Bevin Gangadharan obtained his D.Phil. under the supervision of Prof. Nicole Zitzmann at the University of Oxford where he carried out the first ever gel-based proteomics study to discover novel biomarkers for liver fibrosis. He has more than a decade of experience in proteomics and biomarker discovery and first started in this field in 2000 at Smithkline Beecham looking at depletion of albumin in plasma, an important approach in biomarker discovery. He has published in several peer-reviewed journals and has two patents on novel biomarkers for liver fibrosis. He is on the editorial board for Biomarker Research and gives proteomics lectures to students in the Department of Biochemistry at the University of Oxford.

Liver biopsy is the reference standard for assessing liver fibrosis and no reliable non-invasive diagnostic approach is available to discriminate between the intermediate stages of fibrosis. Therefore suitable serological biomarkers of liver fibrosis are urgently needed. We used proteomics to identify novel fibrosis biomarkers in hepatitis C patients with different degrees of liver fibrosis. Proteins in plasma samples from healthy control individuals and patients with hepatitis C virus (HCV) induced cirrhosis were analysed using a proteomics technique: two dimensional gel electrophoresis (2-DE). This technique separated the proteins in plasma samples of control and cirrhotic patients and by visualizing the separated proteins we were able to identify proteins which were increasing or decreasing in hepatic cirrhosis. Identified markers were validated across all Ishak fibrosis stages and compared to the markers used in FibroTest, Enhanced Liver Fibrosis (ELF) test, Hepascore and FIBROSpect by Western blotting. Forty four candidate biomarkers for hepatic fibrosis were identified of which 20 were novel biomarkers of liver fibrosis. Western blot validation of all candidate markers using plasma samples from patients across all Ishak fibrosis scores showed that the markers which changed with increasing fibrosis most consistently included lipid transfer inhibitor protein, complement C3d, corticosteroid-binding globulin, apolipoprotein J and apolipoprotein L1. These five novel fibrosis markers which are secreted in blood showed a promising consistent change with increasing fibrosis stage when compared to the markers used for the FibroTest, ELF test, Hepascore and FIBROSpect. These markers are currently being further validated using a large clinical cohort. This study identifies 20 novel fibrosis biomarker candidates. The proteins identified may help to assess hepatic fibrosis and eliminate the need for invasive liver biopsies.

Stephen Mortlock is the Global Infectious Diseases and Microbiology Liaison at the Quest Diagnostics Laboratory in Heston UK. Prior to joining Quest Diagnostics, Stephen was the Chief Microbiologist at the Shaukat Khanum Memorial Cancer Hospital and Research Centre in Lahore, Pakistan and was awarded a DSc for his work setting up an antenatal screening programme for the local population. Stephen and colleagues have published over 40 papers on an eclectic mix of subjects from enteric pathogens, food science and the use of saliva for screening for HIV and cotinine. Stephen has also worked for the Health Protection Agency in the UK and spent time in both Africa and the Middle East.

Background: Many people consult their GP for upper gastrointestinal (GI) symptoms, which are often associated with pain or burning and discomfort in the abdomen and range from heartburn and acid regurgitation to nausea and vomiting. Historically, all of these symptoms have been grouped together under the single term ‘dyspepsia’, defined as having one or more symptoms of epigastric pain, burning, postprandial fullness, or early satiation. While gastric or oesophageal cancer is an unusual finding in patients with dyspepsia, excluding malignancy is a common reason for performing endoscopy.
Methods: Quest Diagnostics has been offering the GastroPanel assays for those patients who have been referred to the walk-in clinic complaining of ‘dyspepsia’. This is a set of three assays (Pepsinogen I, Gastrin 17 and Helicobacter pylori) and the results use an algorithm which can provide information about the stomach health and about the function of the stomach mucosa.
Results: Of all the samples tested nearly 70% showed no abnormalities and were reported as ‘normal function of gastric mucosa’. These patients would be classed as having functional dyspepsia. Thirty-six samples were positive for Helicobacter pylori and the remaining samples had a variety of abnormal results.
Conclusion: Dyspepsia is a common problem seen both by primary care physicians and gastroenterologists. Using the results from the serological analysis of the patients’ serum the clinician can delineate between gastric atrophy and a normal health stomach usually without the need to refer the patient for endoscopy.

Teresa García-Berrocoso received her M.Sc. in Biology in 2007 and M.Sc. in Biochemistry in 2008 from the Universitat de Barcelona (Spain). In 2011 she held a research stay in the Biomedical Proteomics Research Group at the University of Geneva (Switzerland) under the supervision of Prof. Jean-Charles Sanchez. Recently, she obtained her Ph.D. in Internal Medicine at Universitat Autònoma de Barcelona (Spain) on “Identification and use of prognostic biomarkers in ischemic stroke” directed by Dr. Joan Montaner. Her scientific career is focused on the application of biomarkers to different indications regarding human cerebral infarction. She supervises the website http://stroke-biomarkers.com, has published 17 original articles (4 of them as first author), a patent and some reviews in stroke biomarkers. She has participated in several national and international congresses and collaborates as a reviewer in European Journal of Neurology and Translational Proteomics. She is member of the International Biomarker in Cerebrovascular Diseases study-group, which was created in 2012, and from the Spanish Proteomics Society.

Background: Cerebral infarction or stroke is a leading cause of death and disability worldwide. Our research group firstly described changes in the proteome of human brain after ischemic stroke, which allow the discovery of new blood biomarkers to predict long-term functional disability. Further analysis of the components of the neurovascular unit, such as neurons and brain vessels, could contribute to a better understanding of the pathophysiology of stroke and might show up specific therapeutic targets or biomarkers.
Objective: To identify differences of protein expression in neurons and brain vessels after ischemic stroke.
Methods: Brain slices from infarcted and healthy contralateral areas of 7 patients who died following an ischemic stroke were visualized by immunofluorescence staining with NeuN (Chemicon) for neurons and with Ulex europaea agglutinin-I (UEA-I, Sigma) for vessels. By means of laser microdissection (LMD6000, Leica) we obtained both neurons and vessels from each sample in 60 μL of 0.1% Rapigest SF surfactant (Waters). Cell lysates underwent sonication, trypsin digestion, acidification (for Rapigest removal) and purification on C18 microspin columns. ESI LTQ-OT mass spectrometry was performed on a LTQ Orbitrap Velos (Thermo Electron) equipped with a NanoAcquity LC system from Waters. Gas-phase fractionation (GPF) for data-dependent analysis was performed with 4 injections of each sample with m/z ranges for precursor ions selection of 400-520, 515-690, 685-979 and 974-2000 Thomsons. Protein identification was done using Mascot and quantification and statistical comparison was performed with Progenesis® LC-MS v4.0 software, after automatic alignment of all runs and combination of the four fractions of each sample. PANTHER was used to classify proteins with Gene Ontology terms. Ingenuity Pathway Analysis (IPA) database was searched to find altered pathways after ischemia.
Results: A total of 768 proteins in neurons and 1078 proteins in vessels were identified and quantified from brain sections of ischemic stroke patients. Of these, 45.7% and 48.3% were identified with at least two peptides, respectively. When paired infarcted and contralateral areas were compared, 58 proteins in neurons and 24 proteins in vessels were found to be differentially expressed (p ≤ 0.05, fold-change ≥ 2, and peptide count ≥ 2). PANTHER analysis revealed that most proteins are related to metabolic or binding processes. In particular, proteins quantified in neurons were involved in membrane trafficking whereas in vessels they were associated with nucleic acid binding. IPA analysis of proteins from neurons showed their involvement in neurological diseases and in cell-to-cell signaling and interaction processes. In contrast proteins from brain vessels were mainly related to cell death and survival.
Conclusions: We have described changes in protein levels of human neurons and vessels after cerebral ischemia, with differential findings regarding the type of cell. If confirmed, these results could contribute to the molecular knowledge of stroke pathology and maybe highlight candidates to be further explored as therapeutic targets or biomarkers for the diagnosis or prognosis of stroke.

Víctor Llombart graduated in Universitat Rovira i Virgili (URV) (Tarragona, Spain) in the specialty of Biotechnology in 2010. One year later, he obtained a Master of Science Degree in Immunology in the Universitat Autònoma de Barcelona (UAB) and Universitat de Barcelona (UB) (Barcelona, Spain). During 2011, he collaborated with the Neuroimmunology Research group of Vall d’Hebrón Institute of Research (VHIR) (Barcelona, Spain). Since 2012 he has been developing his Ph.D. in Internal Medicine in Neurovascular Research Laboratory from VHIR. His Ph.D. is focused on the discovery of new potential biomarkers for the diagnosis of stroke through proteomic based techniques such as Stable Isotope Labelling in Cell Culture (SILAC) and Matrix-Assisted Laser Dessorption/Ionization imaging by Mass Spectrometry (MALDI-IMS). He has published 4 original articles (1 of them as first author), 1 review, developed a patent and collaborated in the public website http://stroke-biomarkers.com. He has also collaborated as a reviewer in the journal Translational Proteomics, and has participated in Spanish Society of Neurology with an oral communication and a poster in 2012 and 2013 respectively.

Introduction: Ischemic stroke is a cerebrovascular disease characterized by the disruption of blood flow into the brain due to an arterial occlusion. Depending on the source of the clot that blocks blood flow, ischemic stroke etiologies can be classified as cardioembolic (CE), large artery atherthrombotic (LAA), small vessel disease (SVD) or undetermined (UND). The use of biomarkers (proteins or genes) might facilitate the accurate identification of each etiology thus contributing to prescribe the most appropriated secondary treatment in order to avoid recurrences. Our aim was to identify differentially expressed genes associated with CE or LAA etiology in ischemic stroke patients.
Methods: Gene expression discovery phase: Eight LAA and 17 CE stroke patients matched by age and neurological severity were included in the discovery phase. All of them underwent reperfusion therapy. Pure RNA was isolated from white blood cells fraction from blood samples (RiboPure and GLOBINclear kits, Ambion, USA). RNA concentration was determined by RiboGreen (Invitrogen, USA) and pooled by mixing equal amounts of RNA from different patients with the same stroke etiology in each pool. Four LAA and 7 CE pools were obtained with a RIN number of 5-7 (Bioanalyzer 2100 platform, Agilent, UK). RNA was retrotranscribed to cDNA (Ovation Pico WTA, Nugen, USA) and hybridized in GeneChip Human Exon 1.0 ST Arrays (Affimetrix, USA). Gene expression validation phase: Differentially expressed genes (p-value≤0.005 and logFC ≥ |1|) were selected as candidates to be analyzed by RT-PCR using TaqMan probes (Applied Biosystems, USA) in individual samples from an independent cohort of 23 LAA and 46 CE stroke patients matched by age and neurological severity. Results are expressed as Relative Quantification (RQ=2-ΔΔCt) to endogenous genes GAPDH and PPIA, and a calibrator sample. Circulating protein analysis: Genes that showed the same trend in the validation phase were analyzed at a protein level through ELISA assay (Cusabio) in 33 CE, 17 LAA, 10 small vessel disease (SVD) and 8 healthy controls. Pearson-Chi squared test was used for the Univariant analyisis and logistic regressions were performed for the multivariate analysis using RQ levels as cut-off points for considered genes. Integrated discrimination improvement (IDI) was employed to compare predictive models in etiological classification. A p-value<0.05 was considered statistically significant and p-value<0.2 a statistical trend.
Results: The genes SIRPB2, DYRK2, NECAP1, IFIT2, CCDC75, IFIT1 were upregulated in CE whereas GOLPH3L, CYB5a, CPN1 and TMEFF1 were upregulated in LAA stroke patients (p-value≤0.005 and logFC ≥ |1|). These were mainly related to processes of carbohydrate metabolism and inflammation processes. After RT-PCR analysis GOLPH3L was significantly upregulated in LAA (p<0.05); and CYB5a and TMEFF1 showed the same trend (p<0.5). Only IFIT1 was upregulated in CE (p<0.5). After logistic regression multivariate analysis, GOLPH3L>0.4524, CYB5a>3.2453 and dyslipidemia remained baseline predictors of LAA etiology (OR: 3.919 [1.092-14.068], p=0.036; OR: 2.620 [0.699-9.829], p=0.153; and OR: 2.849 [0.915-8.870], p=0.071), respectively). Moreover, GOLPH3L>0.4524 and CYB5a>3.2453 helped to distinguish LAA from CE etiology (Integrated Discrimination Index 13%, p=0.002). No difference was found when circulating related protein concentration was analyzed in CE, LAA, SVD patients and healthy controls.
Conclusion: Gene expression analysis is an interesting approach to identify potential new biomarkers or therapeutic targets related to ischemic stroke etiology. However, improvements in this strategy are needed such as gene expression analysis in specific cell subpopulations or at later time-points to avoid acute phase processes. Although the pooling strategy reduces interindividual variability a validation step in individual samples is needed. The presence of common underlying pathophysiologic processes is an important challenge to deal with in new ischemic stroke etiology biomarkers discovery.

Photodynamic therapy is an effective method in the diagnosis and treatment of various tumors, but still the detailed molecular mechanisms and pathways are not well known. In response to the cytotoxic influence of PDT, cells activate and inhibit different proteins that prevent them from reactive oxygen species (ROS). It has been reported the oxidative stress-associated Src activator/Homo sapiens chromosome 9 open reading frame 10 protein (Ossa/C9orf10) protects cancer cells from an oxidative stress-induced apoptosis bySrc family kinases activation. The main aim of the present study was investigating whether the PDT leads to Ossa expression and if this may be connected with co-expression of c-Src tyrosine kinase. Additionally, apoptosis and necrosis following an oxidative stress caused by PDT on cancer cells were analyzed. For these purpose, Ossa and c-Src proteins were examined in MCF-7 human breast cancer cellsafter 5-aminilevulinic acid-mediated photodynamic therapy.The effects of PDT on the expression of the above proteins were detected in different time points (0, 7, 18, 24 hours after irradiation) using immunohistochemistry and Western blot analysis.Our results showed a high expression of Ossa at early time interval after PDT, which was accompanied by a low expression of c-Src tyrosine kinase. This relation between expression of two above proteinsindicate that Ossa could protect cancer cells from PDT through activation of c-Src in response to an oxidative stress. In conclusion, analyzed proteins may be targets to achieve better efficiency of PDT treatment.

Kamila Dus-Szachniewicz studied Molecular Biology at the University of Wrocław, Poland and in 2008-2009 at the University of Ferrara, Italy. Since 2010, she is Ph.D. student in the Department of Pathology at the Wrocław Medical University, Poland. In 2011-2012 she worked as fellow at the Department of Proteomics and Signal Transduction at the Max-Planck Institute of Biochemistry (Martinsried, Germany) where she identified candidate biomarkers forcolonic adenoma and colorectal cancer. Her current research consist in the proteomiccharacterization and validation of novel potential colorectal cancer markers for early diagnosis and molecular therapeutics target.

Mass spectrometry (MS)-based oncoproteomics is a rapidly developing method for the large scale analysis of the proteomes of human samples. It is believed that progress in oncoproteomics is likely to result in the discovery of cancer biomarkers. Searching for proteins specific to the particular type of cancer requires a comprehensive analysis of numerous samples. Tissue material, in addition to body fluids and cell lines, is an invaluable source of information on the proteome of healthy and sick individuals. An important advantage of the use of tissue material in clinical proteomics is the ability to identify "in situ" the changes caused by cancer. The analysis of pathologically changed tissue and their environment allows the tracking of the series of changes at the protein level occurring during tumorgenesis, tumor progression and metastasis. Additionally, the characteristics of tumors protein composition allows knowledge about the cancer development and biology to be deepened. The recent approach was to analyze the proteome of formalin fixed and paraffin embedded colonic adenomas, colorectal cancer and adjacent normal tissues in order to identify the panel of new colorectal cancer biomarkers. Our study resulted in the identification of more than 10.000 proteins from microdissected tissues. The proteomic approach used in this study allowed the evaluation of the abundances of key player proteins in the development and progression of colorectal cancer. Moreover, these observations highlight an improved understanding of the carcinogenesis of colorectal cancer.

Introduction: Serotonin (5-HT) is a naturally occurring vasoactive substance found in the brain, enterochromatin cells and platelets. Elevated levels of 5-HT and its main metabolite – 5-hydroxyindole acetic acid (5-HIAA)– are known to be associated with numerous life-threatening diseases as neurodegenerative disorders or cancer. The assessment of their concentrations in biological fluids and tissues could be helpful for early detection of on-going pathology. Nevertheless, it demands the development of reliable bioanalytical methods for their extraction and separation from biological samples.
Methods: Presented study was focused on establishment of the most accurate extraction and detection conditions for 5-HT and 5-HIAA from biological samples (serum and urine). The main aim was achieved by optimizing extraction of analytes. Various solid phase extraction (SPE) cartridges (C18, C8, HLB and SCN) as well as extraction buffers were tested. Optimal efficiency of extraction was achieved when simple C18 cartridges together with 20 % of methanol and 80% of H2O (pH 4) as eluent were applied. Further optimization of separation conditions of analytes was carried out by use of either high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) with UV/VIS detection. Gained results confirmed that both separation techniques guarantee sensitive and precise determination of 5-HT and 5-HIAA levels in human body fluids in up to 15 min total analysis time.
Results and Discussion: The method has been validated for accuracy, precision, selectivity, linearity, recovery and stability. The limits of detection (LOD) and quantification (LOQ) were satisfactory for both analytes (LOD around 0.5 ng/mL and LOQ 0.25 ng/mL). Linearity was confirmed within a range of 0.5-50 ng/mL with the correlation coefficient greater than 0.9995 in both cases. Comparison of HPLC and CE showed good agreement between the two methods, while different wavelength was applied (200 nm for CE and 220 nm for HPLC). Described methods were successfully applied for the quantification of 5-HT and 5-HIAA in real urine and serum samples derived from healthy controls (man and woman) as well as patients suffering from neuroendocrine tumors (NET). Data revealed significantly higher concentrations of both analytes in patients samples. Therefore, 5-HT and 5-HIAA could be potentially used as a biomarkers for detection of NET. Presented experimental workflow for HPLC or CE of 5-HIAA and 5-HT detection and quantification could facilitate biomarkers’ concentrations assessment from easy accessible patients’ body fluids like urine or blood. That would have the great impact of early cancer detection what is especially important in rare, asymptomatic cancers like neuroendocrine tumors.

Introduction: Prostate cancer (PCa) remains the second most frequent type of cancer in men worldwide. Prostate specific antigen (PSA)-based screening has been able to reduce cancer-related death by only 20%, whereby these are mostly characterized by indolent cancers and over diagnosis has been a huge concern. To date, the key issue in PCa is to differentiate indolent from aggressive cancers with the aim of decreasing potential over-treatment for the disease and to this end, newer biomarkers are needed. Recent advances in the study of proteomics and high throughput technology have led to the discovery of several potential protein biomarkers for PCa. Autoantibodies (AAb) to circulating tumour-derived proteins are spontaneously generated in cancer patients. These tumour-microenvironment changes trigger host-immune responses which produce measurable signals. Hence, AAb signals have the potential to become excellent targets for early detection of cancers and for monitoring efficacy of treatment. In this study, we aimed to identify potential biomarkers among Malaysian prostate cancer patients via a minimally invasive method.
Methods: Using a case-control study design, we profiled for 1636 correctly folded functional proteins in 30 prostate cancer patients and 30 matched controls serum samples using the Oxford Gene Technology’s (Oxford Genomics UK, Ltd) protein array. Penetrance based fold change was calculated to measure the likelihood that a given raw fold change is true. The student t-test was used in order to perform the significance testing. Only proteins with corrected p-values of less than 0.05 were selected. As for functional analysis, proteins with known associations to the disease/condition were identified using a combination of text mining approach from the PubMed database followed by comparisons with KEGG, REACTOME and MalaCards for biological associations.
Results: AAb reactivity for ESRRG was identified to be significantly different (P=0.039) between the subjects with prostate cancer as compared to controls when measured according to the penetrance based fold-changes. Cases exhibited significant fold change difference (3.22-fold change) when compared to matched controls.
Conclusion: The panel of proteins on the array are all closely associated with prostate cancer development, disease progression and play important roles in major biological pathways associated with the disease. As such, AAb corresponding to ESRRG may potentially aid in the early detection of prostate cancer and assist in monitoring progression of disease. This protein could also potentially be a target for immunotherapy.

Gel electrophoresis is used for the protein separation. These gel electrophoresis techniques are played important role in proteomics and genomics. Gel electrophoresis is one of the most important methods for the protein separation. The protein mixtures are separated by isoelectric point and molecular weight in two dimensional gel electrophoresis. Small amount of protein can also be separate with the help of gel electrophoresis. According to the practical procedure each sample was loaded into the wells using a gel loading tip. In the above figure 1 is thermo scientific page rular, there are 10 recombinant unstained protein markers are shown and they have molecular mass between 10KDa to 170 KDa. In the figure 2, at the right side of the first lane found some visible band, which is ladder. In the next three line is control sample, there was no bands visible. Than next three line are treated sample, where some bands are visible. With the help of the figure 1 is the standard, comparing these figure with figure 1, it may be molecular mass of protein around 40KDa. In all the three treated sample biomarker bisphenol A is present. These presences of bisphenol A help us to tell that biomarker must have upregulated this protein expression. Bisphenol A is used in the manufacture of polycarbonate plastic. Low amount of bisphenol A in animal and in human may cause endocrine disruption.(Carwile, J et al.,2009) Bisphenol A(BAP) is potential mammary carcinogen. Epoxy resin are made from Bisphenol A. These epoxy resins are used in household product like plastic product and other device like food and beverage cans.(Christopher I.Li 2010) Bisphenol A has estrogenic and anti androgenic effects.(Chamorro-Garcia et al., 2012)

Reshma S. Cherian is currently doing her Ph.D. in Toxicology from Sree Chitra Tirunal Institute for Medical Sciences and Technology (Govt. of India), Trivandrum, Kerala, India. She completed her Post graduation in Biotechnology from Cochin University of Science and Technology, India.

The search for an alternate and reliable ocular irritation test system, to replace in vivo animal testing, has been futile since there is a lack of validation with the in vivo test system. This has led to propose a better understanding of the crucial cellular and molecular changes occurring in in vivo testing. Here, rabbit cornea was treated continuously with 5 known ocular irritants, over a period of 7 days. Scoring of the response was carried out according to OECD 405. After the sub acute exposure, the animals were sacrificed and corneal histological analysis was carried out. Inflammatory biomarkers (cytokines IL-1α, IL-1β, IL-8 and TNF-α) were tested using ELISA. Apart from that, examination of blood parameters, spleenocyte proliferation and analysis of oxidative stress biomarkers in liver and brain were carried out. After treatment, there was inflammation and fluid discharge from the cornea and conjunctiva. Histological analysis shows typical inflamed tissue morphology when compared to. In comparison to control, there was a significant increase in production of inflammatory cytokine. Biochemical and hematological parameters showed a slight increase. Significant reduction in proliferation of spleenocytes was found when treated with the irritant chemicals. There were also slight alterations in oxidative stress parameters of liver and brain as well. It can be concluded that the rabbit corneal cells treated with known ocular irritants showed an increased inflammatory response, which was confirmed by increased cytokine level. Further, there was a variation in blood parameters, oxidative stress in brain and liver and decreased spleenocyte proliferation.

Francesca Rosa is a medical undergraduate student at the School of Medicine, at the University of Genoa, Italy. Her field of research, is on the application of new contrast agent for tumor targeting. Actually, she is involved in a research project about autoimmune disease and the application of Manganese as “imaging biomaerker”. She was recently, awarded with the “Magna Cum Laude” at the European Congress of Radiologist (ECR) in Vienna (6-10 March 2014).

Manganese-enhanced Magnetic Resonance Imaging-(MEMRI) evolved in the late nineties and relies upon the following three main properties of Mn2+: 1) it is a paramagnetic ion and an excellent T1 relaxivity; 2) it is a calcium (Ca2+) analog that can enter excitable cells (voltage-gated calcium receptor and calcium sensing receptor) and, 3) once in the cells Mn2+ can be transported along axons by microtubule-dependent axonal transport. The above mentioned properties of Mn2+ make it a particularly attractive contrast agent for MRI in order to study physiology and physiopathology of:
1.Brain 2.Heart 3.Salivary glands 4.Cancer
Different animal models were prepared to investigate the application of Manganese as contrast agent by using a clinical 3T MR scanner and a prototype birdcage coil. The results of our study highlighted the potential utility of MEMRI as “functional tool” in order to study the physiological activity of the brain, heart and salivary glands and the different expression of calcium sensing receptor in breast and prostate cancer. In conclusion, our results, open the exploration of MEMRI as a new possible “imaging biomarker” of calcium metabolism in human disease.