conferenceseries.com 773rd Conference
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Posters
Proteomics & Bioinformatics 2016
Page 91 Zubida Al-majdoub, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Quantification of drug-metabolising enzymes in human liver microsomes: A comparison between label-free profiling and targeted quantitative analysis
Zubida Al-majdoub University of Manchester, UK
enonbiotic and drug-metabolizing enzymes (DMEs) are involved in the bioconversion of xenobiotics (including drugs, Xsynthetic chemicals and environmental pollutants) into inactive or active metabolites. In pharmacological therapy, bioconversion can either lead to detoxification or activation of the drug, which has implications on treatment effectiveness and toxicity. Quantitative profiling of the drug-metabolising sub-proteome can be used in the characterisation of liver drug metabolism profiles in individual patients which can be a major step towards stratified or personalized medicine. Immunoquantification and targeted proteomics approaches have traditionally been used to determine abundances of CYP and UGT enzymes; however, bias in the determination of absolute protein abundance between laboratories and methods has been demonstrated. This may be due to differences in methodological workflows or the choice of suitable and specific standards. Label-free analysis can provide a venue for a new methodological setup. Advantages of this type of approach include the possibility of quantifying a large number of proteins without the need for specific standards, allowing comprehensive description of dynamic changes of expression in the proteome under study. As an alternative approach to previously used methods, we aimed to apply a label-free proteomic strategy to quantify and assess the absolute expression of several CYP and UGT enzymes in microsomal fractions extracted from 27 human livers which have previously been characterised.
Biography
Zubida Al-majdoub has completed her Under-graduate studies in Tripoli University, Libya and worked as Teaching Assistant at the same university. She has completed her MPhil in Medicinal Chemistry from School of Pharmacy, University of Manchester, followed by PhD in Quantitative Proteomics under the supervision of Dr. Jill Barber and Professor Simon Gaskell. She is a Research Associate at School of Pharmacy. Her research work focuses on “Quantification of transporters in human brain”.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 92 Valerie Morineaux et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Mass spectrometry in forensic identification of two main potential agents of bioterrorism and warfare: Botulinum toxins and ricin
Valerie Morineaux, J Enche and D Hilaire Direction générale de l’Armement, France
rotein toxins such as botulinum neurotoxins and plant toxin ricin are considered as potential agents for bioterrorism and Pwarfare. Botulinum neurotoxins are produced by Clostridium botulinum commonly found in plants, soils, water and in the intestinal tract of animals. They act by blocking the release of acetylcholine, the principal neurotransmitter at neuromuscular junctions, causing muscle paralysis. Botulinum toxins are among the most poisonous substances. Ricin, on the other hand, is produced by the seeds of castor bean plant (Ricinus communis) which is used to process the castor oil. Ricin is very toxic against eukaryotic cells by inhibiting the protein synthesis and causing cell death. Unambiguous identification of these toxins is required not only for the implementation of effective countermeasures in case of terrorism event but also for law enforcement. This is the reason why we have developed a specific strategy for the detection and identification of botulinum toxins and ricin in complex matrices, by LC-QqQ-MS/MS method operating in Multiple Reaction Monitoring (MRM) mode. In order to be compatible with complicated samples, the mass spectrometry analysis was coupled with an immunocapture step. This method was successfully applied to the identification of botulinum toxins type A subtypes and ricin from complex matrices.
Biography
Valerie Morineaux is an Engineer in the Toxicology department, DGA, regarding NRBC (Nuclear, Radioactive, Biology and Chemical) risks. She has completed her PhD from DGA and Pasteur Institute. She has been working for several years on “The development of analytical methods in toxin identification”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 93 Zdenka Kristofikova et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Nitric oxide synthases and acute sleep deprivation
Zdenka Kristofikovaand Jana Sirova National Institute of Mental Health, Czech Republic
myloid beta peptide and protein tau play a role in the development of Alzheimer disease. It is suggested that normal aging Ais the main risk factor and chronic sleep deprivation is the contributing risk factor here (e.g. via oxidative stress-evoked changes in amyloid beta and protein tau). A great attention is also focused on nitric oxide directly involved in sleep-wake cycle and Alzheimer disease pathogenesis. The purpose of the study is to evaluate changes in activities of nitric oxide synthases (neuronal, endothelial and inducible) in the right and left cortex of young or old rats exposed to increased locomotion (control experiments for the non- specific effects of the apparatus) or acute sleep deprivation (24 hours). In future, results will be compared with those obtained on young or old rats exposed to chronic sleep deprivation. We used Rat Forced Exercise Bed model 80805A*C apparatus (Campden Instruments Ltd.) based on the rotational movement of the activity wheel. Experiments were performed on young (3-4 months) and old (11-12 months) male Wistar rats. Activities of neuronal and endothelial synthases were significantly decreased in old compared to young control rats. The activity of inducible synthase was decreased in the left cortex of young rats exposed to increased locomotion. On the contrary, the activity of inducible synthase was increased in old rats exposed to acute sleep deprivation. The previous analysis of human autoptic brains revealed increased activities of all synthases especially in the left side of people with Alzheimer disease. It seems that acute sleep deprivation can evoke some changes in old rats similar to those seen in Alzheimer disease.
Biography
Zdenka Kristofikova is interested in problems of Alzheimer disease and normal aging for a long time. She focusses especially on “The analysis of the human or rat brain tissue and of cerebrospinal fluid”. Her main aims are “Evaluations of new animal models of Alzheimer disease or of new biomarkers of Alzheimer disease sensitive to changes in early stages of the disease”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 94 Ramona Bosch, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Expression and purification of membrane scaffold proteins for the design of discoidal phospholipid bilayer nanoparticles
Ramona Bosch University of Hohenheim, Germany
he expression and purification of recombinant proteins is an everyday business. Years ago, however, protein production was Tdifficult, time consuming and remained mostly in the domain of experts. The progress of simple, commercially available systems made the technology more widespread and led to an increase in protein production. In this work, a derivate of the human apolipoprotein A-1, the Membrane Scaffold Protein (MSP) is produced biotechnologically in a lab scale bioreactor. The MSP is a genetically engineered protein which has the notable feature to self-assemble into discoidal nanoparticles in the presence of synthetic phospholipids. These so-called nano discs have become increasingly important in the last few years e.g., for the study of membrane-associated proteins. In previous studies, nano discs are assembled by adding a micelles-organized detergent-phospholipid mixture to an aqueous system containing the MSPs. Upon removal of detergent, 10 nm diameter particles are formed. Unfortunately, the resulting nano discs are disordered in this solution and therefore further processing into an ordered and directed membrane cannot be easily achieved. The aim of this work is to create biomimetic membranes consisting of cross-linked nano discs e.g., the translocon SecYEG as part of an embedded protein complex for an active biological transport of potential target proteins. Now, the approach should be replaced by nano patterning of the nanoparticles. It is assumed that the phospholipids interact with the positive charge of a gold lamella and this effect leads to planar aligned nano discs. In next phase, the discs will be cross linked via cysteines which are located in the membrane scaffold proteins. These cysteines serve as cross-links for the disulfide bonds. Then, the gold lamella must be removed and the newly synthesized nano disc-membrane can be coated onto an ultrafiltration membrane for more stability. The resulting membrane allows for a directed investigation of the function of membrane proteins. Therefore, biomimetic membranes consisting of cross-connected nano discs have a high potential to serve as excellent biotechnological tools for research on membrane-associated proteins as well as in method development for selective separation or transport of biomolecules.
Biography
Ramona Bosch completed her studies in Biology with Specialization in Molecular Biology and Microbiology at the Karlsruhe Institute of Technology, Germany. During her Diploma thesis, she investigated the efficiency and classification of antimicrobial substances against Pseudomonas aeruginosa and Staphylococcus aureus. Subsequently, she started her Doctoral studies at the University of Hohenheim, Institute of Food Science and Biotechnology, Department of Bioprocess Engineering. Her research focusses on “The development of biotechnological processes including all process steps (upstream processing, bio-production, downstream processing) which are necessary for the industrial production of biotechnological products”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 95 Marta Stasiak et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Proteomic analysis of endothelial to mesenchymal transition controlled by snail
Marta Stasiak, Katarzyna Gawrys, Marcin Popielarski, Radoslaw Bednarek and Maria Swiatkowska Medical University of Lodz, Poland
olecular mechanism of fibrosis is characterized by the accumulation of a collagen-rich extracellular matrix (ECM) and Mis a feature of a number of fibrotic diseases as well as the stroma of many solid tumors. Recent studies have shown that endothelial cells are capable of undergoing endothelial to mesenchymal transition (EndMT) and generates fibrotic tissue. This mechanism causes fibrosis in organs such as kidney, lung and heart. Our studies demonstrated that overexpression of transcription factor snail1 can initialize EndMT in HMEC-1 cells. We observed the loss of endothelial markers (VE-cadherin) with the simultaneous gain of mesenchymal markers (FSP-1, SMA-α and vimentin). Differential proteomics performed for a global quantitative comparison of two proteomes with Orbitrap Velos mass spectrometers and iTRAQ- a labeling-based method analysis of HMEC-1 displayed a total number of 2145 proteins, among those three were overexpressed and 19 were down- regulated after overexpressed snail1 in endothelial cells. Moreover, the reduced expression of plectin was observed. Heretofore, only one patient with mutation in the plectin gene was previously reported whose case was diagnosed with cardiomyopathy and heart fibrosis. Our experiments concerning early stages of EndMT revealed decreased expression of matrin3, calmodulin and an isoform 1 of nucleophosmin. These data are similar to quantitative proteomic analysis of protein expression profile in a prostate cancer EMT model.
Biography
Marta Stasiak has completed her PhD in Medical Biology in 2007 at Medical University of Lodz. She was a Post-doctoral Visiting Fellow at the Laboratoire de Biochimie Médicale et de Biologie Université de Reims Champagne Ardenne Moléculaire, Reims, France and in the Division of Hematology, Children’s Hospital of Philadelphia, Philadelphia, USA. She is currently an Assistant Professor in Department of Cytobiology and Proteomics, Medical University of Lodz, Poland.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 96 Jeong-Su Lee, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
An analysis of norovirus genome using next-generation sequencing
Jeong-Su Lee National Institute of Food and Drug Safety Evaluation, Republic of Korea
o overcome the technological constraints of the Sanger sequencing, next generation sequencing (NGS) technologies Twere developed. It has become very rapidly a standard tool in pathogen discovery. In recent years, NGS techniques are increasingly being used for outbreak monitoring, metagenomic studies and virus detection. Incorporating deep sequencing techniques into virus diagnostics on clinical samples and foods samples that can be detected and at the same time provides additional information on the characterization of the detected viruses. The objective of this study was development of detection method for NoV GII.4 using NGS. Alignment of the norovirus sequences from underground water samples was performed with CLC Workbench software packgea and DNAstar softare. Similarity, and capsid epitope analyses detected GII.4 Sydney strains by NGS data. An amino acid sequence of the epitopes of the capsid P2 domain was same amino acid in Sydney 2012 strains. NGS technologies will provide an information for epidemiologic studies of NoV.
Biography
Jeong-Su Lee has completed his PhD from Chung-Buk University, Republic of Korea. He is currently working as a Scientific Researcher for the Division of Microbiology, Ministry of Food and Drug Safety. His main research include the study for prevention of infectious diseases and control any public health related issues in Korea.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 97 Katarzyna Gawrys et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Identification and characterization of proteoglycans in early stages of fibrosis
Katarzyna Gawrys, Marcin Popielarski, Radoslaw Bednarek, Maria Swiatkowska and Marta Stasiak Medical University of Lodz, Poland
ibrosis is a pathological process characterized by the production of excessive amount of connective tissue and deregulated Fextracellular matrix (ECM) production results in scarring and thickening of the tissue. ECMs secrete molecules that determine the cell microenvironment and are composed of a dynamic glycoproteins, collagens and proteoglycans (PGs). Proteoglycans are a key component of extracellular matrix, filling the space between the fibrous proteins. They modulate cell migration, cellular adhesion and proliferation. Proteoglycans have significant functional and structural roles in fibrosis. Aim of this study is showing role of proteoglycans in the early stages of fibrotic process. To investigate it, we used endothelial cells enriched in Snail. This model was obtained in two different ways: Human microvascular endothelial cell (HMEC-1) was stimulated with profibrotic transforming growth factor-β1 (TGF-β1) or was transfected with transcription factor: Snail. This study describes changes in the proteoglycans deposition in fibrosis. We determined the in vitro regulation of proteoglycans, such as: Lumican, versican, fibromodulin in response to TGF-β1 in HMEC-1 and in cells with Snail overexpressing. The investigation the role of proteoglycans in fibrosis could offer new insights in the pathogenesis of this disease.
Biography
Katarzyna Gawrys obtained Bachelor’s degree in Medical Biotechnology at Medical University of Silesia in 2010 and a Master’s degree in Medical Biotechnology at Jagiellonian University in Kraków, Poland in 2012. She held numerous practices in research units and has participated in many conferences and specialized trainings. She currently works at Medical University of Lodz, Department of Cytobiology and Proteomics.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 98 Sung-Fang Chen, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry
Sung-Fang Chen National Taiwan Normal University, Taiwan
rostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for Pdistinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs comprised of native proPSA as well as truncated proPSA forms [-2] proPSA, [-5] proPSA and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids owing to its high sensitivity, specificity, simplicity and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are most complex biological fluids, the immunoprecipitation workflow is optimized to achieve sufficient sensitivity, efficiencies of protein purification with immuno affinity depletion. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at ng/mL levels corresponding to a concentration six orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers including the mature and precursor forms of PSA can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.
Biography
Sung-Fang Chen completed his PhD at Cleveland State University (CSU) (a joint program in the field of Clinical-Bioanalytical Chemistry at Lerner Research Institute of the Cleveland Clinic) in 2000. After having a Post-doctoral training at the Institute of Chemistry, Academia Sinica, Taiwan, he took a position of Research Manager at the Biomedical Engineering Center, Industrial Technology Research Institute in 2002. He joined National Taiwan Normal University as a Faculty member in 2009 and has been an Associate Professor since 2013.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 99 Alena Dmitrievna Volyntceva, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Molecular studies of scorpion toxins interactions with calcium-activated potassium channels
Alena Dmitrievna Volyntceva Lomonosov Moscow State University, Russia
he calcium-activated potassium channels (KCa channels) are widely expressed in the organism. Several studies demonstrated Tthe potential role of KCa3.1 channel blockage as a therapeutic strategy. Knowledge of the molecular aspects of blockers binding process is an important step in designing highly efficient and selective ligands. Aim of this study was an interface analysis in complexes of hybrid channel KcsA-KCa3.1 with peptide blockers agitoxin, charybdotoxin and maurotoxin. KcsA- KCa3.1 chimera represents KcsA channel with P-loop taken from the human KCa3.1. 3D structure was generated by homology modeling using complex of mutated KcsA channel with charybdotoxin (pdb-code 2A9H) as a template. Molecular dynamic simulation was performed using Gromacs software. Optimal conformations of toxins in channel binding site were chosen from the trajectories. Hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxins and hybrid channel were evaluated using program Platinum and APBS software package. Contacts energy characteristics evaluation showed that maurotoxin was the most effective blocker of KCa3.1 channel among these toxins. This result is in good agreement with the experimental data. Knowledge of channel-toxins interfaces allowed us to propose amino acid mutations in toxins to increase binding affinity and selectivity. Results of the conducted investigation are of great interest for drug development.
Biography
Alena Dmitrievna Volyntceva is a Post-graduate student of Chair of Bioengineering at the Faculty of Biology, Lomonosov Moscow State University. She is the Junior Reseacher and has been investigating receptor-ligand interactions for six years in the Laboratory of Molecular Dynamics and Molecular Modeling. Her current interests includes “Study of potassium channels interactions with scorpion toxins”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 100 Kyu-Heon Kim, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Development of RNA standardized controls for quantitative detection of rotavirus and astrovirus by realtime RT-PCR
Kyu-Heon Kim Ministry of Food & Drug Safety, Republic of Korea
otavirus and astrovirus are important human pathogens that cause epidemic acute viral gastroenteritis. These viruses are Rrelated to viruses as the causative agents in outbreaks of gastroenteritis. The objectives of in this study, we developed the RNA standardized control for the real-time PCR detection of rotavirus and astrovirus. Real-time PCR assays showing high sensitivity are being currently used for the detection of foodborne viruses. However, the standardized controls indispensible for the confirmation and quantification is absent. We developed the standardized controls for the real-time PCR detection of rotavirus and astrovirus with the supply of the standardized control. To RNA synthesis of standardized positive controls of rotavirus and astrovirus from pBHA plasmid vector, we conducted the T7 promotor tagging PCR and in vitro transcription. Detection limit of the real-time PCR controls was estimated by endpoint detection of synthesis RNA and their serial 10- fold dilutions. The detection limit of rotavirus and astrovirus by real-time PCR was confirmed up to 10 copy and 100 copy, respectively. And the values of realtime-PCR R2 analysed a 0.99% in the all realtime-PCR standardized controls. Development of standardized controls for real-time PCR of rotavirus and astrovirus be used to products in order to investigation of foodborne outbreak.
Biography
Kyu-Heon Kim has completed his PhD from Chung-Buk National University, Republic of Korea. He is currently working as a Scientific Researcher for the Division of Foodborne Disease Prevention and Surveillance, Ministry of Food and Drug Safety. His main research include the study for prevention of infectious diseases and control any public health related issues in Korea.
Notes:
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 101 Marlon Henrique e Silva Cardoso et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Comparative NanoUPLC-MSE analysis between magainin I-susceptible and -resistant Escherichia coli strains
Marlon Henrique e Silva Cardoso, Keyla C de Almeida, Elizabete de S Cândido, André M Murad, Simoni C Dias and Octávio L Franco Universidade de Brasília, Brazil
n recent years, the antimicrobial peptides (AMPs) have been prospected and designed as a promise of new alternatives Ito conventional antibiotics. Indeed, this class of pharmacological molecules has presented great potential action toward susceptible and resistant bacterial strains, in both their free-floating and biofilm lifestyles. However, increasing reports have reported the mechanisms by which bacteria resist AMP administration. Thus, here we performed a comparative proteomic
study by using the total bacterial lysate of magainin I-susceptible and –resistant E. coli strains. After nanoUPLC-MSE analyses, we identified 742 proteins distributed among the experimental groups, 427 proteins being identified in theE. coli (ATCC 8739) group, 664 in the E. coli susceptible (control) group and 651 in the E. coli resistant group. These proteins were also separated into 5 biological classes using KEGG orthology, being 73% related to metabolism. Also, 25 proteins were differentially expressed in the resistant strains, 10 proteins being upregulated, including outer membrane proteins (OMPs), periplasmic oligopeptide-binding protein (OppA) and zinc resistance-associated proteins (ZraP) and 15 downregulated, such as chaperone protein DnaK, glutaminase 1 (glsA1), glucosamine-6-phosphate deaminase (NagB) and NAD(P)H dehydrogenase (WrbA). Moreover, 60 exclusive proteins were identified in the magainin I-resistant strains, among which biofilm formation (YoaB), cell wall (DblB) formation and multidrug efflux pump (AcrA) proteins could be observed. Thus, data here reported show that several metabolic pathways have been related with E. coli resistance to cationic AMPs, revealing the crucial role of multiple “omics” studies in order to elucidate the global molecular mechanisms involved in this resistance.
Biography
Marlon Henrique e Silva Cardoso is a PhD student of Molecular Pathology at the University of Brasília, Brazil. He obtained his BSc in Biology/Biotechnology at the Catholic University of Brasília, Brazil. In 2012, he became a Member of the Center for Proteomics and Biochemical Analyses, coordinated by Prof. Dr. Octávio Luiz Franco. In 2014, he was invited by Dr. Franco to join the S-Inova Biotech as the Responsible for the Department of Structural Bioinformatics and Computational Biophysics. Since 2013, he has published more than 10 articles (7 during an year as a PhD student) in renowned journals and a book chapter.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 102 Minh Tan Nguyen et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Application of N-terminal fusion protein partners for prokaryotic soluble expression of human VEGF165 and its simple purification with maltose binding protein tag
Minh Tan Nguyen1, Martin Krupa1, Bon-Kyung Koo1, Jung-A Song1, Thu Trang Thi Vu1, Bich Hang Do1, Anh Ngoc Nguyen1, Taewook Seo1, Jiwon Yoo 1, Boram Jeong1, Jonghwa Jin2, Kyung Jin Lee1, Heung-Bum Oh1 and Han Choe1 1University of Ulsan, South Korea 2Osong Medical Innovation Foundation, South Korea
uman vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process Hof tumor growth and metastatic dissemination. E. coli is a convenient platform for protein expression. However, attempts to express human VEGF165 (hVEGF) in E. coli result in poorly soluble expression or protein misfolding and aggregation into inclusion bodies, hampering purification. To overcome these obstacles, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI) and the b’a’ domain of PDI (PDIb’a’) were constructed and tested for soluble overexpression of hVEGF in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and this tag significantly increased the protein’s solubility. 0.8 mg of pure hVEGF was successfully purified from 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first study on successfully expressing and purifying soluble hVEGF in E. coli.
Biography
Minh Tan Nguyen completed his Master’s degree in Medical Science from University of Ulsan College of Medicine, Seoul, South Korea, in February 2014. He is currently a PhD student at Physiology Laboratory, University of Ulsan College of Medicine. His research interest includes “Prokaryotic expression and purification of therapeutic proteins, synthetic antibody libraries for phage display”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 103 Katarzyna Gawrys et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Role of fibrosin in the endothelial to mesenchymal transition
Katarzyna Gawrys, Marcin Popielarski, Radoslaw Bednarek, Maria Swiatkowska and Marta Stasiak Medical University of Lodz, Poland
ibrosin, which stimulates synthesis of collagen-rich extracellular matrix (ECM) proteins and induces chemotaxis of Ffibroblasts, might be involved in fibrosis. The term fibrosis describes the accumulation of tough, fibrous scar tissue as a reparative response to injury or damage. Fibrosis occurs when excess fibrous connective tissue and ECM accumulate in an organ or tissue in normal healing or pathological process. Many diseases are caused by tissue fibrosis resulting in chronic inflammation. Endothelial to mesenchymal transition (EndMT) is a process by which endothelial cells convert to a more mesenchymal cell type that can give rise to cells such as fibroblasts. EndMT is a main source of mesenchymal cells participating in the fibrosis. In our research, we analyze early stages of fibrosis which could be initiated by transforming growth factor-beta (TGF-β) and transcription factor such as snail. Our experimental model uses human microvascular endothelial cells (HMEC- 1) stimulated with TGF-β1 or transfected with pcDNA3.1-snail. Profile of endothelial and mesenchymal cell markers were determined to characterize early stage of fibrosis. Bioinformatics transcriptomics analysis of 57716 genes demonstrated up- regulation of fibrosin after TGF-β1 stimulation. We already confirmed it on the transcription and protein level. We observed an increase of fibrosin in HMEC-1 also after transfection with snail. Thus, overexpression of snail in HMEC-1 leads to enhanced expression of fibrosin. In conclusion, our results indicate the regulatory role of fibrosin in fibrotic process. The current results will provide information about fibrosin as a regulatory molecule involved in EndMT and in fibrosis.
Biography
Katarzyna Gawrys completed her Bachelor’s degree in Medical Biotechnology at Medical University of Silesia in 2010 and a Master’s degree in Medical Biotechnology at Jagiellonian University in Kraków, Poland in 2012. She held numerous practices in research units and has participated in many conferences and specialized trainings. She currently works at Medical University of Lodz, Department of Cytobiology and Proteomics.
Notes:
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 104 Jie Ma et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Using entrapment sequences strategy for evaluation of database search engines and quality control methods in shotgun proteomics
Jie Ma1, Xiaodong Feng1, 2, ChengChang1, Tao Chen1 and Yunping Zhu1 1Beijing Proteome Research Center, China 2Chongqing University, China
ith the advance of mass spectrometry and experimental techniques, proteome research has broken through the bottleneck Wof data generation and a huge amount of mass spectrometry (MS) data has been accumulated rapidly in the past few years. Meanwhile, the lack of efficient data analysis and quality control methods has greatly hindered proteome development. Target-decoy searching strategy has become one of the most popular strategies to control the false identification in MS/MS data analysis. While this strategy can estimate the false discovery rate (FDR) within a dataset, it cannot directly evaluate the false positive matches in target identifications. In this study, we developed an improved target-decoy strategy: THe entrapment sequences method, to set up an objective standard to evaluate the performance of quality control methods and database search tools. Using both standard datasets and experimental datasets, we started with a preliminary study of the size of entrapment sequences and found ten times the sample sequences could be a reasonable size of entrapment sequences. Then, we went on to give a definition and equation of estimated FDR and actual FDR. We found the entrapment sequences method can be a good supplement to target-decoy strategy. So, we evaluated the performance of five quality control methods (BuildSummary, PepDistiller, PeptideProphet, FDRAnalysis and ScoreBased Method) and five database search engines (Mascot, X!Tandem, Comet, MS-GF+ and Tide). We demonstrated that the entrapment sequences method could be an excellent strategy to assess each step of the mass spectrometry data analysis process.
Biography
Jie Ma has completed her PhD from Beijing Institute of Radiation Medicine. She is now an Assistant Professor at Beijing Institute of Radiation Medicine. She has published more than 10 papers in reputed journals of Proteomics and Bioinformatics.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 105 Lau, Benjamin Yii Chung et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Proteomic approach to determine the biological variation of oil palm fruit mesocarp
Lau, Benjamin Yii Chung and Ramli, Umi Salamah Malaysian Palm Oil Board, Malaysia
comparative protein expression study has been initiated to determine the metabolic factors controlling the production Aof high-value oleic acid in the oil palm. In order to ensure that any differences in protein expression occurred primarily due to the differential regulation during oleic acid production, the credibility of the examined biological replicates is crucial and need to be established before further exploration. The extend of biological variation among oil palm fruit mesocarps were accentuated using gel-based and gel free proteomic techniques. A comparative protein profile analysis performed on eleven major one-dimensional gel electrophoresis-separated protein bands revealed that the intensities were similar across each of the replicates for low and high oleic acid mesocarps. Subsequently, a two-dimensional gel protein profiles demonstrated minor variations (in the number of protein spots and intensities) between replicates for low and high oleic acid mesocarps. A more in-depth evaluation of the low and high oleic acid replicates was executed with nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS). The evaluation showed that the number of identified proteins for each of the low and high oleic acid replicates differed by 3-14% with low/high oleic acid (LO/HO)1 as the reference replicate. The difference in the number of identified peptides between LO2/LO1 and HO2/HO1 was 72 and 73 peptides, respectively. The number of identified peptides for LO3 varied by 69 peptides when compared to the LO1. Comparison between HO3 and HO1exhibited a difference of 41 peptides. The biological variations of low and high oleic acid replicates were highlightedmore effectively usinga peptide centric approach but not with the relatively less sensitive gel-based proteomic techniques. Theseproteome differences also indicated the presence of biological variations.
Biography
Benjamin Lau has completed his PhD in 2015 from Lincoln University, New Zealand. He is a Proteomics Research Scientist at the Advanced Biotechnology and Breeding Division, MPOB. His works focus on plant (oil palm) proteomics and currently investigating the metabolic factors controlling the yield and high-value fatty acids production in the oil palm.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 106 Hana Tejkalová et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Neonatal immune challenges affect behaviour, cytokine profiles and brain morphology parameters in adult rats
1 2 3 1 Hana Tejkalová , Kačer Petr , Jan Klaschka and Jiří Horáček 1National Institute of Mental Health, Czech Republic 2University of Chemistry and Technology Prague, Czech Republic 3Czech Academy of Sciences, Czech Republic
erinatal immune challenge can induce neurodevelopmental dysfunction, permanent immune dysregulation and abnormal Pbehaviour which have been documented in clinic findings of human neuropsychiatric disorders. The aim of this study was to evaluate the response to an inflammatory stimulus in rats exposed to lipopolysacccharide (LPS) as a model of neonatal subchronic infectional stimulus (realized at postnatal days 5-9) on immune, neurodegerative and behavioural parameters,
which may be relevant to human neurobiology. The series of molecules of interest leukotriene B4 (LTB4), interleukine 4 (IL- 4), interleukine 10 (IL-10) and tumor necrosis factor α (TNFα) were determined by metabolomic and proteomic techniques. An analytical method combining immunomagnetic separation with MALDI-TOF MS determination was used for the
determination of protein biomarkers. The analysis of small molecule biomarkers (leukotriene B4) was realized by LC-ESI-MS/ MS. Our results showed that early immune stimulation evaluated in adult animals alters the levels of leucotrienes ((LTB4), IL4 and leads to neurodegenerative processes and behavioural deviations which are amplified by acute dopaminergic or glutamatergic stimulation. Leucotrienes (LTSB4) and cytokine IL4 were elevated in LPS rats acute exposed to MK-801 or to GBR 12909. Moreover, the behavioral observations documented hyperlocomotion, impairment of prepulse inhibition and cognitive deficit in LPS rats injected with acute NMDA antagonist (i.e., MK-801) or dopamine releaser (GBR 12909). These findings support the crucial pathophysiological role of early immune stimulation in many neuropsychiatric disorders (e.g., schizophrenia, autism).
Biography
Hana Tejkalová completed her PhD from Faculty of Science, Charles University in Prague. She is a Senior Researcher at National Institute of Mental Health (NIMH). She has published nearly 50 papers in reputed journals (total citations 166). Her research activities involve “The use of behaviour in the animal modelling of psychiatric disorders, especially schizophrenia”. She also acted as the Czech representative in FELASA from 2010 until 2014”.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 107 Tao Chen et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
A wiki-based web server for human disease
Tao Chen1, Youhuan Li2, Lei Zou2, Jie Ma1, Dongyan Zhao2 and Yunping Zhu1 1National Center for Protein Sciences, China 2Peking University, China
he massive onslaught of human disease and related genes, proteins and cellular processes can be difficult to manage and Tintegrate. Disease wiki is a wiki-based web server to help the community edit and create any biological knowledge of human diseases. The goal is to provide the research communities with up-to-date disease-related information which can be updated at any time by communities to keep pace with the continuously accumulating knowledge. Disease wiki content currently covers 8,607 diseases, 54,999 relations between diseases and genes, 13,349 genes related with diseases and 20,249 proteins. Each page contains editable tables which can be updated by users to refine any existing content. Each page is also paired with talk page which can support various user comments and discussions. Page content can also be edited, searched, listed or browsed in tree structure. An alternative way users can contribute is to create new disease, new gene, new protein and new relations between terms using pre-defined template and category. Disease wiki uses the same media wiki technology that powers wikipedia, allowing users to contribute at many different levels by editing existing pages, creating new pages or creating new categories to dynamically organize the content. Disease wiki can be accessed online at http://diseasewiki.hupo.org.cn.
Biography
Tao Chen has completed her PhD from National University of Defense Techonolgy. She is now an Assistant Professor at Beijing Institute of Radiation Medicine. She has published more than 10 papers in reputed journals of Computer Science.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 108 Fadzliza Hafiza Ramli et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Profiling of osteosarcoma serum leads to identification of proteins involved in modulation of α4β1 integrin expression in osteosarcoma cells migration
Fadzliza Hafiza Ramli1, Zulaika Roslan1, Azura Mansor2, Mudiana Muhamad1, Nor Faissal Yasin2 and Sharaniza Ab-Rahim3 1 Universiti Teknologi MARA, Malaysia 2University of Malaya, Malaysia
steosarcoma (OS) is a malignant bone tumor that predominantly arises in children and adolescent. Metastasis of OS Ooften leads to fatality and reducing the survival rate of OS patients. The mechanism involved in metastasis often related to cell-to-cell interaction and cell migration, which often involved integrin receptor proteins, cell adhesion molecules and the cytoskeleton structure. Therefore, the aim of this study is to profile the serum proteins isolated from OS patients and identify the cell adhesion molecules that involved in integrin signaling pathway. The work was further carried out to elucidate the expression of α4β1 integrin, tubulin and actin proteins as well as cell migration properties in primary OS cell cultured. Profiling of the OS serum proteins was carried out using iTRAQ analysis. Verification of the protein expression was done using western blot and immunofluorescence analysis. Fibronectin, thrombospondin-1 and vitronectin, involved in integrin signaling pathway was identified from the profile. The expression of integrin protein from the OS primary culture also reveals significant alteration and rearrangement on the cytoskeleton structure in the OS cells. The result showed a significant increase in β1 integrin expression while no significance found between primary OS cells to the control. Immunostaining assessment showed actin expression significantly higher compared to the tubulin expression across all primary OS cells group. This study has confirmed the involvement of integrin in cytoskeletal arrangement for the OS migration. This could be related to the expression of the adhesion molecules (thrombospondin-1, fibronectin and vitronectin) that has been identified in the OS serum protein.
Biography
Fadzliza Hafiza Ramli has completed her Bachelor (Hons) of Biomolecular Science from Universiti Teknologi MARA Malaysia. She then completed her Master’s degree in the field of Medical Sciences (Biochemistry & Molecular Medicine).
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 109 Hélida Monteiro de Andrade et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
A proteomic road to acquire an accurate serological diagnosis for human tegumentary leishmaniasis
Hélida Monteiro de Andrade1, B S S Lima1, S F Pires1, L C Fialho Jr1, E J Oliveira2, R A Machado-de-Avila3, C Chávez-Olórtegui1, A D Chapeaurouge2 and J Perales2 1Federal University of Minas Gerais, Brazil 2FIOCRUZ, Brazil 3Universidade do Extremo Sul Catarinense, Brazil
iagnostic tools are important for clinical management and epidemiological evaluation of Tegumentary (TL) and Visceral D(VL) Leishmaniasis. Serology is not frequently used for the diagnosis of the TL form because low antibody titers and cross-reaction with VL. Therefore, it is crucial to identify specific and immunogenic antigens from species associated with the TL form. Here, we employed a proteomic (DIGE-MS/MS) and an immunoproteomic (western blot-MS/MS) approach coupled to an in silico analysis and identified the most abundant and immunogenic proteins from L. amazonensis, L. braziliensis and L. infantum. Of 16 species specific proteins, nine were from the species causative of the TL form (L. amazonensis and L. braziliensis). In silico analysis revealed 18 B-cell epitopes with 0% similarity to T. cruzi orthologs and therefore, less likely to crossreact with sera of patients with Chagas disease. Two proteins reacted exclusively with serum from TL patients and presented several B-cell epitopes without similarity to T. cruzi orthologs: THe hypothetical protein GI 134063939 and the metallo-peptidase clan MA(E)-family M3. The immunoassay using peptide array revealed nine peptides with strong reactivity to sera from TL patients. These proteins and peptides may be good candidates to improve the specificity and sensibility of serological tests aiming to diagnose the TL of this neglected human disease.
Biography
Hélida Monteiro de Andrade has completed her PhD from Federal University of Minas Gerais (UFMG), Brazil, and Post-doctoral studies from FIOCRUZ, Brazil. She is the Head of Leishmaniasis Laboratory in Parasitology department from UFMG. She has published almost 50 papers in reputed journals in Parasitology and or Proteomic area, 21 of them using proteomic approaches. She has been advisor of more than 30 students in scientific initiation, Master’s, PhD and post-doctoral levels.
Notes:
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 110 Matyushkina Daria, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Proteome reorganization of the bacterium upon invasion of a host cell as a mechanism of adaptation: A Mycoplasma gallisepticum model
Matyushkina Daria Federal Research and Clinical Centre, Russia
hat strategies do bacteria employ for adaptation to its host and are these strategies different for varied hosts? At the Wmoment, there are a lot of studies concerned interaction of bacteria with their hosts but still has not been fully understood, which global changes bacteria undergo during invasion and persistence in the host cell and how these changes depend on the type of host. In this study, we used a Mycoplasma gallisepticum model because members of the genus Mycoplasma are characterized by a reduced genome, the size of which varies from 0.58-2.20 Mb, low content of GC bases, lack a cell wall and many metabolic pathways. All these qualities make M. gallisepticum convenient model for the study of system biology. It was shown using a combination of two-dimensional differential gel electrophoresis, MALDI-MS, LC-MS and MRM methods that M. gallisepticum S6 was capable to switch to another proteome state during the invasion of various eukaryotic host cells (human, chicken and mouse) and maintain that state for several passages. Eukaryotic cells induced similar proteome reorganization of M. gallisepticum during infection, despite different origins of the host cell lines. The most notable changes involved oxidative stress response, glycolysis, translation factors and hemagglutinins of VlhA family. We observed the up-regulation of the two regulatory proteins: SpxA and YebC/PmpR. We constructed SpxA- and YebC/PmpR-overexpressing strains of M. gallisepticum and identified the protein response similar to the one observed in intracellular conditions. We proposed their role as regulators of the adaptation to intracellular environment.
Biography
Matyushkina Daria is a graduate Student in the Laboratory of Proteomic Analysis of the Federal Research and Clinical Centre of Physical-Chemical Medicine (Moscow, Russia) and published 12 papers in reputed journals.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 111 Yi-Li Huang et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Evaluation of disulfide scrambling at various pH on Avastin digestion by mass spectrometry
Chiung-Wen Chang1, Yi-Li Huang1, Shi-Han Jian1, Yu-Hua Lin1, Sheng Yu Huang2, Sung-Fang Chen1 1National Taiwan Normal University, Taiwan 2Mithra Biotechnology Inc., Taiwan
isulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds Dto ensure quality and functions of protein drugs. Protein digestion procedures cannot be avoided for disulfide linkage analysis in conventional manner. In order to preserve enzyme activity during protein digestion, it is commonly carried out at basic environment which increases the possibilities of disulfide bond scrambling. However; when disulfide bond rearrangement occurs, it is not quite easy to differentiate whether by sample itself or digestion process cause the scrambling disulfide linkages. In this study, optimization on digestion pH was realized for the reduction of disulfide bond rearrangement. Three sets of proteases, including trypsin plus Glu-C, thermolysin and Lys-C were used, followed by dimethyl labeling and mass spectrometry for bevacizumab (Avastin) disulfide linkage analysis. There was no scrambled disulfide bond identified at pH 6 when using Lys-C or trypsin plus Glu-C as enzymes. When thermolysin was applied, there were still scrambled disulfide bonds identified either at pH 5, pH 6 or pH 7. Nevertheless, there was fewer scrambled disulfide bonds observed at low pH. All disulfide bonds on bevacizumab can be solved with this approach. The results demonstrated that by choosing the proper enzymes, using lower digestion pH environment could reduce the degree of scrambled disulfide linkages.
Biography
Yi-Li Huang is a Chemistry major master student at National Taiwan Normal University. His research interests focus on analytical chemistry, and proteomics-related mass spectrometry. His research thesis entitled “Evaluation of disulfide scrambling at various pH on Avastin digestion by mass spectrometry” will be presented in this proteomics conference.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 112 Marta Nekulova et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Quantitative analysis of medulloblastoma using tandem mass spectrometry
Marta Nekulova1, Petra Dvorakova2, Zahradnikova M1, Kyr M2, Jezova M2, Pavelka Z2, Slaby O3, Sterba J2, Zitterbart K2 and Hernychova L1 1Masaryk Memorial Cancer Institute, Czech Republic 2University Hospital Brno-Masaryk University, Czech Republic 3Central European Institute of Technology, Czech Republic
edulloblastoma is the most common malignant pediatric brain tumour that is distinguished in four major subgroups. MTherefore, there is an important need to identify clinically reliable protein markers for these subgroups which would be useful for the choice of appropriate treatment. Here, we present a novel proteomic approach for the quantification of proteins obtained from medulloblastoma tissue samples using tandem mass tags (TMT). We analysed medulloblastoma samples of all four known molecular subgroups in clinically standard risk patients. The tissue was lysed using urea buffer, stored at -80°C overnight and centrifuged for 30 minutes. The protein concentration was determined using BCA protein assay kit. Flowingly 100 µg of proteins was digested using filter aided sample preparation (FASP). Observed peptides were labelled using TMT10plex isobaric mass tags and mixed together. Finally, the samples were analysed using Orbitrap Elite coupled with UltiMate 3000 RSLCnano chromatograph and the acquired data processed with Proteome Discoverer 1.4. We obtained a list of quantified proteins from tumour proteome of medulloblastoma. Results has been subjected to statistical and bioinformatic evaluation. This new approach allowed to distinguish proteomic signatures linked to different clinical characterisation within medulloblastoma molecular subgroups.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 113 Fatima Saoud Al-Mohannadi et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Transforming growth factor-beta 1- and insulin-dependent regulation of carcinogenic transformation is in line with proteome profiling and identification of TGF beta and insulin as keynodes of carcinogenic transformation of MCF7 cells
Fatima Saoud Al-Mohannadi1, Reem Saeed Mubarak1, Kah Wai Lin2, Mariya Yakymovych3, Shahab Uddin Khan4 and Serhiy Souchelnytskyi1,2,3 1Qatar University, Qatar 2Karolinska University Hospital, Sweden 3Ludwig Institute for Cancer Research, Sweden 4Hamad Medical Corporation, Qatar
lucose, insulin and transforming growth factor-beta (TGFb) are potent regulators of cell functions. We explored effects Gof glucose, insulin and TGFb1 on human conditionally tumorigenic breast epithelial cells MCF7. To identify the most affected carcinogenic transformation-related cellular function, we performed cell contact inhibition, proliferation, spheroid formation and migration assays. We have also performed a proteome profiling of MCF7 cells undergoing carcinogenic transformation. Systemic analysis of our proteomics data showed that TGFb and insulin signalling are among key regulatory pathways affected in transformation of MCF7 from conditionally tumorigenic into invasive cells. We observed that the high level of glucose and insulin promoted growth and loss of contact inhibition, as observed by overgrowth of cells. Treatment of the cells with TGFb1 led to inhibition of cellular growth and counteracting of the insulin effect. Migration and spheroid formation by MCF7 cells were also affected in the same way, notably, insulin enhanced and TGFb1 inhibited these activities. We observed that the mechanism of interaction between glucose- and TGFb1-dependent mechanisms on cells may include a Smad3/CAGA element-dependent transcriptional regulation, and transcriptional regulation and expression of PAI-1. Thus, we report that TGFb1 counteracted glucose- and insulin-dependent stimulatory effects on MCF7 cells proliferation, loss of contact inhibition, spheroid formation and migration. TGFb and insulin were identified as keynodes in the regulatory network which was built with proteins identified upon proteome profiling of transformation of MCF7 cells. We identified 150 proteins which change their expression upon acquisition of invasive phenotype by MCF7 cells (parental MCF7 vs invasive MCF7 clone c46). We also identified 302 proteins with different expression in invasive MCF7c46 and metastatic MDA-MB-231, and 279 proteins with different expression in non-invasive parental MCF7 and MDA-MB-231 cells. These combinations showed that the invasiveness signature may contain less than 100 proteins, with TGFb and insulin among the keynodes of the proteome- dependent network. Thus, the cell based assays, reporter assays and proteome profiling of MCF7 cells showed that TGFb and insulin signaling are indeed crucial regulators of carcinogenic transformation.
[email protected] [email protected]
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 114 João Lídio da Silva Gonçalves Vianez Júnior et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Whole transcriptome analysis reveals that Zika virus halts cell cycle progression and disrupts neuronal differentiation in human neurospheres
João Lídio da Silva Gonçalves Vianez Júnior1, Janaina Mota de Vasconcelos1, Patricia P Garcez2, 4, Juliana Minardi Nascimento4, 3, Rodrigo Madeiro da Costa4, Rodrigo Delvecchio2, Sandro Patroca da Silva1, Pablo Trindade4, Erick Correia Loiola4, Luiza M Higa2, Juliana S Cassoli3, Gabriela Vitória4, Patricia Sequeira5, Jaroslaw Sochacki4, Renato S Aguiar2, Hellen Thais Fuzii6, Ana M Bispo de Filippis5, Daniel Martins-de-Souza3, Amilcar Tanuri2 and Stevens K Rehen2, 4 1Evandro Chagas Institute, Brazil 2Federal University of Rio de Janeiro, Brazil 3University of Campinas, Brazil 4D’Or Institute for Research and Education, Brazil 5FIOCRUZ, Brazil 6Federal University of Pará, Brazil
razil is facing an unprecedented growth in the number of microcephaly cases in babies. This phenomenon coincided with Bthe recent Zika virus (ZIKV) outbreak in this country. Although the Brazilian Ministry of Health was quick to recognize that ZIKV was probably the cause of microcephaly in newborns, the underlying mechanisms leading to the development of this pathology have not been established. To tackle this problem at the molecular level, we employed whole transcriptome sequencing of human neurospheres derived from neural stem cells exposed to ZIKV isolated in Brazil (Asian genotype). Differential gene expression analysis of control (MOCK) and ZIKV infected neurospheres generated a list of 26 down-regulated and 64 up-regulated genes. Among the up-regulated detected genes, the cyclin-dependent kinase inhibitor 1A (CDKN1A) and the glial fibrillary acidic protein gene (GFAP) were found. CDKN1A prevents the activation of the cyclin E/CDK2 complex, acting as a regulator of cell cycle progression during G1 and GFAP is a known marker of astrocytes. We also observed a decrease in the expression of the neurogenic differentiation 1 gene (NEUROD1) which is directly involved in the neurogenic program. Those findings suggest that ZIKV infection induces cell cycle arrest and inhibits the neuronal differentiation, resulting not only in the reduction of the size but in a deeper disruption of the normal development of the human brain.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 115 Elena Santonico, J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Solution structure of CUBO, a novel domain that preferentially binds the ubiquitin-like protein NEDD8
Elena Santonico University of Rome Tor Vergata, Italy
mong the members of the ubiquitin-like (Ubl) protein family, NEDD8 is the closest in sequence to ubiquitin (58% Aidentity). The two modification mechanisms and their functions, however, are largely distinct and the two molecules are not interchangeable. Selectivity is ensured by a complex network of interactions between modifying enzymes and adaptors, some of which are specific while others are promiscuous. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that exclusively binds NEDD8 has been described so far. Here, we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their selectivity. Many selected domains bind preferentially ubiquitin and to a lesser extent NEDD8. In few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We also identified a unique domain which maps to the carboxyl-end of the protein KHNYN, that has a clear preference for NEDD8 and binds neddylated cullins. Given this ability, we named this domain CUBO (Cullins Binding Domain). We present here the solution structure of CUBO domain in isolation and in complex with NEDD8. The model for the KHNYN-NEDD8 interaction is based on the NMR structure of the isolated domain, mutational analysis and chemical shift perturbations (CSPs) upon binding. The CUBO domain of KHNYN is to date the first example of NEDD8-binding protein that preferentially recognizes neddylated substrates.
Biography
Elena Santonico has completed her PhD and Post-doctoral studies at Tor Vergata University. She is a Research Scientist at the Department of Biology. Her research topic is mainly focused on “Studying the function of ubiquitin and Ub-like binding domains in proteolysis”.
Notes:
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 116 conferenceseries.com 773rd Conference
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
e-Posters
Proteomics & Bioinformatics 2016
Page 117 Gabriel Monteiro da Silva et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Virtual screening of HPV’s oncoprotein E6 and rational ligand design
Gabriel Monteiro da Silva, Elvira Tamarozzi and Silvana Giuliatti University of São Paulo, Brazil
he viral oncoprotein E6 is related with the development of cervical cancer in HPV positive patients. E6 has been reported Tto bind to TNF-α, p53, p600 and p21, blocking the traditional pathways to apoptosis and leading to unregulated cell growth. This work aimed to use computational methods such as virtual screening (both target-based and ligand-based), molecular dynamics simulation, toxicity prediction and rational ligand design in order to search for potential inhibitors to human papillomavirus E6 oncoprotein. We also conducted a parallel study using other in silico methods such as binding site prediction and pharmacophore identification in order to thoroughly uncover the oncoprotein’s properties. The most common European variants of E6 oncoproteins, previously modeled by Tamarozzi & Giuliatti (2015), were submitted to target-based virtual screening by using GOLD and Autodock Vina software and the molecules within the FDA-approved database as ligand pool. The highest scoring protein/ligand complexes were submitted to molecular dynamics simulation using GROMACS and then underwent visual analysis in both PyMOL and Chimera in order to evaluate their molecular interactions with the active site of E6. Finally, we performed theoretical toxicity predictions and pharmacokinetics simulations through a series of open- source web servers in order to select only the safest ligands which were used as pivot molecules for the rational design of hybrids novel molecules that combine the desired characteristics of the best ranked ligands, leading us to a considerable pool of potential inhibitors to oncoprotein E6.
Biography
Gabriel Monteiro da Silva is a Master’s Student at University of São Paulo. Under the guidance of Professor Silvana Giuliatti, he presented an oral session about Virtual Screening at EUROGIN 2015 multidisciplinary congress and has extensive experience in “Deploying in silico methods aimed towards the inhibition of HPV’s oncogenic viral proteins”.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 118 Dubovskaya L V et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Blood plasma proteomic profiling of breast cancer patients for biomarkers discovery
Dubovskaya L V and Bakakina Y S National Academy of Sciences of Belarus, Belarus
reast cancer remains one of the most common women oncological diseases in the world. The success of therapy depends on Bearly diagnostics and efficient treatment. Early diagnostics of breast cancer is complicated by asymptomatic development of the disease and the lack of reliable markers. Proteomics is shown to be an effective approach for discovery of disease biomarkers. Two dimensional (2D) gel-electrophoresis represents a powerful and widely used proteomic method for the analysis of complex protein mixtures. The aim of the study was to find unique biomarkers for early diagnostics and monitoring of breast cancer using proteomic analysis. Blood plasma samples from women with clinically and histological confirmed breast cancer, fibroadenoma and healthy donors were used in the study. The subtypes of malignant breast tumors were based on the standard histological criteria. Blood plasma proteins were separated by 2D gel-electrophoresis and identified by MALDI- TOF mass-spectrometry. It was found that blood plasma proteomic profiles were different for patients with various molecular subtypes of breast cancer. Identified differences were evident in the appearance of additional new proteins and changes in the expression of proteins present in norm. MALDI-TOF mass-spectrometry revealed the panel of proteins, namely clusterin, α2- HS-glycoprotein, haptoglobin α1 and serum amyloid A, which reflects a degree of tumor malignancy and varies depending on the molecular subtypes of breast cancer. Current data provides an advance to develop supplemental methods for non-invasive preclinical diagnostics and to reduce the risk of the disease progression.
Biography
Dubovskaya L V has completed her PhD from the Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus. She is the Director of the Institute of Biophysics and Cell Engineering of National Academy of Sciences of Belarus. She has published more than 35 papers in reputed journals and has been serving as a Peer Review Member of repute.
Notes:
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
Page 119 Holyavka M G et al., J Proteomics Bioinform 2016, 9:11(Suppl) conferenceseries.com http://dx.doi.org/10.4172/0974-276X.C1.092
7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Virtual screening and experimental testing of high affinity ligands for immobilization of some proteases
Holyavka M G1, Kondratyev M S2, Koroleva V A1 and Artyukhov V G1 1Voronezh State University, Russia 2Russian Academy of Sciences, Russia
t is known that the immobilization of the enzyme on an insoluble carrier solves several important problems in medicine: IPreparing prolonged action formulations due to stabilization and increase half-life of the enzyme; a possibility of obtaining the directed delivery of the drug solution, and its diffusion into the body and; directed regulation of optimums of preparation operation (temperature optimum, pH optimum). Molecules of trypsin (pdb 3UY9), papain (pdb 9PAP) and collagenase (pdb 2CLT) taken from the database of protein structures (PDB) was used as protein models. Virtual screening of ligands for immobilization of some proteases for medical application was performed. The set of ligands included high-molecular compounds (poly-cation and poly-anion exchange fibers) and chitosan. Based on the comparative analysis of the total energy values, the localization of the ligand binding sites and several literature data, we made several suppositions concerning the mechanisms of interaction of the suggested matrices for the immobilization with enzyme molecules and the structural features of such complexes. The adsorptive method of enzyme immobilization on the above matrices proposed here allowed us to keep the enzyme’s initial catalytic activity up to 70% for trypsin, papain and collagenase.
Biography
Holyavka M G is a Senior Researcher and Associate Professor in the Department of Biophysics and Biotechnology at Voronezh State University, Russia. She received her Bachelor’s in Science in 2005 and her Master’s in 2007. She has completed her PhD in the year 2010. She is currently working on structural & functional properties of homogeneous and heterogeneous biocatalysts on the basis of hydrolytic enzymes.
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J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Accepted Abstracts
Proteomics & Bioinformatics 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Effects of metal oxide nanomaterials on cultured human cells: A redox proteomic investigation
David Sheehan, Arjun Prakash Sridharan, Hugh Doyle and Yao Zhu University College Cork, Ireland
anomaterials are an important category of emerging environmental threat given that they are being used in ever-increasing Namounts and for an ever-wider range of applications. Nanoparticles, a sub-set of nanomaterials, are particles of average diameter < 100 nm and are readily prepared from metal oxides. Metal oxide nanoparticles are often used in sun creams and
other topical preparations. In this study the sensitivity of a panel of human cell lines (HEk, HeLa and A172) to TiO2, CuO2 and
ZnO2 nanoparticles was assessed. Cytotoxicity was determined using both neutral red retention and the MTT assays. These
suggested that cells had much lower LC50 with ZnO2 and CuO2 than with TiO2 and that both assays gave comparable results. A
more detailed study was made of CuO2 effects on HeLa cells using redox proteomics. Thiol-containing proteins were labelled with 5-iodoacetamido-fluorescein (IAF) and fluorescence images were obtained in a Typhoon scanner. Gels were then stained with colloidal coomassie and all images were analysed with SameSpots image analysis software. Differentially expressed and selectively oxidised proteins were identified by peptide mass fingerprinting by LC-tandem MS. Our data suggested that >20
individual proteins are selectively oxidised in response to CuO2. [email protected]
Phosphorylation of activation loop induces structural changes in c-Src tyrosine kinase leading to a conformational switch to the ATP binding conformation in the presence of the ligand
Encarna Pucheta-Martínez, Nicola D’Amelio and Francesco Luigi Gervasio University College London, UK
yrosine kinases play a crucial role in tumor formation and are the most frequently mutated protein family in cancer. The Tfirst viral oncogenic protein discovered, c-Src is involved in metastasis and is mutated in 50% of colon, liver, lung, breast and pancreas tumors. Understanding its conformational dynamics is important to address its activation mechanism and also help the design of more selective inhibitors as in the case of the anti-leukemic drug imatinib (Gleevec). Upon phosphorylation, various conserved structural elements including the activation loop, switch from an inactive to an active form able to bind ATP and phosphorylate a substrate in a cellular signaling process leading to cell replication. Crystal structures of their catalytic domain suggest that phosphorylation restrains the motion of flexible parts, in particular the activation loop which becomes visible in the crystals of the phosphorylated form. In this work, we show how phosphorylation drastically changes the dynamics of the C-lobe in c-Src by NMR analysis, a phenomenon totally invisible by crystallographic data. Flexibility in this region may have the biological role of preparing the structure to harbor the substrate. Besides this effect, signals from other parts of the protein, most likely the activation loop, become visible in agreement with a reduced conformational variability suggested by crystallographic data. NMR also shows that these modifications deeply impact on the mechanism of action through changing the binding affinity for ATP or ADP. In fact, while both the un-phosphorylated and the phosphorylated forms promptly bind ATP, de-phosphorylation greatly reduces the affinity for ADP, as its binding would impede the kinase to work efficiently. We also show that the conformation required for the binding of ATP and ADP to c-Src is already present in the absence of the ligand (independently on the phosphorylation state) and the interaction involves a process of conformational selection rather than induced fit.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Battling antimicrobial resistance where it counts: An extended proteome research of five bacterial species
Gilberto Igrejas1, 2,Tiago Santos1, Catarina Marinho1 and Patrícia Poeta2 1University of Trás-os-Montes, Portugal 2Universidade Nova de Lisboa, Portugal
roteomics is the large-scale study of proteins and it is often considered the next step in the study of biological systems. PUnlike the genome, the proteome is dynamic: It varies according to cell type and its functional state and the evaluation of protein profiles in response to various stress mechanisms, such as sensitivity to antibiotics or modifications related to antimicrobial resistance which represents a valid and integrating approach for the development of new therapeutic strategies. Antimicrobial resistance presents a significant challenge to scientists in the field of infectious diseases. The identification of protein determinants for resistance not only provides biomarkers for resistance to a particular drug but also aids in the understanding of the mechanisms of antibiotic function and resistance. The full knowledge of how antibiotics resistance evolves and is transmitted between potential hosts of different ecosystems takes on great importance. The functional genomics and proteomics unit based at the University of Trás-os-Montes and Alto Douro (UTAD) in Vila Real, Portugal, has recently completed 10 years of proteomic research related to antimicrobial resistance. During this time, five different bacterial species and 32 bacterial strains were studied, isolated from clinical and wildlife samples, 2770 protein spots were characterized through 2-DE and MALDI-TOF MS, and 392 proteins identified by shotgun proteomics (LC-MS/MS). The group has accomplished the evaluation of ESBL-producing Escherichia coli strain protein profiles, proteome comparison of vancomycin-resistant Enterococcus spp. strains compared to the proteome of the same strain without antimicrobial stress, whole proteome analysis of quinolone-resistant Salmonella strains, a sub-proteome analysis of a methicillin-resistant Staphylococcus aureus strain and several other proteomic approaches which we intend to overview in this review with the intention of connecting the dots between this large protein database and other antimicrobial resistance published results and determining a metabolic pathway which results in antimicrobial behavior.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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Multi-omics profiling of tumor interstitial fluid of breast cancer patients: A novel resource to identify cancer biomarkers for prognostic classification and detection
Irina Gromova Danish Cancer Society Research Center, Denmark
reast cancer (BC) is one of the most common cancers with millions of cases each year world-wide. Recent studies have Brevealed extensive genetic diversity and intra-tumor heterogeneity even within the same BC subtype. BC heterogeneity that is determined by complex clonal and spatial tumor organization severely affects key biological pathways thus challenging BC diagnostic classification and treatment. Despite tremendous efforts, no robust blood markers for BC patients have been identified so far, mainly due to current underestimation of BC complexity and high dilution factor in blood. The levels of disease biomarkers in local tumor microenvironment are estimated to be several orders higher than in blood and thus, the analysis of lesion-proximal fluids have become one of the most promising strategies for identification of potential candidates for non-invasive biomarkers. In this respect, tumor interstitial fluid (TIF), watery phase that is formed largely in solid tumor interstitium, comprising all biocompounds externalized from tumor mass is a novel, unique and valuable source for biomarker discovery. In the course of the study, we applied multiple–omics technologies, such as different proteomics platforms and glycomics screening, for the profiling of biomolecules externalized directly from the tumor and its microenvironment. Representative sets of tumors of multiple BC subtypes, matched TIFs, and blood samples obtained from high risk breast cancer patients with complete clinical and histopathological records including characterization of immune-infiltrates were analyzed. Such integrative approach allows revealing interconnection of BC biomarkers and their associated specific subtypes depending on the spatial tumor organization.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Identification of novel biomarker candidates for hypertrophic cardiomyopathy and other cardiovascular diseases leading to heart failure
Jiri Stulik University of Defense-FMHS, Czech Republic
n-depth proteome discovery analysis represents new strategy in an effort to identify novel reliable specific protein markers Ifor hypertrophic cardiomyopathy and other life threatening cardiovascular diseases. To systematically identify novel protein biomarkers of cardiovascular diseases with high mortality, we employed an iTRAQ-based quantitative proteome technology to make comparative analysis of plasma samples obtained from patients suffering from non-obstructive hypertrophic cardiomyopathy, stable dilated cardiomyopathy, aortic valve stenosis, chronic stable coronary artery disease and stable arterial hypertension. We found 128 plasma proteins whose abundances were uniquely regulated among the analyzed cardiovascular pathologies. Most of them have not been described yet. Additionally, application of statistical exploratory analyses of the measured protein profiles indicated the relationship in pathophysiology of the examined cardiovascular pathologies.
Role of post-translational modifications by ubiquitin family of proteins in resistance mechanisms of pancreatic cancer cells
Philippe Soubeyran Cancer Research Center of Marseille, France
ancreatic cancer remains nowadays one of the deadliest forms of cancer. This situation is partly due to the fact that these Ptumors become rapidly resistant to any kind of therapies. Our aim is to identify new resistance mechanisms which would explain the multi-resistant phenotype of pancreatic cancer cells. Chemotherapies trigger cellular stress responses that help the cell to survive. These responses are based on the post-translational modification (PTMs) of key components of these pathways. Among all possible PTMs, modifications mediated by ubiquitin family members appear to play major roles in these processes but, so far, have not been really studied in this context. Therefore, we intent to identify alterations of the ubiquitin and ubiquitin-like pathways and to determine which ones are involved in resistance mechanisms. To this end, we use cell lines expressing tagged version of each ubiquitin and ubiquitin-like studied, to specifically purify modified proteins and identify them by tandem mass spectrometry. Hence, we have established the PTMs profiles of pancreatic cancer cells, treated and not treated with chemotherapeutic drugs, resistant or not to them. We could observe that chemotherapeutic treatment, as well as acquisition of the resistant phenotype, is associated with an important modulation of PTMs profiles. Among those, we could validate the role of the modification of one candidate regarding the survival of the cell challenge by gemcitabine. Hence, studying alterations of PTMs mediated by the ubiquitin family of proteins associated with cancer resistance has the potential to reveal important new molecular mechanisms involved in the phenomenon.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Proteomics atlas of cancer cells’ death and survival
Roman A Zubarev Karolinska Institutet, Sweden
ass-spectrometry based proteomics is widely used to profile proteome changes in cancer cells in response to different Manticancer treatments. The proteome changes are expected to follow one of the established death pathways. Until 2008, three such mechanisms have been widely recognized. In 2012, the Nomenclature Committee on Cell Death (NCCD) has listed 13 distinct mechanisms of cellular death. However, in the 2015 NCCD report this classification has effectively been disavowed, because new research has shown that programmed death pathways previously believed to be unidirected are actually reversible. To differentiate between different modes of cell death, we mapped the proteome changes occurring in three attached cell lines treated with 50 different anticancer agents. Upon 24 hours incubation at a dose at which 50% cells are tested as dead, the still- attached cells and the floating cells were separately collected with the floating population showing the majority of dead cells while the attached cells consist of preferentially living cells. Proteome comparison of the surviving and dying cells with the untreated cells reveals the specific mechanisms of drug action as well as the pathways of death and survival that are common for all tested cell lines and drugs. Thus created proteomics atlas of cancer cell death and survival will serve as a reference in fundamental studies as well as in drug development.
Introducing epigenomics in systems biology: Cross-talk between cell signal transduction and epigenetic mechanisms
Simone Sidoli, Pau Pascual Garcia, Katarzyna Kulej, Maya Capelson and Benjamin A Garcia University of Pennsylvania, USA
ntegrating omics strategies is becoming a new frontier in systems biology, since disciplines like genomics Iand proteomics are now established. However, the traditional view of how these disciplines interplay, i.e., genomics→transcriptomics→proteomics→metabolomics, is too static to exhaustively represent a biological system. Events like early response to stimulus (protein phosphorylation) and structural gene regulation (epigenetic mechanisms) must enter in the equation. We investigated the development of larvae from Drosophila melanogaster upon treatment with kinase inhibitors. By integrating proteomics, phosphoproteomics, histone modification analysis and chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) we reconstructed links between drug treatment and phenotypic abnormalities during development. Larvae from wild type (OregonR) hatched and grew from eggs laid on food w/o inhibitors for the kinases EGFR and c-Met. Considering the phosphoproteome as indicative of early response to stimulus we characterized pathways of proteins with regulated phosphorylations connecting the inhibitor target with nuclear receptors and histone modifier enzymes. Specifically, we found down-regulated phosphosites in both inhibitor treatments on the ecdysone nuclear receptor and the interacting trithorax complex, which last catalyzes methylation on histone H3 lysine 4 (H3K4me). This modification, enriched in actively transcribed genes, globally decreased upon inhibitor treatment. ChIP-seq analysis mapped H3K4me on genes coding for proteins involved in translational initiation in wild type, which we found expressed in lower abundance in treated larvae. Collectively, our preliminary data indicate how drug treatment might be related to developmental abnormalities (slower growth), using epigenomics to link early response to proteome regulation.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Microbial impact on bile acid metabolism in the disease state using UPLC-TMS
Susan Joyce1,2 1APC Microbiome Institute, Ireland 2University College Cork, Ireland
ile acid signatures can be used as indicators of metabolic status and of gut signaling and health. We detect these steroid Bmolecules in different biological samples by UPLC-MS. Bile salts are conjugated bile acid (BA) moieties that are synthesized in the liver from cholesterol. They are now regarded as gut signaling hormones and are recognized as directing gene expression both locally and through cross-talk with the other tissues, mainly the liver. Following entry to the GI tract, microbial enzymes modify bile salts-bile salt hydrolase (BSH) enzymes and bile acids-bile acid inducible (Bai) enzymes in a spatial and temporal dependent manner. Hence, the gut microbiota is responsible for the range and the diversity of bile acids and salts and therefore microbial directed bile acid metabolism can play a central role in directing metabolic processes. While representatives of all the main phyla carry BSH, a property of gut associated bacteria only, these enzymes range in activity from none to very active and they show strain specific and different substrate specificities. Here, we present snapshot studies where we examine a range of bile acid altering activity in preclinical models of gut disease and in the disease state (Obesity, Short bowel Syndrome, Colitis) and their influence on host gene expression and gut health.
The regulatory potential of protein post-translational modifications in chromatin biology and gene expression investigated by MS-based proteomics
Tiziana Bonaldi European Institute of Oncology, Italy
hromatin is a highly dynamic, well-organized and yet ill-defined protein-DNA-RNA structure that controls various DNA- Cdependent processes. A large number of site-specific post-translational modifications of histones (hPTMs) contribute to the maintenance and modulation of chromatin plasticity, gene activation, DNA replication and repair and a variety of other biological processes and disease states. The observation of the diversity, frequency and co-occurrence of histone modifications at distinct genomic loci led to the notion that these marks create a molecular barcode, read by effector proteins that translate it into a specific transcriptional state, or process, on the underlying DNA. However, the molecular details of its working mechanisms are only partially characterized. More recently, various technological progresses have enabled the detection of these PTMs on an increasing number of non-histone proteins, involved in a variety of biological processes. Recent achievements made Mass Spectrometry (MS) and quantitative proteomics excellent tools to help understanding how histone and non- histonic PTMs mediate the structural-functional state of chromatin. My team contributed to the field by setting-up distinct MS-proteomics strategies, combined with various biochemical methods of enrichment of chromatin and extra-chromatin proteins, to investigate chromatin plasticity and nuclear dynamics governed by post-translational modifications. The talk will offer an overview of the MS-proteomics strategies developed to gain insights into chromatin biology, with emphasis on: THe proteomic dissection of chromatin regulatory regions; the hPTMs-analysis of clinical specimens and the recent achievements on the methyl-proteome profiling and its impact in DDR and miRNA biogenesis
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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Discover the underlying mechanism in diabetic cardiac dysfunction by integrating knowledge from genomic and genetic studies
Zhengyuan Xia, Zipeng Liu and Junwen Wang University of Hong Kong, Hong Kong
yocardial infarction (MI) is a major cause of sudden death and one of the most common perioperative complications Mprevalent in diabetes mellitus. The underlying biological process is different from that in non-diabetes partially due to increased oxidative stress in diabetes. Cardioprotective interventions that are effective in non-diabetic patients lose their effectiveness in diabetic patients, which exacerbates the susceptibility of diabetic hearts to myocardial ischemia reperfusion injury (IRI). However, the mechanism is still largely unclear. The rapid evolution of genomic and genetic approaches, such as microarray and genome-wide association study (GWAS), provides additional insights into complex disease studies. Here, by combining gene co-expression network analysis from a set of microarray profiling and MI/type 2 diabetes (T2D) associated gene sets from GWAS, we built a transcription factor (TF) based regulatory network to explore the pathological behavior. The resulting network using this combination method was validated by high enrichment in several well-documented pathways of diabetic cardiac pathology (e.g. PI3K/Akt and Jak/Stat3 signaling pathway) and was also significantly improved than that using only genomic or genetic data individually. This TF-based network also revealed numbers of previously unreported protein interactions linking distinct pathways, among which we verified a relation between Stat3 and Hif-1α in diabetic myocardial IRI model. Thus, our study showed potency of combining knowledge from genomic and genetic studies in discovering the hidden mechanism in diabetic cardiac dysfunction.
Studying mtDNA replication using in vitro reconstituted systems
Jay P Uhler University of Gothenburg, Sweden
itochondria are essential for the production of cellular energy and contain their own genomes (mtDNA) that encode M13 subunits of the respiratory chain, 22 tRNAs and 2 rRNAs. The remaining >1500 mitochondrial proteins are nuclear encoded including those required for mtDNA maintenance. The human mitochondrial genome is an approximately 16.5kb circular molecule; mutations in which can lead to mitochondrial disease. mtDNA is replicated by a dedicated mitochondrial replication machinery that includes the replicative DNA polymerase POLγ, the TWINKLE helicase and mitochondrial single stranded DNA binding protein. However, many other DNA replication factors remain to be identified and studied. Moreover, the regulation of mtDNA replication initiation, elongation and termination are not yet fully understood. To help address these unresolved questions, our research group reconstituted a minimal mitochondrial replisome in vitro over 10 years ago. This in vitro mtDNA replication system is based on purified recombinant proteins and specifically designed DNA templates. Based on this approach, many basic mechanisms have been addressed such as leading/lagging strand replication and RNA priming/ removal. The system also enables in vitro modeling of mitochondrial diseases by using disease-based mutant proteins or by reconstituting aberrant replication processes. Most recently, the system has been useful in performing screens to uncover the effects of various drug compounds on mtDNA replication.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Targeted lipidomics: Analytical approach for the seperation of lipids by using UPLC-ESI/MS/MS in serum samples of diabetes mellitus pateints
Smita Panchal Indian Institue of Toxicology and Research, India
n the present study, an ionic liquid based vortex assisted surfactant-enhanced emulsification microextraction (IL-VASEME) Imethod followed by Plackett-Burmann Design (PBD) and Central Composite Design (CCD) using liquid chromatography- electrospray mass spectrometry (LC-ESI-MS/MS) has been applied for the determination of fatty acids, triglycerides and phospholipids in serum samples of healthy and diabetic subjects. Study afforded the separation of these lipid classes in a single run with Rt of 5 min. The method was statistically optimized to reduce the extraction time. The extraction parameters further were optimized by design of experiment (DOE) approach. The ionic liquid, 1-butyl-3-methylimmidazolium
hexafluorophosphate (BMIMPF6) was used as an extraction solvent, while surfactant Triton X-100 was used as an emulsifying agent. Statistical method, PBD screened the most significant factors such as ionic liquid volume, surfactant strength and pH for optimizing conditions for the separation of lipids. The screened factor values were based on the CCD, which was optimized as 45 µL of ionic liquid, 7.5 pH and 1.25% of surfactant strength for extraction of lipids. The limit of detection (LOD) and limit of quantification (LOQ) were 0.012-0.034 ng/mL and 0.046-0.114 ng/mL respectively. The recovery of lipids was in the range of 90.9-114%. The intraday and interday precision in the serum sample ranged between 1.42-4.48% and 3.75-10.8% respectively. The study revealed that the IL-VASEME method was comparatively more sensitive with other conventional methods for the separation of both polar and non-polar lipids in single step.
Structure based pharmacophore modeling for design of riboswitch based potent inhibitors for Vibrio cholerae
Somdutt Mujwar Maulana Azad National Institute of Technology, India
holera pandemics are caused by facultative pathogenic Vibrio cholerae bacteria persisting in the countries having warmer Cclimatic conditions as well as the presence of large water bodies with huge amount of organic matter, it is responsible for the millions of deaths annually. Presently, the available therapy for cholera is Oral Rehydration Therapy (ORT) with an antibiotic drug. Excessive utilization of life saving antibiotics drugs leads to the development of resistance by the infectious microorganism against the antibiotic drugs resulting in loss of effectiveness of these drugs. Also, many side effects are associated with the use of these antibiotic drugs. This riboswitch is explored as an alternative drug target for Vibrio cholerae bacteria to overcome the problem of drug resistance as well as side effects associated with the antibiotics drugs. The bacterial riboswitch is virtually screened with 24407 ligands to get possible drug candidates. The 10 ligands showing best binding with the riboswitch are selected to design a pharmacophore which can be utilized to design lead molecules by using the phenomenon of bioisosterism.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Sensitivity of Palb2-null tumor cells to an oxidative stress inducing agent
Yanhong Gao Chinese PLA General Hospital, China
ALB2 gene mutations, as the newly discovered breast cancer associated gene, has brought new direction for the prevention Pand treatment of breast cancer. To better understand the function of PALB2 and whether it can be used for drug, we generated p53-single-null (as control) and Palb2; p53-double-null cell lines from the mouse mammary tumors obtained and we found that Palb2-null tumor cells were hypersensitive to DNA damaging agents in previous study. To explore new ways to selectively kill Palb2-null tumor cells, we tested the potential of targeting oxidative stress in the cells. For this purpose, we chose phenethyl isothiocyanate (PEITC) and L-sulforaphane. We tested the sensitivity of five different Palb2-null tumor cell lines and three different control lines to the drugs. Cells were seeded in 96 well plates and treated with different concentrations of the two drugs for 72 hours. Then, cell viability was measured by CellTiterGlo® assay. Comparing with L-sulforaphane, we found Palb2-null tumor cells were hypersensitive to PEITC. PEITC is a natural compound rich in vegetables such as watercress and broccoli, etc. PEITC has long been known to possess anti-cancer activity, has been extensively studied. According to the result, it is raising an tempting prospect of preventing or treating PALB2-associated cancers with the inexpensive drug.
J Proteomics Bioinform 2016 Proteomics & Bioinformatics 2016 Volume 9, Issue 11(Suppl) ISSN: 0974-276X JPB, an open access journal October 24-26, 2016
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7th International Conference on Proteomics & Bioinformatics October 24-26, 2016 Rome, Italy
Quantitative proteomic analysis of prostate cancer for biomarker discovery and drug target identification
Amilcar Flores Morales University of Copenhagen, Denmark
linical management of prostate cancer (PCa) is marred by excessive diagnose and treatment of indolent tumors and also Cby paucity of effective therapies for patients harboring metastatic, castration resistant tumors. Proteins are the essential effectors of cellular functions and the targets of most of the currently used or newly developed drugs. Genetic analysis alone cannot be used to predict protein function and concentration changes because protein function is regulated at the translation and postranslational levels (e.g., by ubiquitination, phosphorylation, proteolysis, etc.). Therefore, system level quantitative proteomic analysis can reveal novel information that could be used in the identification of biomarkers or viable drug targets to address important clinical needs. Our investigations have been motivated by our limited understanding of the proteome changes associated with PCa initiation and progression. We will present the results of the most extensive investigations of the prostate cancer proteome to date. We have implemented a stable isotope based methodology to identify and quantify 6000 proteins in minute amounts of formalin fixed paraffin embedded tissue samples and apply it to the analysis of malignant (n=28) and non-malignant (n=8) tissue from prostatectomy samples and bone metastases (n=25). We will describe protein, pathways and processes that are de-regulated at different stages of disease progression. To demonstrate the translational value of this type of analysis, we identified a novel prognostic biomarker, proNPY that can select patients who are at increased risk death among those harboring low Gleason score tumors. The prognostic value of this biomarker was assessed in two independent large cohorts of patients managed by watchful waiting.
Biography
Amilcar Flores Morales completed his PhD in Chemistry from National University of Colombia and Post-doctoral studies from the Karolinska Institute where he became an Associate Professor in 2006. In 2009, he became a Professor of Molecular Endocrinology at the University of Copenhagen. He has a long standing interest in “The application of system-wide molecular profiling (omics) to the characterization of endocrine tumors with a focus on prostate cancer”.
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