15.
    High Throughput Identification of Plasma Proteomic Signature of Inflammatory Bowel Disease.
    Antonio F Di Narzo, Carrie Brodmerkel, Shannon E Telesco, Carmen Argmann, Lauren A Peters, Katherine Li, Brian Kidd, Joel Dudley, Judy Cho, Eric E Schadt, Andrew Kasarskis, Radu Dobrin, Ke Hao.
       Background: The molecular etiology of inflammatory bowel disease (IBD) and its two subtypes, ulcerative colitis (UC) and Crohn's disease (CD), have been carefully investigated at genome and transcriptome levels. Recent advances in high throughput proteome quantification has enabled comprehensive large scale plasma proteomics studies of IBD.
    Methods: The study used two cohorts: (1) CERTIFI-cohort: 42 sample from the CERTIFI trial of anti TNFα refractory CD patients; (2) PROgECT-UNITI-HC cohort: 46 UC samples PROgECT study, 84 CD samples of UNITI I and UNITI II, and 72 healthy controls recruited in Mount Sinai Hospital, New York, USA. Plasma proteome for these two cohorts was quantified using high throughput platforms.
    Results: On PROgECT-UNITI-HC cohort, we measured a total of 1,310 proteins. 493 proteins showed different plasma levels in IBD patients versus controls at 10% FDR, among which 11, proteins had a fold change greater than 2. Proteins up-regulated in IBD were enriched for immunity functionality while the proteins down-regulated in IBD were enriched for nutrition and metabolism. Proteomic profiles were very similar between UC and CD. On CERTIFI cohort, 1,014 proteins were measured, and found plasma protein level had little correlation with blood or intestine transcriptome.
    Conclusions: We report the largest proteomics study to date on IBD and controls. A large proportion of plasma proteins are altered in IBD, revealing insights on the disease etiology indicating a potential for biomarker discovery.
    DOI:  https://doi.org/10.1093/ecco-jcc/jjy190
  15. Genome Med. 2018 Nov 15. 10(1): 83
    Computational analysis reveals histotype-dependent molecular profile and actionable mutation effects across cancers.
    Daniel Heim, Grégoire Montavon, Peter Hufnagl, Klaus-Robert Müller, Frederick Klauschen.
       BACKGROUND: Comprehensive mutational profiling data now available on all major cancers have led to proposals of novel molecular tumor classifications that modify or replace the established organ- and tissue-based tumor typing. The rationale behind such molecular reclassifications is that genetic alterations underlying cancer pathology predict response to therapy and may therefore offer a more precise view on cancer than histology. The use of individual actionable mutations to select cancers for treatment across histotypes is already being tested in the so-called basket trials with variable success rates. Here, we present a computational approach that facilitates the systematic analysis of the histological context dependency of mutational effects by integrating genomic and proteomic tumor profiles across cancers.
    METHODS: To determine effects of oncogenic mutations on protein profiles, we used the energy distance, which compares the Euclidean distances of protein profiles in tumors with an oncogenic mutation (inner distance) to that in tumors without the mutation (outer distance) and performed Monte Carlo simulations for the significance analysis. Finally, the proteins were ranked by their contribution to profile differences to identify proteins characteristic of oncogenic mutation effects across cancers.
    RESULTS: We apply our approach to four current proposals of molecular tumor classifications and major therapeutically relevant actionable genes. All 12 actionable genes evaluated show effects on the protein level in the corresponding tumor type and showed additional mutation-related protein profiles in 21 tumor types. Moreover, our analysis identifies consistent cross-cancer effects for 4 genes (FGFR1, ERRB2, IDH1, KRAS/NRAS) in 14 tumor types. We further use cell line drug response data to validate our findings.
    CONCLUSIONS: This computational approach can be used to identify mutational signatures that have protein-level effects and can therefore contribute to preclinical in silico tests of the efficacy of molecular classifications as well as the druggability of individual mutations. It thus supports the identification of novel targeted therapies effective across cancers and guides efficient basket trial designs.
    Keywords:  Cancer; Genomics; Pan-cancer analysis; Proteomics; Targeted cancer therapy
    DOI:  https://doi.org/10.1186/s13073-018-0591-9
  16. J Proteome Res. 2018 Nov 16.
    Accelerated Barocycler Lysis and Extraction (ABLE) sample preparation for clinical proteomics by mass spectrometry.
    Natasha Lucas, Andrew B Robinson, Maiken Marcker Espersen, Sadia Mahboob, Dylan Xavier, Jing Xue, Rosemary L Balleine, Anna deFazio, Peter G Hains, Phillip J Robinson.
      We have developed a streamlined proteomic sample preparation protocol termed Accelerated Barocycler Lysis and Extraction (ABLE), that substantially reduces the time and cost of tissue sample processing. ABLE is based on pressure cycling technology (PCT) for rapid tissue solubilisation and reliable, controlled proteolytic digestion. Here, a previously reported PCT based protocol was optimised using 1-4 mg biopsy punches from rat kidney. The tissue denaturant urea was substituted with a combination of sodium deoxycholate (SDC) and N-propanol. ABLE produced comparable numbers of protein identifications in half the sample preparation time, being ready for MS injection in three hours compared with six hours for the conventional urea based method. To validate ABLE, it was applied to a diverse range of rat tissues (kidney, lung, muscle, brain, testis), human HEK 293 cell lines and human ovarian cancer samples, followed by SWATH-mass spectrometry (SWATH-MS). There were similar numbers of quantified proteins between ABLE-SWATH and the conventional method, with greater than 70% overlap for all sample types, except muscle (58%). The ABLE protocol offers a standardised, high-throughput, efficient and reproducible proteomic preparation method, that when coupled with SWATH-MS, has the potential to accelerate proteomics analysis to achieve a clinically relevant turn-around-time.
    DOI:  https://doi.org/10.1021/acs.jproteome.8b00684
  17. Cells. 2018 Nov 15. pii: E213. [Epub ahead of print]7(11):
    Proteomic Signatures of Clostridium difficile Stressed with Metronidazole, Vancomycin, or Fidaxomicin.
    Sandra Maaß, Andreas Otto, Dirk Albrecht, Katharina Riedel, Anke Trautwein-Schult, Dörte Becher.
      The anaerobic pathogen Clostridium difficile is of growing significance for the health care system due to its increasing incidence and mortality. As C. difficile infection is both supported and treated by antibiotics, a deeper knowledge on how antimicrobial agents affect the physiology of this important pathogen may help to understand and prevent the development and spreading of antibiotic resistant strains. As the proteomic response of a cell to stress aims at counteracting the harmful effects of this stress, it can be expected that the pattern of a pathogen's responses to antibiotic treatment will be dependent on the antibiotic mechanism of action. Hence, every antibiotic treatment is expected to result in a specific proteomic signature characterizing its mode of action. In the study presented here, the proteomic response of C. difficile 630∆erm to vancomycin, metronidazole, and fidaxomicin stress was investigated on the level of protein abundance and protein synthesis based on 2D PAGE. The quantification of 425 proteins of C. difficile allowed the deduction of proteomic signatures specific for each drug treatment. Indeed, these proteomic signatures indicate very specific cellular responses to each antibiotic with only little overlap of the responses. Whereas signature proteins for vancomycin stress fulfil various cellular functions, the proteomic signature of metronidazole stress is characterized by alterations of proteins involved in protein biosynthesis and protein degradation as well as in DNA replication, recombination, and repair. In contrast, proteins differentially expressed after fidaxomicin treatment can be assigned to amino acid biosynthesis, transcription, cell motility, and the cell envelope functions. Notably, the data provided by this study hint also at so far unknown antibiotic detoxification mechanisms.
    Keywords:  2D PAGE; Clostridiodes difficile; antibiotics; protein synthesis; proteomics
    DOI:  https://doi.org/10.3390/cells7110213
  18. JCI Insight. 2018 Nov 15. pii: 122703. [Epub ahead of print]3(22):
    Targeting the CALR interactome in myeloproliferative neoplasms.
    Elodie Pronier, Paolo Cifani, Tiffany R Merlinsky, Katharine Barr Berman, Amritha Varshini Hanasoge Somasundara, Raajit K Rampal, John LaCava, Karen E Wei, Friederike Pastore, Jesper Lv Maag, Jane Park, Richard Koche, Alex Kentsis, Ross L Levine.
      Mutations in the ER chaperone calreticulin (CALR) are common in myeloproliferative neoplasm (MPN) patients, activate the thrombopoietin receptor (MPL), and mediate constitutive JAK/STAT signaling. The mechanisms by which CALR mutations cause myeloid transformation are incompletely defined. We used mass spectrometry proteomics to identify CALR-mutant interacting proteins. Mutant CALR caused mislocalization of binding partners and increased recruitment of FLI1, ERP57, and CALR to the MPL promoter to enhance transcription. Consistent with a critical role for CALR-mediated JAK/STAT activation, we confirmed the efficacy of JAK2 inhibition on CALR-mutant cells in vitro and in vivo. Due to the altered interactome induced by CALR mutations, we hypothesized that CALR-mutant MPNs may be vulnerable to disruption of aberrant CALR protein complexes. A synthetic peptide designed to competitively inhibit the carboxy terminal of CALR specifically abrogated MPL/JAK/STAT signaling in cell lines and primary samples and improved the efficacy of JAK kinase inhibitors. These findings reveal what to our knowledge is a novel potential therapeutic approach for patients with CALR-mutant MPN.
    Keywords:  Drug therapy; Hematology; Oncology; Proteomics; Signal transduction
    DOI:  https://doi.org/10.1172/jci.insight.122703
  19. Microb Pathog. 2018 Nov 10. pii: S0882-4010(18)31037-4. [Epub ahead of print]126 205-211
    Identification of factors involved in Enterococcus faecalis biofilm under quercetin stress.
    Shariq Qayyum, Divakar Sharma, Deepa Bisht, Asad U Khan.
      Enterococcus faecalis is a gram positive enteric commensal bacteria or opportunistic pathogen and its infection involves biofilm formation. Quercetin, a plant origin polyphenol was found to inhibit E. faecalis biofilm. Crystal violet assay, SEM and CLSM microscopy confirmed biofilm inhibition by quercetin. Proteomics was used to elucidate the changes occurred in bacterial cell by quercetin treatment. 2D-Electrophorosis and MALDI-TOF analysis revealed that nineteen proteins were differentially expressed in quercetin treated sample. Glycolytic pathways, protein translation-elongation pathways and protein folding pathways were under differential expression after treatment. Real Time-PCR (RT-PCR) validated the proteomic data at genomic level except for the translation elongation factor G which showed opposite data to proteomics. Protein-protein interaction networks constructed using STRING 10.0 demonstrated strong connection of translation-elongation proteins with many important proteins. The results of the comparative analysis indicate that quercetin exerts its inhibitory effect by disturbing glycolytic, protein translation-elongation and protein folding pathways. This disturbs bacterial physiology and stops transition of planktonic cells to biofilm state.
    Keywords:  Biofilm; Enterococcus faecalis; Protein-protein interactions; Proteomics; Quercetin
    DOI:  https://doi.org/10.1016/j.micpath.2018.11.013
  20. Anal Chem. 2018 Nov 15.
    Targeted Quantitative Profiling of GTP-binding Proteins in Cancer Cells Using Isotope-Coded GTP Probes.
    Rong Cai, Ming Huang, Yinsheng Wang.
      GTP-binding proteins play important roles in many essential biological processes, including cell signaling, trafficking and protein synthesis. To assess quantitatively these proteins at the whole proteome level, we developed a high-throughput targeted proteomic method based on the use of isotope-coded GTP probes and multiple-reaction monitoring (MRM) analysis. Targeted proteins were labeled with desthiobiotin-GTP probes, digested with trypsin, and the ensuing desthiobiotin-conjugated peptides were enriched with streptavidin beads for LC-MS/MS analysis. We also established a Skyline MRM library based on shotgun proteomic data acquired for 12 different human cell lines. The library contained 605 tryptic peptides derived from 217 GTP-binding proteins, representing approximately 60% of the annotated human GTP-binding proteome. By using this library, in conjunction with isotope-coded GTP probes and scheduled LC-MRM analysis, we investigated the differential expression of GTP-binding proteins in a pair of primary/metastatic colon cancer cell lines SW480/SW620. We were able to quantify 97 GTP-binding proteins, and we further validated the differential expression of several GTP-binding proteins by Western blot analysis. Together, we developed a facile targeted quantitative proteomic method for the high-throughput analysis of GTP-binding proteins, and applied the method for probing the altered expression of these proteins involved in colon cancer metastasis.
    DOI:  https://doi.org/10.1021/acs.analchem.8b03727
  21. World J Surg. 2018 Nov 13.
    Proteomic Identification of Biomarkers Associated with Eating Control and Bariatric Surgery Outcomes in Patients with Morbid Obesity.
    Carmen Rodríguez-Rivera, Carmen Pérez-García, José Ramón Muñoz-Rodríguez, Marta Vicente-Rodríguez, Filomena Polo, Rhian-Marie Ford, Esperanza Segura, Alberto León, Elisabet Salas, Luis Sáenz-Mateos, Carmen González-Martín, Gonzalo Herradón, Luis Beato-Fernández, Jesús Martín-Fernández, Luis F Alguacil.
       BACKGROUND: The current therapeutics of morbid obesity could be significantly improved after the identification of novel biomarkers associated with the food addiction endophenotype of obesity and with bariatric surgery outcomes.
    METHODS: We applied differential expression proteomics and enzyme-linked immunosorbent confirmatory assays to identify (a) proteins that varied according to loss of control over eating in morbidly obese patients and (b) proteins that varied between normoweight controls and patients before and 1 year after bariatric surgery.
    RESULTS: Clusterin was the only protein that consistently varied according to eating control in patients. Patients showed increased levels of serum amyloid P protein, apolipoprotein A4, serotransferrin, complement factors B and C3 and haptoglobin with respect to controls; the levels of all these proteins tended to return to control values 1 year after surgery. In contrast, apolipoprotein A1 and transthyretin were initially downregulated in patients and were scarcely changed by surgery. Leucine-rich alpha-2-glycoprotein was markedly increased in patients only after surgery.
    CONCLUSIONS: Clusterin could be of interest as a putative biomarker for food addiction diagnosis in people with morbid obesity. In addition, postsurgical normalization of the proteins initially dysregulated in obese subjects might help monitor clinical improvements after surgery, while lasting or newly detected alterations (i.e., those affecting transthyretin and leucine-rich alpha-2-glycoprotein) could reflect partial refractoriness and/or contribute to the early prediction of clinical problems.
    DOI:  https://doi.org/10.1007/s00268-018-4851-z
  22. Lung Cancer. 2018 Nov;pii: S0169-5002(18)30571-3. [Epub ahead of print]125 157-163
    Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer.
    Dijana Djureinovic, Tea Dodig-Crnković, Cecilia Hellström, Georg Holgersson, Michael Bergqvist, Johanna S M Mattsson, Fredrik Pontén, Elisabeth Ståhle, Jochen M Schwenk, Patrick Micke.
       OBJECTIVES: Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies.
    MATERIALS AND METHODS: To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases.
    RESULTS: Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%-4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients.
    CONCLUSION: We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets.
    Keywords:  Adenocarcinoma; Cancer immunity; Lung cancer; MAGE; Squamous cell cancer; Tumor markers
    DOI:  https://doi.org/10.1016/j.lungcan.2018.09.012
  23. J Cell Physiol. 2018 Nov 11.
    Integrative proteomics and immunochemistry analysis of the factors in the necrosis and repair in acetaminophen-induced acute liver injury in mice.
    Qin Feng, Ningwei Zhao, Wenkai Xia, ChengJie Liang, Guoxin Dai, Jian Yang, Jingxia Sun, Lanying Liu, Lan Luo, Jie Yang.
      Acetaminophen (APAP) overdose-induced acute liver injury (AILI) is a significant clinical problem worldwide, the hepatotoxicity mechanisms are well elucidated, but the factors involved in the necrosis and repair still remain to be investigated. APAP was injected intraperitoneally in male Institute of Cancer Research (ICR) mice. Quantitative proteome analysis of liver tissues was performed by 2-nitrobenzenesulfenyl tagging, two-dimensional-nano high-performance liquid chromatography separation, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Diffrenetial proteins were verified by the immunochemistry method. 36 and 44 differentially expressed proteins were identified, respectively, at 24 hr after APAP (200 or 300 mg·kg -1 ) administration. The decrease in the mitochondrial protective proteins Prdx6, Prdx3, and Aldh2 accounted for the accumulation of excessive reactive oxygen species (ROS) and aldehydes, impairing mitochondria structure and function. The Gzmf combined with Bax and Apaf-1 jointly contributed to the necrosis. The blockage of Stat3 activation led to the overexpression of unphosphorylated Stat3 and the overproduction of Bax. The overexpression of unphosphorylated Stat3 represented necrosis; the alternation from Stat3 to p-Stat3 in necrotic regions represented hepatocytes from death to renewal. The high expressions of P4hα1, Ncam, α-SMA, and Cygb were involved in the liver repair, they were not only the markers of activated HSC but also represented an intermediate stage of hepatocytes from damage or necrosis to renewal. Our data provided a comprehensive report on the profile and dynamic changes of the liver proteins in AILI; the involvement of Gzmf and the role of Stat3 in necrosis were revealed; and the role of hepatocyte in liver self-repair was well clarified.
    Keywords:  Stat3; acetaminophen; liver repair; necrosis; proteomics
    DOI:  https://doi.org/10.1002/jcp.27397
  24. Microbes Infect. 2018 Nov 13. pii: S1286-4579(18)30168-0. [Epub ahead of print]
    A proteomics approach for the identification of species-specific immunogenic proteins in the Mycobacterium abscessus complex.
    Mathis Steindor, Vanesa Nkwouano, Anja Stefanski, Kai Stuehler, Thomas Richard Ioerger, David Bogumil, Marc Jacobsen, Colin Rae Mackenzie, Rainer Kalscheuer.
      The Mycobacterium abscessus complex can cause fatal pulmonary disease, especially in cystic fibrosis patients. Diagnosing M. abscessus complex pulmonary disease is challenging. Immunologic assays specific for M. abscessus are not available. In this study seven clinical M. abscessus complex strains and the M. abscessus reference strain ATCC19977 were used to find species-specific proteins for their use in immune assays. Six strains showed rough and smooth colony morphotypes simultaneously, two strains only showed rough mophotypes, resulting in 14 separate isolates. Clinical isolates were submitted to whole genome sequencing. Proteomic analysis was performed on bacterial lysates and culture supernatant of all 14 isolates. Species-specificity for M. abscessus complex was determined by a BLAST search for proteins present in all supernatants. Species-specific proteins underwent in silico B- and T-cell epitope prediction. All clinical strains were found to be M. abscessus ssp. abscessus. Mutations in MAB_4099c as a likely genetic basis of the rough morphotype were found in six out of seven clinical isolates. 79 proteins were present in every supernatant, of which 12 are exclusively encoded by all members of M. abscessus complex plus M. immunogenum. In silico analyses predicted B- and T-cell epitopes in all of these 12 species-specific proteins.
    Keywords:  Mycobacterium abscessus; cystic fibrosis; epitopes; non tuberculous mycobacteria; proteomics
    DOI:  https://doi.org/10.1016/j.micinf.2018.10.006
  25. PLoS One. 2018 ;13(11): e0207405
    Discovering proteins for chemoprevention and chemotherapy by curcumin in liver fluke infection-induced bile duct cancer.
    Jarinya Khoontawad, Kitti Intuyod, Rucksak Rucksaken, Nuttanan Hongsrichan, Chawalit Pairojkul, Porntip Pinlaor, Thidarut Boonmars, Chaisiri Wongkham, Alun Jones, Jordan Plieskatt, Jeremy Potriquet, Jeffrey M Bethony, Jason Mulvenna, Somchai Pinlaor.
      Modulation or prevention of protein changes during the cholangiocarcinoma (CCA) process induced by Opisthorchis viverrini (Ov) infection may become a key strategy for prevention and treatment of CCA. Monitoring of such changes could lead to discovery of protein targets for CCA treatment. Curcumin exerts anti-inflammatory and anti-CCA activities partly through its protein-modulatory ability. To support the potential use of curcumin and to discover novel target molecules for CCA treatment, we used a quantitative proteomic approach to investigate the effects of curcumin on protein changes in an Ov-induced CCA-harboring hamster model. Isobaric labelling and tandem mass spectrometry were used to compare the protein expression profiles of liver tissues from CCA hamsters with or without curcumin dietary supplementation. Among the dysregulated proteins, five were upregulated in liver tissues of CCA hamsters but markedly downregulated in the CCA hamsters supplemented with curcumin: S100A6, lumican, plastin-2, 14-3-3 zeta/delta and vimentin. Western blot and immunohistochemical analyses also showed similar expression patterns of these proteins in liver tissues of hamsters in the CCA and CCA + curcumin groups. Proteins such as clusterin and S100A10, involved in the NF-κB signaling pathway, an important signaling cascade involved in CCA genesis, were also upregulated in CCA hamsters and were then suppressed by curcumin treatment. Taken together, our results demonstrate the important changes in the proteome during the genesis of O. viverrini-induced CCA and provide an insight into the possible protein targets for prevention and treatment of this cancer.
    DOI:  https://doi.org/10.1371/journal.pone.0207405
  26. Mol Oncol. 2018 Nov 15.
    O-GlcNAc-induced nuclear translocation of hnRNP-K is associated with progression and metastasis of cholangiocarcinoma.
    Chatchai Phoomak, Dayoung Park, Atit Silsirivanit, Kanlayanee Sawanyawisuth, Kulthida Vaeteewoottacharn, Marutpong Detarya, Chaisiri Wongkham, Carlito B Lebrilla, Wongkham Sopit.
      O-GlcNAcylation is a key post-translational modification that modifies the functions of proteins. Associations between O-GlcNAcylation, shorter survival of cholangiocarcinoma (CCA) patients and increased migration/invasion of CCA cell lines have been reported. However, the specific O-GlcNAcylated proteins (OGPs) that participate in promotion of CCA progression are poorly understood. OGPs were isolated from human CCA cell lines, KKU-213 and KKU-214, using a click chemistry-based enzymatic labeling system, identified using LC-MS/MS, and searched against an OGP database. From the proteomic analysis, a total of 21 OGPs related to cancer progression were identified, of which 12 have not been previously reported. Among these, hnRNP-K, a multi-faceted RNA and DNA binding protein known as a pre-mRNA-binding protein, was one of the most abundantly expressed, suggesting its involvement in CCA progression. O-GlcNAcylation of hnRNP-K was further verified by anti-OGP/anti-hnRNP-K immunoprecipitations and sWGA pull-down assays. The perpetuation of CCA by hnRNP-K was evaluated using siRNA, which revealed modulation of cyclin D1, XIAP, EMT markers, MMP2 and MMP7 expression. In native CCA cells, hnRNP-K was primarily localized in the nucleus; however, when O-GlcNAcylation was suppressed hnRNP-K was retained in the cytoplasm. These data signify an association between nuclear accumulation of hnRNP-K and the migratory capabilities of CCA cells. In human CCA tissues, expression of nuclear hnRNP-K was positively correlated with high O-GlcNAcylation levels, metastatic stage, and shorter survival of CCA patients. This study demonstrates the significance of O-GlcNAcylation on the nuclear translocation of hnRNP-K and its impact on the progression of CCA. This article is protected by copyright. All rights reserved.
    Keywords:  O-GlcNAcylated proteins; bile duct cancer; heterogeneous nuclear ribonucleoprotein-K; metastasis
    DOI:  https://doi.org/10.1002/1878-0261.12406
  27. BMC Microbiol. 2018 Nov 13. 18(1): 185
    MALDI-TOF mass spectrometry-based serotyping of V. parahaemolyticus isolated from the Zhejiang province of China.
    Ping Li, Wenwen Xin, Susu Xia, Yun Luo, Zhongwen Chen, Dazhi Jin, Shan Gao, Hao Yang, Bin Ji, Henghui Wang, Yong Yan, Lin Kang, Jinglin Wang.
       BACKGROUND: Vibrio parahaemolyticus is as an important food-borne pathogen circulating in China. Since 1996, the core serotype has become O3:K6, which has specific genetic markers. This serotype causes the majority of outbreaks worldwide. Until now, nearly 21 serotypes were considered as serovariants of O3:K6. Among these, O4:K68, O1:K25 and O1:KUT have caused pandemic outbreaks. O4:K8, a serovariant of O3:K6, has become the second most dominant serotype circulating in China after O3:K6. In this study, we report the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze and characterize 146 V. parahaemolyticus isolates belonging to 23 serotypes.
    RESULTS: Upon mass spectral analysis, isolates belonging to O4:K8 formed a distinct group among the five main pandemic groups (O3:K6, O4:K8, O4:K68, O1:K25 and O1:KUT). Two major protein peaks (m/z 4383 and 4397) were significantly different between serotype O4:K8 and the four other pandemic strains. Both of these peaks were present in 32 out of 36 O4:K8 isolates, but were absent in 105 out of 110 non-O4:K8 isolates. These peaks were also absent in all 74 pandemic serotypes (O3:K6, O4:K68, O1:K25 and O1:KUT).
    CONCLUSION: Our results highlight the threat of O4:K8 forming a distinct group, which differs significantly from pandemic serotypes on the proteomic level. The use of MALDI-TOF MS has not been reported before in a study of this nature. Mass spectrum peaks at m/z 4383 and 4397 may be specific for O4:K8. However, we cannot conclude that MALDI-TOF MS can be used to serotype V. parahaemolyticus.
    Keywords:  Biomarkers; MALDI-TOF MS; O4:K8; Vibrio parahaemolyticus
    DOI:  https://doi.org/10.1186/s12866-018-1328-z
  28. Molecules. 2018 Nov 09. pii: E2929. [Epub ahead of print]23(11):
    Integrated Proteomics and Lipidomics Investigation of the Mechanism Underlying the Neuroprotective Effect of N-benzylhexadecanamide.
    Yanyan Zhou, Hongjie Wang, Feifei Guo, Nan Si, Adelheid Brantner, Jian Yang, Lingyu Han, Xiaolu Wei, Haiyu Zhao, Baolin Bian.
      Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP⁺)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP⁺-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein⁻lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases.
    Keywords:  N-benzylhexadecanamide; lipidomics; neuroprotective effect; neurotransmitter; proteomics
    DOI:  https://doi.org/10.3390/molecules23112929
  29. Front Mol Neurosci. 2018 ;11 371
    No Plasmatic Proteomic Signature at Clinical Disease Onset Associated With 11 Year Clinical, Cognitive and MRI Outcomes in Relapsing-Remitting Multiple Sclerosis Patients.
    Claire Bridel, Anand J C Eijlers, Wessel N van Wieringen, Marleen Koel-Simmelink, Cyra E Leurs, Menno M Schoonheim, Joep Killestein, Charlotte E Teunissen.
      Background: The clinical course of relapsing-remitting multiple sclerosis (RRMS) is highly heterogeneous and prognostic biomarkers at time of diagnosis are lacking. Objective: We investigated the predictive value of the plasma proteome at time of diagnosis in RRMS patients. Methods: The plasma proteome was interrogated using a novel aptamer-based proteomics platform, which allows to measure the levels of a predefined set of 1310 proteins. Results: In 67 clinically and radiologically well characterized RRMS patients, we found no association between the plasma proteome at diagnosis and clinical, cognitive or MRI outcomes after 11 years. Conclusions: Proteomics studies on cerebrospinal fluid may be better suited to identify prognostic biomarkers in early RRMS.
    Keywords:  MRI; cognition; multiple sclerosis; prognosis; proteomics
    DOI:  https://doi.org/10.3389/fnmol.2018.00371
  30. BMC Biochem. 2018 Nov 12. 19(1): 9
    Effect of IAPP on the proteome of cultured Rin-5F cells.
    Samaneh Miraee-Nedjad, Paul F G Sims, Jean-Marc Schwartz, Andrew J Doig.
       BACKGROUND: Islet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of β islet cells, though the underlying molecular events leading from IAPP deposition to β cell death are still largely unknown.
    RESULTS: We used OFFGEL™ proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells. The OFFGEL™ methodology is highly effective at generating quantitative data on hundreds of proteins affected by IAPP, with its accuracy confirmed by In Cell Western and Quantitative Real Time PCR results. Combining data on individual proteins identifies pathways and protein complexes affected by IAPP. IAPP disrupts protein synthesis and degradation, and induces oxidative stress. It causes decreases in protein transport and localization. IAPP disrupts the regulation of ubiquitin-dependent protein degradation and increases catabolic processes. IAPP causes decreases in protein transport and localization, and affects the cytoskeleton, DNA repair and oxidative stress.
    CONCLUSIONS: Results are consistent with a model where IAPP aggregates overwhelm the ability of a cell to degrade proteins via the ubiquitin system. Ultimately this leads to apoptosis. IAPP aggregates may be also toxic to the cell by causing oxidative stress, leading to DNA damage or by decreasing protein transport. The reversal of any of these effects, perhaps by targeting proteins which alter in response to IAPP, may be beneficial for type II diabetes.
    Keywords:  Amylin; Mass spectrometry; Pathway analysis; Protein-protein interactions; Proteomics; Type 2 diabetes
    DOI:  https://doi.org/10.1186/s12858-018-0099-3
  31. Sci Rep. 2018 Nov 13. 8(1): 16728
    Proteomics reveals ablation of PlGF increases antioxidant and neuroprotective proteins in the diabetic mouse retina.
    Madhu Sudhana Saddala, Anton Lennikov, Dennis J Grab, Guei-Sheung Liu, Shibo Tang, Hu Huang.
      Placental growth factor (PlGF or PGF), a member of the vascular endothelial growth factor (VEGF) sub-family, plays a crucial role in pathological angiogenesis and inflammation. However, the underlying molecular mechanisms that PlGF mediates regarding the complications of non-proliferative diabetic retinopathy (DR) remain elusive. Using an LC-MS/MS-based label-free quantification proteomic approach we characterized the alterations in protein expression caused by PlGF ablation in the retinas obtained from C57BL6, Akita, PlGF-/- and Akita.PlGF-/- mice. After extraction and enzymatic digestion with Trypsin/LysC, the retinal proteins were analyzed by Q-Exactive hybrid Quadrupole-Orbitrap mass spectrometry. Differentially expressed proteins (DEPs) were identified in four comparisons based on Z-score normalization and reproducibility by Pearson's correlation coefficient. The gene ontology (GO), functional pathways, and protein-protein network interaction analysis suggested that several proteins involved in insulin resistance pathways (Gnb1, Gnb2, Gnb4, Gnai2, Gnao1, Snap2, and Gngt1) were significantly down-regulated in PlGF ablated Akita diabetic mice (Akita.PlGF-/- vs. Akita) but up-regulated in Akita vs. C57 and PlGF-/- vs. C57 conditions. Two proteins involved in the antioxidant activity and neural protection pathways, Prdx6 and Map2 respectively, were up-regulated in the Akita.PlGF-/- vs. Akita condition. Overall, we predict that down-regulation of proteins essential for insulin resistance, together with the up-regulation of antioxidant and neuroprotection proteins highlight and epitomize the potential mechanisms important for future anti-PlGF therapies in the treatment of DR.
    DOI:  https://doi.org/10.1038/s41598-018-34955-x
  32. Front Cell Infect Microbiol. 2018 ;8 357
    Increased Virulence of Bloodstream Over Peripheral Isolates of P. aeruginosa Identified Through Post-transcriptional Regulation of Virulence Factors.
    Caitríona Hickey, Bettina Schaible, Scott Nguyen, Daniel Hurley, Shabarinath Srikumar, Séamus Fanning, Eric Brown, Bianca Crifo, David Matallanas, Siobhán McClean, Cormac T Taylor, Kirsten Schaffer.
      The factors influencing the virulence of P. aeruginosa in the development of invasive infection remain poorly understood. Here, we investigated the role of the host microenvironment in shaping pathogen virulence and investigated the mechanisms involved. Comparing seven paired genetically indistinguishable clinical bloodstream and peripheral isolates of P. aeruginosa, we demonstrate that isolates derived from bloodstream infections are more virulent than their peripheral counterparts (p = 0.025). Bloodstream and peripheral isolates elicited similar NF-kB responses in a THP-1 monocyte NF-kappaB reporter cell line implicating similar immunogenicity. Proteomic analysis by mass spectrometry identified multiple virulence and virulence-related factors including LecA and RpoN in significantly greater abundance in the bacterial supernatant from the bloodstream isolate in comparison to that from the corresponding peripheral isolate. Investigation by qPCR revealed that control of expression of these virulence factors was not due to altered levels of transcription. Based on these data, we hypothesize a post-transcriptional mechanism of virulence regulation in P. aeruginosa bloodstream infections influenced by surrounding microenvironmental conditions.
    Keywords:  bloodstream; infection; proteomics; pseudomonas; virulence
    DOI:  https://doi.org/10.3389/fcimb.2018.00357
  33. Anal Chem. 2018 Nov 15.
    A Targeted Quantitative Proteomic Analysis Revealed Roles of Small GTPases in Acquired Tamoxifen Resistance in MCF-7 Cells.
    Ming Huang, Yinsheng Wang.
      Development of tamoxifen resistance remains a tremendous challenge for the treatment of estrogen receptor (ER)-positive breast cancer. Small GTPases of the Ras superfamily play crucial roles in intracellular trafficking and cell signaling, and aberrant small GTPase signaling are implicated in many types of cancer. In this study, we employed a targeted quantitative proteomic approach, which relies on stable isotope labeling by amino acid in cell culture (SILAC), gel fractionation, and scheduled multiple-reaction monitoring (MRM) analysis, to assess the differential expression of small GTPases in wild-type and tamoxifen-resistant MCF-7 cells. The method displayed superior sensitivity and reproducibility over the shotgun proteomic approach, and it facilitated the quantification of 96 small GTPases. Among them, 13 and 10 proteins were significantly down- and up-regulated (with > 1.5-fold change), respectively, in the tamoxifen-resistant relative to the parental line. In particular, we observed a significant down-regulation of RAB31 in tamoxifen-resistant cells, which, in combination with bioinformatic analysis and downstream validation experiments, supported a role of RAB31 in tamoxifen resistance in ER-positive breast cancer cells. Together, our result demonstrated that the targeted proteomic method constitutes a powerful approach for revealing the role of small GTPases in therapeutic resistance.
    DOI:  https://doi.org/10.1021/acs.analchem.8b04526
  34. Liver Int. 2018 Nov 16.
    Genetic determinants of steatosis and fibrosis progression in pediatric non-alcoholic fatty liver disease.
    Christian A Hudert, Silvia Selinski, Birgit Rudolph, Hendrik Bläker, Christoph Loddenkemper, Ria Thielhorn, Nikolaus Berndt, Klaus Golka, Cristina Cadenas, Jörg Reinders, Stephan Henning, Philip Bufler, Peter L M Jansen, Hermann-Georg Holzhütter, David Meierhofer, Jan G Hengstler, Susanna Wiegand.
       BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children and adolescents today. In comparison to adult disease, pediatric NAFLD may show a periportal localization, which is associated with advanced fibrosis. This study aimed to assess the role of genetic risk variants for histologic disease pattern and severity in childhood NAFLD.
    METHODS: We studied 14 single nucleotide polymorphisms (SNP) in a cohort of 70 adolescents with biopsy-proven NAFLD. Genotype was compared to an adult control cohort (n=200) and analyzed in relation to histologic disease severity and liver tissue proteomics.
    RESULTS: Three of the 14 SNPs were significantly associated with pediatric NAFLD after FDR adjustment, rs738409 (PNPLA3, P=2.80×10-06 ), rs1044498 (ENPP1, P=0.0091) and rs780094 (GCKR, P=0.0281). The severity of steatosis was critically associated with rs738409 (OR=3.25; 95% CI: 1.72-6.52, FDR adjusted P=0.0070). The strongest variants associated with severity of fibrosis were rs1260326, rs780094 (both GCKR) and rs659366 (UCP2). PNPLA3 was associated with a portal pattern of steatosis, inflammation and fibrosis. Proteome profiling revealed decreasing levels of GCKR protein with increasing carriage of the rs1260326/rs780094 minor alleles and down-regulation of the retinol pathway in rs738409 G/G carriers. Computational metabolic modelling highlighted functional relevance of PNPLA3, GCKR and UCP2 for NAFLD development.
    CONCLUSIONS: This study provides evidence for the role of PNPLA3 as a determinant of portal NAFLD localization and severity of portal fibrosis in children and adolescents, the risk variant being associated with an impaired hepatic retinol metabolism. This article is protected by copyright. All rights reserved.
    Keywords:  genetics; metabolic modelling; pediatric NAFLD; proteomics
    DOI:  https://doi.org/10.1111/liv.14006
  35. Methods Mol Biol. 2018 ;1819 153-173
    Computational and Experimental Approaches to Predict Host-Parasite Protein-Protein Interactions.
    Yesid Cuesta-Astroz, Guilherme Oliveira.
      In host-parasite systems, protein-protein interactions are key to allow the pathogen to enter the host and persist within the host. The study of host-parasite molecular communication improves the understanding the mechanisms of infection, evasion of the host immune system and tropism across different tissues. Current trends in parasitology focus on unraveling host-parasite protein-protein interactions to aid the development of new strategies to combat pathogenic parasites with better treatments and prevention mechanisms. Due to the complexity of capturing experimentally these interactions, computational approaches integrating data from different sources (mainly "omics" data) become key to complement or support experimental approaches. Here, we focus on the application of experimental and computational methods in the prediction of host-parasite interactions and highlight the potential of each of these methods in specific contexts.
    Keywords:  Computational biology; Host–parasite interactions; Parasitology; Protein–protein interactions; Proteomics; Secretome; Systems biology
    DOI:  https://doi.org/10.1007/978-1-4939-8618-7_7
  36. Am J Respir Crit Care Med. 2018 Nov 13.
    Tumor-Derived Autoantibodies Identify Malignant Pulmonary Nodules.
    Kristin J Lastwika, Julia Kargl, Yuzheng Zhang, Xiaodong Zhu, Edward Lo, David Shelley, Jon J Ladd, Wei Wu, Paul Kinahan, Sudhakar N J Pipavath, Timothy W Randolph, Melissa Shipley, Paul D Lampe, A McGarry Houghton.
       RATIONALE: Screening for non-small cell lung cancer is associated with earlier diagnosis and reduced mortality but also increased harm due to invasive follow up of benign pulmonary nodules. Lung tumorigenesis activates the immune system, components of which could serve as tumor specific biomarkers.
    OBJECTIVES: To profile tumor-derived autoantibodies as peripheral biomarkers of malignant pulmonary nodules.
    METHODS: High-density protein arrays were used to define the specificity of autoantibodies isolated from B cells of 10 resected lung tumors. These tumor-derived autoantibodies were also examined as free or complexed to antigen in the plasma of the same 10 patients and matched benign nodule controls. Promising autoantibodies were further analyzed in an independent cohort of 250 nodule positive patients.
    MEASUREMENTS AND MAIN RESULTS: Thirteen tumor B cell-derived autoantibodies isolated ex vivo showed ≥50% sensitivity and ≥70% specificity for lung cancer. ‎In plasma, 11/13 autoantibodies were present both complexed to and free from antigen. In the larger validation cohort, 5/13 tumor-derived autoantibodies remained significantly elevated in cancers. A combination of 4 of these autoantibodies could detect malignant nodules with an AUC=0.74 and had an AUC=0.78 in a sub-cohort of indeterminate (8-20mm in the longest diameter) pulmonary nodules.
    CONCLUSIONS: Our novel pipeline identifies tumor-derived autoantibodies that could effectively serve as blood biomarkers for malignant pulmonary nodule diagnosis. This approach has future implications for both a cost-effective and non-invasive approach to determine nodule malignancy for widespread LDCT screening.
    Keywords:  Autoantibodies; Lung cancer early detection
    DOI:  https://doi.org/10.1164/rccm.201804-0628OC
  37. PLoS One. 2018 ;13(11): e0207371
    C/VDdb: A multi-omics expression profiling database for a knowledge-driven approach in cardiovascular disease (CVD).
    Marco Fernandes, Alisha Patel, Holger Husi.
      The cardiovascular disease (C/VD) database is an integrated and clustered information resource that covers multi-omic studies (microRNA, genomics, proteomics and metabolomics) of cardiovascular-related traits with special emphasis on coronary artery disease (CAD). This resource was built by mining existing literature and public databases and thereafter manual biocuration was performed. To enable integration of omic data from distinct platforms and species, a specific ontology was applied to tie together and harmonise multi-level omic studies based on gene and protein clusters (CluSO) and mapping of orthologous genes (OMAP) across species. CAD continues to be a leading cause of death in the population worldwide, and it is generally thought to be an age-related disease. However, CAD incidence rates are now known to be highly influenced by environmental factors and interactions, in addition to genetic determinants. With the complexity of CAD aetiology, there is a difficulty in research studies to elucidate general elements compared to other cardiovascular diseases. Data from 92 studies, covering 13945 molecular entries (4353 unique molecules) is described, including data descriptors for experimental setup, study design, discovery-validation sample size and associated fold-changes of the differentially expressed molecular features (p-value<0.05). A dedicated interactive web interface, equipped with a multi-parametric search engine, data export and indexing menus are provided for a user-accessible browsing experience. The main aim of this work was the development of a data repository linking clinical information and molecular differential expression in several CVD-related traits from multi-omics studies (genomics, transcriptomics, proteomics and metabolomics). As an example case of how to query and identify data sets within the database framework and concomitantly demonstrate the database utility, we queried CAD-associated studies and performed a systems-level integrative analysis. URL: www.padb.org/cvd.
    DOI:  https://doi.org/10.1371/journal.pone.0207371
  38. Cancer Cell. 2018 Nov 12. pii: S1535-6108(18)30467-7. [Epub ahead of print]34(5): 807-822.e7
    KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism.
    Angelina V Vaseva, Devon R Blake, Thomas S K Gilbert, Serina Ng, Galen Hostetter, Salma H Azam, Irem Ozkan-Dagliyan, Prson Gautam, Kirsten L Bryant, Kenneth H Pearce, Laura E Herring, Haiyong Han, Lee M Graves, Agnieszka K Witkiewicz, Erik S Knudsen, Chad V Pecot, Naim Rashid, Peter J Houghton, Krister Wennerberg, Adrienne D Cox, Channing J Der.
      Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3β signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.
    Keywords:  EGFR; ERK; ERK5; FBXW7; KRAS; MYC; PI3K; SRC; pancreatic cancer
    DOI:  https://doi.org/10.1016/j.ccell.2018.10.001
  39. J Cell Biochem. 2018 Nov 11.
    Epitope-based immunoinformatics approach on RNA-dependent RNA polymerase (RdRp) protein complex of Nipah virus (NiV).
    Lavanya Ravichandran, Arthi Venkatesan, J Febin Prabhu Dass.
      Persistent outbreaks of Nipah virus (NiV) with severe case fatality throw a major challenge on researchers to develop a drug or vaccine to combat the disease. With little knowledge of its molecular mechanisms, we utilized the proteome data of NiV to evaluate the potency of three major proteins (phosphoprotein, polymerase, and nucleocapsid protein) in the RNA-dependent RNA polymerase complex to count as a possible candidate for epitope-based vaccine design. Profound computational analysis was used on the above proteins individually to explore the T-cell immune properties like antigenicity, immunogenicity, binding to major histocompatibility complex class I and class II alleles, conservancy, toxicity, and population coverage. Based on these predictions the peptide 'ELRSELIGY' of phosphoprotein and 'YPLLWSFAM' of nulceocapsid protein were identified as the best-predicted T-cell epitopes and molecular docking with human leukocyte antigen-C (HLA-C*12:03) molecule was effectuated followed by validation with molecular dynamics simulation. The B-cell epitope predictions suggest that the sequence positions 421 to 471 in phosphoprotein, 606 to 640 in polymerase and 496 to 517 in nucleocapsid protein are the best-predicted regions for B-cell immune response. However, the further experimental circumstance is required to test and validate the efficacy of the subunit peptide for potential candidacy against NiV.
    Keywords:  Nipah virus; epitope-based vaccine design; molecular docking; molecular dynamics; nucleocapsid protein; phosphoprotein and polymerase
    DOI:  https://doi.org/10.1002/jcb.27979
  40. J Acquir Immune Defic Syndr. 2018 Nov 05.
    S100a14 is Increased in Activated Nk Cells and Plasma of HIV-Exposed Seronegative People Who Inject Drugs and Promotes Monocyte-Nk crosstalk.
    Krystal Colón, David W Speicher, Peter Smith, Mack Taylor, David S Metzger, Luis J Montaner, Costin Tomescu.
       BACKGROUND: HIV-exposed sero-negative people who inject drugs (HESN-PWID) have been shown to have increased NK cell and myeloid activation when compared to control donors.
    METHODS: We investigated potential mechanisms maintaining NK activation by conducting quantitative proteome comparisons of NK cells from HESN-PWID subjects and control donors. Proteins upregulated in NK cells were measured in the plasma of HESN-PWID subjects by ELISA and further investigated for their ability to induce innate immune activation in vitro.
    RESULTS: The NK cell proteome comparison showed markedly higher levels of Interferon-stimulated proteins and S100 proteins, including S100A14. Consistent with these results, we observed significantly higher levels of S100A14 in the plasma of HESN-PWID subjects compared to controls (p=0.033, n=25). In vitro, the addition of recombinant S100A14 protein significantly activated NK cells in a PBMC mixture (p=0.011, n=9), but not purified NK cells alone. Treatment of purified monocytes with recombinant S100A14 protein induced secretion of TNF-alpha and led to significantly higher NK CD69 activation (p=0.0156, n=7) in a co-culture through a TLR4 dependent interaction.
    CONCLUSIONS: Our study identified S100A14 as a novel protein increased within NK cells and plasma of HESN-PWID subjects with the capacity to sustain NK activation through TLR4-dependent activation of myeloid cells.
    DOI:  https://doi.org/10.1097/QAI.0000000000001911
  41. J Ethnopharmacol. 2018 Nov 13. pii: S0378-8741(18)31260-1. [Epub ahead of print]
    Quantitative proteomics reveal antidepressant potential protein targets of xiaochaihutang in corticosterone induced model of depression.
    Kuo Zhang, Meiyao He, Dongmei Su, Xing Pan, Yuting Li, Haotian Zhang, Jingyu Yang, Chunfu Wu.
       ETHNOPHARMACOLOGICAL RELEVANCE: Xiaochaihutang (XCHT), one of famous Chinese herbal prescription for treating Shaoyang symptom, has been used successfully in depressive disorders for many years. Our laboratory has demonstrated that XCHT remarkably alleviated various depressive behaviors induced by several depressive animal models, but previous studies only focused on one or several protein targets, lacked dynamic change and interrelation of proteins. Therefore, potential protein targets and mechanisms are required further systematic investigation.
    AIM OF THE STUDY: To discover and assess the differentially expressed proteins (DEPs) of hippocampus after oral administration of XCHT in corticosterone (CORT) induced model of depression by using isobaric tags for relative and absolute quantification (iTRAQ) analysis.
    MATERIALS AND METHODS: The antidepressant effects of XCHT were assessed by two behavioral despair models (forced swimming test and tail suspension test) in CORT induced model of depression. The DEPs of hippocampus after XCHT treatment were investigated by iTRAQ analysis. Potential protein targets and mechanisms were assessed by gene ontology (GO), Kyoto encyclopedia of gene and genomes (KEGG) and protein-protein interaction (PPI) network.
    RESULTS: Our data demonstrated XCHT could significantly improve depressive behaviors. A total of 241 DEPs were identified after XCHT treatment, including 68 up regulation and 173 down regulation proteins. GO enrichment results indicated that XCHT mainly regulated intracellular structural proteins involved in cellular response to stress, transferase activity and steroid hormone. KEGG enrichment analysis showed that endocytosis might be the principal pathway of XCHT on depression. PPI analysis predicted cell division cycle and apoptosis regulator protein 1 (Ccar1) and Calretinin (Calb2) might play the central roles in XCHT's antidepressant network.
    CONCLUSION: Our results indicate that XCHT plays the important roles in antidepressant action by restoring DEPs, which results in the dysregulation of hippocampal neurogenesis, neurotransmitter and steroid hormone. The current results wish to provide novel perspectives for revealing the potential protein targets of XCHT on depression.
    Keywords:  Baicalein (PubChem CID: 5281605); Baicalin (PubChem CID: 64982); Diphenhydramine (PubChem CID: 3100); Ginsenoside Rg1 (PubChem CID: 441923); Liquiritin (PubChem CID: 503737); Saikosaponin a (PubChem CID: 167928); Verapamil (PubChem CID: 2520); Wogonin (PubChem CID: 5281703); Wogonoside (PubChem CID: 29927693); Xiaochaihutang; antidepressant; differentially expressed protein; proteomics
    DOI:  https://doi.org/10.1016/j.jep.2018.11.020
  42. Methods. 2018 Nov 09. pii: S1046-2023(18)30114-2. [Epub ahead of print]
    HiPLA: High-throughput imaging Proximity Ligation Assay.
    Leonid A Serebryannyy, Tom Misteli.
      Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize changes to protein interactomes. Using HiPLA with a library of antibodies, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. In combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions were accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.
    Keywords:  High-throughput imaging; Hutchinson-Gilford progeria syndrome; Lamin proteomics; Proximity ligation
    DOI:  https://doi.org/10.1016/j.ymeth.2018.11.004
  43. Exp Mol Pathol. 2018 Nov 08. pii: S0014-4800(18)30282-X. [Epub ahead of print]
    Molecular biomarkers of Graves' ophthalmopathy.
    Christine M Longo, Paul J Higgins.
      Graves' ophthalmopathy (GO), a complication of Graves' disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-β1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-β1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy.
    Keywords:  Biomarkers; Fibrosis; Graves' disease; Inflammatory cytokines; Orbitopathy; PAI-1; Plasmin cascade; SERPINs; TGF-β; Tissue remodeling
    DOI:  https://doi.org/10.1016/j.yexmp.2018.11.004
  44. Parasit Vectors. 2018 Nov 12. 11(1): 584
    Trichinella britovi muscle larvae and adult worms: stage-specific and common antigens detected by two-dimensional gel electrophoresis-based immunoblotting.
    Sylwia Grzelak, Bożena Moskwa, Justyna Bień.
       BACKGROUND: Trichinella britovi is the second most common species of Trichinella that may affect human health. As an early diagnosis of trichinellosis is crucial for effective treatment, it is important to identify sensitive, specific and common antigens of adult T. britovi worms and muscle larvae. The present study was undertaken to uncover the stage-specific and common proteins of T. britovi that may be used in specific diagnostics.
    METHODS: Somatic extracts obtained from two developmental stages, muscle larvae (ML) and adult worms (Ad), were separated using two-dimensional gel electrophoresis (2-DE) coupled with immunoblot analysis. The positively-visualized protein spots specific for each stage were identified through liquid chromatography-tandem mass spectrometry (LC-LC/MS).
    RESULTS: A total of 272 spots were detected in the proteome of T. britovi adult worms (Ad) and 261 in the muscle larvae (ML). The somatic extracts from Ad and ML were specifically recognized by T. britovi-infected swine sera at 10 days post infection (dpi) and 60 dpi, with a total of 70 prominent protein spots. According to immunoblotting patterns and LC-MS/MS results, the immunogenic spots recognized by different pig T. britovi-infected sera were divided into three groups for the two developmental stages: adult stage-specific proteins, muscle larvae stage-specific proteins, and proteins common to both stages. Forty-five Ad proteins (29 Ad-specific and 16 common) and thirteen ML proteins (nine ML-specific and four common) cross-reacted with sera at 10 dpi. Many of the proteins identified in Ad (myosin-4, myosin light chain kinase, paramyosin, intermediate filament protein B, actin-depolymerizing factor 1 and calreticulin) are involved in structural and motor activity. Among the most abundant proteins identified in ML were 14-3-3 protein zeta, actin-5C, ATP synthase subunit d, deoxyribonuclease-2-alpha, poly-cysteine and histide-tailed protein, enolase, V-type proton ATPase catalytic and serine protease 30. Heat-shock protein, intermediate filament protein ifa-1 and intermediate filament protein B were identified in both proteomes.
    CONCLUSIONS: To our knowledge, this study represents the first immunoproteomic identification of the antigenic proteins of adult worms and muscle larvae of T. britovi. Our results provide a valuable basis for the development of diagnostic methods. The identification of common components for the two developmental stages of T. britovi may be useful in the preparation of parasitic antigens in recombinant forms for diagnostic use.
    Keywords:  2-DE; Adult worm; Immunoblotting; Mass spectrometry; Muscle larvae; Trichinella britovi
    DOI:  https://doi.org/10.1186/s13071-018-3177-x
  45. BMC Cancer. 2018 Nov 12. 18(1): 1096
    Calcium-activated chloride channel regulator 1 as a prognostic biomarker in pancreatic ductal adenocarcinoma.
    Dingyuan Hu, Daniel Ansari, Qimin Zhou, Agata Sasor, Katarzyna Said Hilmersson, Monika Bauden, Yi Jiang, Roland Andersson.
       BACKGROUND: In a previous study utilizing mass spectrometry-based proteomics, we identified calcium-activated chloride channel regulator 1 (CLCA1) as a potential tumor suppressor in pancreatic cancer and the expression was inversely correlated with patient survival. The aim of the study was to further validate the prognostic significance of CLCA1 in pancreatic cancer.
    METHODS: CLCA1 expression was evaluated with tissue microarrays and immunohistochemistry in 140 patients with pancreatic ductal adenocarcinoma that underwent surgical resection at Skåne University Hospital, Sweden. Kaplan-Meier and Cox proportional hazards modeling were used to explore the association between CLCA1 and clinicopathological factors and survival.
    RESULTS: CLCA1 expression was denoted as positive in 90 tumors (64.3%), with positive staining being limited to the tumor cells. There were no significant association between CLCA1 expression and established clinicopathological parameters. Low CLCA1 expression correlated significantly with shorter disease-free survival (11.9 vs 17.5 months, P = 0.042). Multivariable Cox regression analysis confirmed the results (HR 0.61, 95% CI-0.40-0.92, P = 0.019).
    CONCLUSIONS: Low CLCA1 expression is an independent factor of poor disease-free survival in pancreatic cancer.
    Keywords:  CLCA1; Calcium-activated chloride channel regulators; Pancreatic ductal adenocarcinoma; Survival
    DOI:  https://doi.org/10.1186/s12885-018-5013-2
  46. Adv Parasitol. 2018 ;pii: S0065-308X(18)30050-2. [Epub ahead of print]102 45-72
    Opisthorchis viverrini Proteome and Host-Parasite Interactions.
    Sutas Suttiprapa, Javier Sotillo, Michael Smout, Watcharapol Suyapoh, Sujittra Chaiyadet, Timir Tripathi, Thewarach Laha, Alex Loukas.
      The omics technologies have improved our understanding of the molecular events that underpin host-parasite interactions and the pathogenesis of parasitic diseases. In the last decade, proteomics and genomics in particular have been used to characterize the surface and secreted products of the carcinogenic liver fluke Opisthorchis viverrini and revealed important roles for proteins at the host-parasite interface to ensure that the flukes can migrate, feed and reproduce in a hostile environment. This review summarizes the advances made in this area, primarily focusing on discoveries enabled by the publication of the fluke secreted proteomes over the last decade. Protein families that will be covered include proteases, antioxidants, oncogenic proteins and the secretion of exosome-like extracellular vesicles. Roles of these proteins in host-parasite interactions and pathogenesis of fluke-induced hepatobiliary diseases, including cholangiocarcinogenesis, are discussed. Future directions for the application of this knowledge to control infection and disease will also be discussed.
    Keywords:  Cholangiocarcinoma; Extracellular vesicle; Helminth; Host–parasite interaction; Opisthorchis viverrini; OvES
    DOI:  https://doi.org/10.1016/bs.apar.2018.06.002
  47. Future Microbiol. 2018 Nov 15.
    Kaurenoic acid and its sodium salt derivative: antibacterial activity against Porphyromonas gingivalis and their mechanism of action.
    Carlos Hg Martins, Fariza Abrão, Thaís S Moraes, Pollyanna F Oliveira, Denise C Tavares, Lizandra G Magalhães, Fábio C Galvão, Rodrigo Cs Veneziani, Sérgio R Ambrósio.
       AIM: To evaluate the antibacterial activity of 12 kaurane-type diterpenes against a panel of bacteria that cause endodontic infection.
    METHODS & MATERIALS: We conducted tests against bacteria in the planktonic or in the sessile mode, cytotoxic assays for the most promising compounds against human normal lung fibroblast cells, and Porphyromonas gingivalis (ATCC 33277) proteomic analysis.
    RESULTS & CONCLUSION: Kaurenoic acid and its salt exhibited satisfactory antibacterial action against the evaluated bacteria. Proteomic analysis suggested that these compounds might interfere in bacterial metabolism and virulence factor expression. Kaurane-type diterpenes are an important class of natural products and should be considered in the search for new irrigating solutions to treat endodontic infections.
    Keywords:  P. gingivalis; antibacterial activity; antibiofilm; cytotoxic assay; kaurane-type diterpenes; proteomic
    DOI:  https://doi.org/10.2217/fmb-2018-0140
  48. J Microbiol Methods. 2018 Nov 12. pii: S0167-7012(18)30591-8. [Epub ahead of print]
    Rapid detection of colistin-resistant Klebsiella pneumoniae using MALDI-TOF MS peak-based assay.
    Cesira Giordano, Simona Barnini.
      Carbapenemase-producing Klebsiella pneumoniae has become a worldwide recognized cause of nosocomial infections and requires urgent public health attention. The main reason of this concern is the increasing resistance to all the last-resort antibiotics, including colistin. The ideal methodology for colistin susceptibility testing still remains undefined. However, the emergence of colistin as one of the last-option treatments requires a reliable method to determine the susceptibility profile of resistant isolates. The aim of the present study was to evaluate the impact of detecting colistin resistance in Klebsiella pneumoniae isolates using MALDI-TOF MS in clinical routine practice. For this reason, 139 isolates of K. pneumoniae were collected during 2015-2017 from patients hospitalized at Pisa University Hospital. Colonies suspected to be colistin resistant were identified using MALDI-TOF MS (Bruker Daltonik GmbH, Bremen, Germany) following a protein extraction protocol. Strains were previously wholly genome sequenced. To create a customized database entry and to generate classifying algorithm models, 1.112 mass spectra were collected. In relation to their mass signals and intensities, a two dimensional peak distribution was created. The recognition capability of the algorithm based on two manually selected mass peaks was 91,8%, while cross validation was 87,6%. The proportion of correctly classified colistin-resistant K. pneumoniae was 91% and colistin-susceptible was 73%. The emergence of colistin-resistant Gram-negative organisms has a dramatic impact on patient outcomes. Our study, based on MALDI-TOF MS technology, offers rapid preliminary results on colistin resistance profile coupled with bacteria identification.
    Keywords:  Colistin resistance; Genetic algorithm; MALDI-TOF MS; Mass peaks; Proteome
    DOI:  https://doi.org/10.1016/j.mimet.2018.11.008
  49. Nucleic Acids Res. 2018 Nov 10.
    dbPTM in 2019: exploring disease association and cross-talk of post-translational modifications.
    Kai-Yao Huang, Tzong-Yi Lee, Hui-Ju Kao, Chen-Tse Ma, Chao-Chun Lee, Tsai-Hsuan Lin, Wen-Chi Chang, Hsien-Da Huang.
      The dbPTM (http://dbPTM.mbc.nctu.edu.tw/) has been maintained for over 10 years with the aim to provide functional and structural analyses for post-translational modifications (PTMs). In this update, dbPTM not only integrates more experimentally validated PTMs from available databases and through manual curation of literature but also provides PTM-disease associations based on non-synonymous single nucleotide polymorphisms (nsSNPs). The high-throughput deep sequencing technology has led to a surge in the data generated through analysis of association between SNPs and diseases, both in terms of growth amount and scope. This update thus integrated disease-associated nsSNPs from dbSNP based on genome-wide association studies. The PTM substrate sites located at a specified distance in terms of the amino acids encoded from nsSNPs were deemed to have an association with the involved diseases. In recent years, increasing evidence for crosstalk between PTMs has been reported. Although mass spectrometry-based proteomics has substantially improved our knowledge about substrate site specificity of single PTMs, the fact that the crosstalk of combinatorial PTMs may act in concert with the regulation of protein function and activity is neglected. Because of the relatively limited information about concurrent frequency and functional relevance of PTM crosstalk, in this update, the PTM sites neighboring other PTM sites in a specified window length were subjected to motif discovery and functional enrichment analysis. This update highlights the current challenges in PTM crosstalk investigation and breaks the bottleneck of how proteomics may contribute to understanding PTM codes, revealing the next level of data complexity and proteomic limitation in prospective PTM research.
    DOI:  https://doi.org/10.1093/nar/gky1074
  50. Sci Rep. 2018 Nov 12. 8(1): 16671
    Identification of DKK-1 as a novel mediator of statin effects in human endothelial cells.
    Marta Pontremoli, Maura Brioschi, Roberta Baetta, Stefania Ghilardi, Cristina Banfi.
      This study shows that DKK-1, a member of the Dickkopf family and a regulator of the Wnt pathways, represents a novel target of statins which, through the inhibition of HMG-CoA reductase and of non-steroidal isoprenoid intermediates, exert extra-beneficial effect in preventing atherosclerosis beyond their effect on the lipid profile. We found that atorvastatin downregulates DKK-1 protein (-88.3 ± 4.1%) and mRNA expression (-90 ± 4.2%) through the inhibition of Cdc42, Rho and Rac geranylgeranylated proteins. Further, a combined approach based on the integration of label-free quantitative mass spectrometry based-proteomics and gene silencing allowed us to demonstrate that DKK-1 itself mediates, at least in part, statin effects on human endothelial cells. Indeed, DKK-1 is responsible for the regulation of the 21% of the statin-modulated proteins, which include, among others, clusterin/apoJ, plasminogen activator inhibitor type 1 (PAI-1), myristoylated alanine-rich C-kinase substrate (MARCKS), and pentraxin 3 (PTX3). The Gene Ontology enrichment annotation revealed that DKK-1 is also a potential mediator of the extracellular matrix organization, platelet activation and response to wounding processes induced by statin. Finally, we found that plasma level of DKK-1 from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) was lower (-42 ± 23%) than that of control animals. Thus, DKK-1 is not only a target of statin but it directly regulates the expression of molecules involved in a plethora of biological functions, thus expanding its role, which has been so far restricted mainly to cancer.
    DOI:  https://doi.org/10.1038/s41598-018-35119-7
  51. Proteomes. 2018 Nov 12. pii: E47. [Epub ahead of print]6(4):
    Paraquat-Mediated Oxidative Stress in Anopheles gambiae Mosquitoes Is Regulated by An Endoplasmic Reticulum (ER) Stress Response.
    Brian B Tarimo, Henry Chun Hin Law, Dingyin Tao, Rebecca Pastrana-Mena, Stefan M Kanzok, Joram J Buza, Rhoel R Dinglasan.
      Paraquat is a potent superoxide (O₂-)-inducing agent that is capable of inducing an oxidative imbalance in the mosquito midgut. This oxidative imbalance can super-stress the malaria parasite, leading to arrested development in the mosquito midgut and reduced transmission. While several studies have explored the effect of paraquat on malaria parasites, a fundamental understanding of the mosquito response to this compound remains unknown. Here, we quantified the mosquito midgut proteomic response to a paraquat-laced sugar meal, and found that An. gambiae midguts were enriched in proteins that are indicative of cells under endoplasmic reticulum (ER) stress. We also carried out qRT-PCR analyses for nine prominent thioredoxin (Trx) and glutathione (GSH)-dependent genes in mosquito midguts post P. falciparum blood meal ingestion to evaluate the concordance between transcripts and proteins under different oxidative stress conditions. Our data revealed an absence of significant upregulation in the Trx and GSH-dependent genes following infected blood meal ingestion. These data suggest that the intrinsic tolerance of the mosquito midgut to paraquat-mediated oxidative stress is through an ER stress response. These data indicate that mosquitoes have at least two divergent pathways of managing the oxidative stress that is induced by exogenous compounds, and outline the potential application of paraquat-like drugs to act selectively against malaria parasite development in mosquito midguts, thereby blocking mosquito-to-human transmission.
    Keywords:  Anopheles gambiae; endoplasmic reticulum stress; malaria; oxidative stress; paraquat; transmission-blocking
    DOI:  https://doi.org/10.3390/proteomes6040047
  52. Neurobiol Aging. 2018 Oct 17. pii: S0197-4580(18)30376-2. [Epub ahead of print]74 77-89
    Image-guided phenotyping of ovariectomized mice: altered functional connectivity, cognition, myelination, and dopaminergic functionality.
    Cynthia Anckaerts, Jaana van Gastel, Valerie Leysen, Rukun Hinz, Abdelkrim Azmi, Pascal Simoens, Disha Shah, Firat Kara, An Langbeen, Peter Bols, Charlotte Laloux, Vincent Prevot, Marleen Verhoye, Stuart Maudsley, Annemie Van der Linden.
      A large proportion of the population suffers from endocrine disruption, e.g., menopausal women, which might result in accelerated aging and a higher risk for developing cognitive disorders. Therefore, it is crucial to fully understand the impact of such disruptions on the brain to identify potential therapeutic strategies. Here, we show using resting-state functional magnetic resonance imaging that ovariectomy and consequent hypothalamus-pituitary-gonadal disruption result in the selective dysconnectivity of 2 discrete brain regions in mice. This effect coincided with cognitive deficits and an underlying pathological molecular phenotype involving an imbalance of neurodevelopmental/neurodegenerative signaling. Furthermore, this quantitative mass spectrometry proteomics-based analysis of molecular signaling patterns further identified a strong involvement of altered dopaminergic functionality (e.g., DAT and predicted upstream regulators DRD3, NR4A2), reproductive signaling (e.g., Srd5a2), rotatin expression (rttn), cellular aging (e.g., Rxfp3, Git2), myelination, and axogenesis (e.g., Nefl, Mag). With this, we have provided an improved understanding of the impact of hypothalamus-pituitary-gonadal dysfunction and highlighted the potential of using a highly translational magnetic resonance imaging technique for monitoring these effects on the brain.
    Keywords:  Aging; Behavior; Cognition; Dopamine; Functional connectivity; Mass spectrometry; Myelination; Ovariectomy; Quantitative proteomics; Resting-state fMRI
    DOI:  https://doi.org/10.1016/j.neurobiolaging.2018.10.012
  53. J Autoimmun. 2018 Nov 09. pii: S0896-8411(18)30622-X. [Epub ahead of print]
    Prognostic models in primary biliary cholangitis.
    Laura Cristoferi, Alessandra Nardi, Vincenzo Ronca, Pietro Invernizzi, George Mells, Marco Carbone.
      Risk prediction modelling is important to better understand the determinants of the course and outcome of PBC and to inform the risk across the disease continuum in PBC enabling risk-stratified follow-up care and personalised therapy. Current prognostic models in PBC are based on treatment response to ursodeoxycholic acid because of the well-established relationship between alkaline phosphatase on treatment and long-term outcome. In addition, serum alkaline phosphatase correlates with ductular reaction and biliary metaplasia, which are hallmark of biliary injury. Considering the waiting time for treatment failure in high-risk patients is not inconsequential, efforts are focused on bringing forward risk stratification at diagnosis by predicting treatment response at onset. There is a need for better prognostic variables that are central to the disease process. We should take an integrative approach that incorporates multiple layers of information including genetic and environmental influences, host characteristics, clinical data, and molecular alterations for risk assessments. Biomarker discovery has an accelerated pace taking advantage of the emergence of large-scale omics platforms (genomics, epigenomics, transcriptomics, proteomics, metabolomics, and others) and whole-genome sequencing. In the digital era, applications of artificial intelligence, such as machine learning, can support the computing power required to analyse the vast amount of data produced by omics. The information is then used for the development of personalised risk prediction models that through clinical trials and hopefully industry partnerships can guide risk management strategies. We are facing an unprecedented opportunity for the integration of molecular diagnostics into the clinic, which promotes progress toward the personalised management of patients with PBC.
    Keywords:  Alkaline phosphatase; Personalised medicine; Primary biliary cholangitis; Prognostic models; Risk prediction
    DOI:  https://doi.org/10.1016/j.jaut.2018.10.024
  54. Mol Cell. 2018 Oct 30. pii: S1097-2765(18)30802-5. [Epub ahead of print]
    MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis.
    Manpreet Kalkat, Diana Resetca, Corey Lourenco, Pak-Kei Chan, Yong Wei, Yu-Jia Shiah, Natasha Vitkin, Yufeng Tong, Maria Sunnerhagen, Susan J Done, Paul C Boutros, Brian Raught, Linda Z Penn.
      Transforming members of the MYC family (MYC, MYCL1, and MYCN) encode transcription factors containing six highly conserved regions, termed MYC homology boxes (MBs). By conducting proteomic profiling of the MB interactomes, we demonstrate that half of the MYC interactors require one or more MBs for binding. Comprehensive phenotypic analyses reveal that two MBs, MB0 and MBII, are universally required for transformation. MBII mediates interactions with acetyltransferase-containing complexes, enabling histone acetylation, and is essential for MYC-dependent tumor initiation. By contrast, MB0 mediates interactions with transcription elongation factors via direct binding to the general transcription factor TFIIF. MB0 is dispensable for tumor initiation but is a major accelerator of tumor growth. Notably, the full transforming activity of MYC can be restored by co-expression of the non-transforming MB0 and MBII deletion proteins, indicating that these two regions confer separate molecular functions, both of which are required for oncogenic MYC activity.
    Keywords:  BioID; MYC; TFIIF; TRRAP; cancer; histone acetylation; mass spectrometry; protein interactions; transcriptional elongation; transformation
    DOI:  https://doi.org/10.1016/j.molcel.2018.09.031
  55. Nat Commun. 2018 Nov 13. 9(1): 4770
    Proteome-wide analysis of USP14 substrates revealed its role in hepatosteatosis via stabilization of FASN.
    Bin Liu, Shangwen Jiang, Min Li, Xuelian Xiong, Mingrui Zhu, Duanzhuo Li, Lei Zhao, Lili Qian, Linhui Zhai, Jing Li, Han Lu, Shengnan Sun, Jiandie Lin, Yan Lu, Xiaoying Li, Minjia Tan.
      Ubiquitin-specific protease 14 (USP14) is one of the major proteasome-associated deubiquitinating enzymes critical for proteome homeostasis. However, substrates of USP14 remain largely unknown, hindering the understanding of its functional roles. Here we conduct a comprehensive proteome, ubiquitinome and interactome analysis for USP14 substrate screening. Bioinformatics analysis reveals broad new potential roles of USP14, especially in lipid and carbohydrate metabolism. Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14. USP14 directly interacts with and increases FASN stability. As a result, overexpression of USP14 promotes liver triglyceride accumulation in C57BL/6 mice, whereas genetic ablation or pharmacological inhibition of USP14 ameliorates hepatosteatosis, hyperglycemia and insulin resistance in obese mice. In conclusion, our findings reveal for the first time an indispensable role of USP14 in hepatosteatosis through FASN stabilization.
    DOI:  https://doi.org/10.1038/s41467-018-07185-y
  56. PLoS Negl Trop Dis. 2018 Nov 16. 12(11): e0006963
    Identification and characterization of Loa loa antigens responsible for cross-reactivity with rapid diagnostic tests for lymphatic filariasis.
    Marla I Hertz, Hugues Nana-Djeunga, Joseph Kamgno, Abdel Jelil Njouendou, Valerine Chawa Chunda, Samuel Wanji, Amy Rush, Peter U Fischer, Gary J Weil, Philip J Budge.
      The Global Program to Eliminate Lymphatic Filariasis (LF) relies on rapid diagnostic tests (RDTs) to determine where annual mass drug administration for LF is required and when it can be stopped. These tests detect a Wuchereria bancrofti glycoprotein in the blood of infected persons via a carbohydrate moiety recognized by the monoclonal antibodies AD12 and DH6.5. Loiasis cross-reactivity with LF RDTs has recently been recognized as a serious obstacle to LF elimination in loiasis-endemic areas. To better understand the nature of this cross-reactivity, we used the DH6.5 antibody to immunoaffinity purify Loa loa antigens from the sera of individuals with a positive RDT due to loiasis. Immunoblot analysis revealed many circulating AD12/DH6.5-reactive antigens, and proteomic analysis identified multiple L. loa proteins in LF RDT-positive loiasis sera. These included both secreted and somatic proteins, suggesting that they may be released by dying L. loa adult worms and/or microfilariae. Unlike the single high molecular weight W. bancrofti circulating filarial antigen that is reliably present in the blood of persons with bancroftian filariasis, reactive L. loa antigens appeared to be only transiently present in the blood of a subset of persons with loiasis. These key differences between the circulating antigens of W. bancrofti and L. loa can be used to differentiate positive results generated by both species and may lead to improved diagnostic tests for LF and loiasis.
    DOI:  https://doi.org/10.1371/journal.pntd.0006963
  57. Nature. 2018 Nov 14.
    Autophagy maintains tumour growth through circulating arginine.
    Laura Poillet-Perez, Xiaoqi Xie, Le Zhan, Yang Yang, Daniel W Sharp, Zhixian Sherrie Hu, Xiaoyang Su, Anurag Maganti, Cherry Jiang, Wenyun Lu, Haiyan Zheng, Marcus W Bosenberg, Janice M Mehnert, Jessie Yanxiang Guo, Edmund Lattime, Joshua D Rabinowitz, Eileen White.
      Autophagy captures intracellular components and delivers them to lysosomes, where they are degraded and recycled to sustain metabolism and to enable survival during starvation1-5. Acute, whole-body deletion of the essential autophagy gene Atg7 in adult mice causes a systemic metabolic defect that manifests as starvation intolerance and gradual loss of white adipose tissue, liver glycogen and muscle mass1. Cancer cells also benefit from autophagy. Deletion of essential autophagy genes impairs the metabolism, proliferation, survival and malignancy of spontaneous tumours in models of autochthonous cancer6,7. Acute, systemic deletion of Atg7 or acute, systemic expression of a dominant-negative ATG4b in mice induces greater regression of KRAS-driven cancers than does tumour-specific autophagy deletion, which suggests that host autophagy promotes tumour growth1,8. Here we show that host-specific deletion of Atg7 impairs the growth of multiple allografted tumours, although not all tumour lines were sensitive to host autophagy status. Loss of autophagy in the host was associated with a reduction in circulating arginine, and the sensitive tumour cell lines were arginine auxotrophs owing to the lack of expression of the enzyme argininosuccinate synthase 1. Serum proteomic analysis identified the arginine-degrading enzyme arginase I (ARG1) in the circulation of Atg7-deficient hosts, and in vivo arginine metabolic tracing demonstrated that serum arginine was degraded to ornithine. ARG1 is predominantly expressed in the liver and can be released from hepatocytes into the circulation. Liver-specific deletion of Atg7 produced circulating ARG1, and reduced both serum arginine and tumour growth. Deletion of Atg5 in the host similarly released circulating arginine and suppressed tumorigenesis, which demonstrates that this phenotype is specific to autophagy function rather than to deletion of Atg7. Dietary supplementation of Atg7-deficient hosts with arginine partially restored levels of circulating arginine and tumour growth. Thus, defective autophagy in the host leads to the release of ARG1 from the liver and the degradation of circulating arginine, which is essential for tumour growth; this identifies a metabolic vulnerability of cancer.
    DOI:  https://doi.org/10.1038/s41586-018-0697-7
  58. Viruses. 2018 Nov 13. pii: E629. [Epub ahead of print]10(11):
    ISG15, a Small Molecule with Huge Implications: Regulation of Mitochondrial Homeostasis.
    Manuel Albert, Martina Bécares, Michela Falqui, Carlos Fernández-Lozano, Susana Guerra.
      Viruses are responsible for the majority of infectious diseases, from the common cold to HIV/AIDS or hemorrhagic fevers, the latter with devastating effects on the human population. Accordingly, the development of efficient antiviral therapies is a major goal and a challenge for the scientific community, as we are still far from understanding the molecular mechanisms that operate after virus infection. Interferon-stimulated gene 15 (ISG15) plays an important antiviral role during viral infection. ISG15 catalyzes a ubiquitin-like post-translational modification termed ISGylation, involving the conjugation of ISG15 molecules to de novo synthesized viral or cellular proteins, which regulates their stability and function. Numerous biomedically relevant viruses are targets of ISG15, as well as proteins involved in antiviral immunity. Beyond their role as cellular powerhouses, mitochondria are multifunctional organelles that act as signaling hubs in antiviral responses. In this review, we give an overview of the biological consequences of ISGylation for virus infection and host defense. We also compare several published proteomic studies to identify and classify potential mitochondrial ISGylation targets. Finally, based on our recent observations, we discuss the essential functions of mitochondria in the antiviral response and examine the role of ISG15 in the regulation of mitochondrial processes, specifically OXPHOS and mitophagy.
    Keywords:  OXPHOS; interferon; mitochondria; mitophagy; ubiquitin-like modification
    DOI:  https://doi.org/10.3390/v10110629
  59. Transpl Int. 2018 Nov 13.
    Urinary proteomics: fancy gadgetry or a clinically useful diagnostic instrument? The end-user's perspective.
    George S Reusz.
      Late deterioration of kidney function is a major challenge in transplantation medicine, thus early detection of subclinical kidney damage is of outstanding importance. Conventional laboratory parameters such as declining kidney function assessed by serum creatinine or creatinine clearance and proteinuria are late indicators of an already established damage. While urine properties have long been the subject of medical investigations, classical parameters based on physico-chemical properties and microscopic analysis remain far from helpful for establishing a refined etiological diagnosis and generally do not indicate the stage of kidney disease. This article is protected by copyright. All rights reserved.
    Keywords:  Biomarker; chronic active antibody-mediated rejection (cABMR); proteomics
    DOI:  https://doi.org/10.1111/tri.13374
  60. ACS Nano. 2018 Nov 16.
    Cell-Membranes Immunotherapy Based on Natural Killer Cell-Membranes-Coated Nanoparticles for Effective Inhibition of Primary and Abscopal Tumor Growth.
    Guanjun Deng, Zhihong Sun, Sanpeng Li, Xinghua Peng, Wenjun Li, Lihua Zhou, Yifan Ma, Ping Gong, Lintao Cai.
      Developing effective immunotherapies with low toxicity and high tumor specificity is the ultimate goal in the battle against cancer. Here, we reported a cell-membranes immunotherapy strategy that was able to eliminate primary tumors and inhibited distant tumors by using NK cell-membranes-cloaked photosensitizer TCPP-loaded nanoparticles (NK-NPs). Proteomic profiling of NK cell-membranes was performed through shotgun proteomics, and we found that NK cell-membranes enabled the NK-NPs to target tumors and could induce or enhance pro-inflammatory M1-macrophages polarization to produce antitumor immunity. The TCPP loaded in NK-NPs could induce cancer cells death through photodynamic therapy and consequently enhanced the antitumor immunity efficiency of the NK cell-membranes. The results confirmed that NK-NPs selectively accumulated in the tumor and were able to eliminate primary tumor growth and produce an abscopal effect to inhibit distant tumors. This cell-membranes immunotherapeutic approach offers a strategy of tumor immunotherapy.
    DOI:  https://doi.org/10.1021/acsnano.8b05292
  61. Front Microbiol. 2018 ;9 2577
    MALDI-TOF MS Applications to the Detection of Antifungal Resistance: State of the Art and Future Perspectives.
    Walter Florio, Arianna Tavanti, Emilia Ghelardi, Antonella Lupetti.
      MALDI-TOF MS technology has made possible revolutionary advances in the diagnosis of infectious diseases. Besides allowing rapid and reliable identification of bacteria and fungi, this technology has been recently applied to the detection of antimicrobial resistance. Several approaches have been proposed and evaluated for application of MALDI-TOF MS to antimicrobial susceptibility testing of bacteria, and some of these have been or might be applied also to yeasts. In this context, the comparison of proteomic profiles of bacteria/yeasts incubated with or without antimicrobial drugs is a very promising method. Another recently proposed MALDI-TOF MS-based approach for antifungal susceptibility testing is the application of the semi-quantitative MALDI Biotyper antibiotic susceptibility test rapid assay, which was originally designed for antimicrobial susceptibility testing of bacteria, to yeast isolates. Increasingly effective and accurate MS tools and instruments as well as the possibility to optimize analytical parameter settings for targeted applications have generated an expanding area in the field of clinical microbiology diagnostics, paving the way for the development and/or optimization of rapid methods for antifungal susceptibility testing in the near future. In the present study, the state of the art of MALDI-TOF MS applications to antifungal susceptibility testing is reviewed, and cutting-edge developments are discussed, with a particular focus on methods allowing rapid detection of drug resistance in pathogenic fungi causing systemic mycoses.
    Keywords:  MALDI-TOF mass spectrometry; antifungal susceptibility testing; antimicrobial resistance; blood culture; rapid AFST
    DOI:  https://doi.org/10.3389/fmicb.2018.02577
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