Targeted Metabolomics Using the UPLC/MS-based AbsoluteIDQ ...
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Scientific Program Scientific Program VI International Symposium on OMICS and Bioinformatics VI International Symposium on OMICS and Bioinformatics Iberostar Laguna Azul Hotel, Cuba th st October 20 - 23
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Scientific ProgramScientific ProgramVI International Symposium on OMICS and BioinformaticsVI International Symposium on OMICS and Bioinformatics
Iberostar Laguna Azul Hotel, Cuba
th stOctober 20 - 23
INDEX
Scientific Program. Oral Presentations...............................................1
Oral Presentations. Abstracts..............................................................9
Scientific Program. Poster Presentations.........................................41
Poster Presentations. Abstracts........................................................46
Participants List..................................................................................79
NOTEBOOK..........................................................................................85
Scientific program Oral Presentations
1
thOCTOBER 20
OPENING SESSION
17:00-17:15 Welcome remarks and presentation of the
Scientific Program
Dr. Luis Javier González, Head of System Biology
Department, CIGB
17:15-18:00 Opening lecture: Progress on the Biomedical
Research pipeline at the Center for Genetic
Engineering & Biotechnology, Havana, Cuba
Dr. Gerardo Guillén Nieto, Director of Biomedical
Research
18:00-18:45 Opening lecture: 3D genome structure analysis by
high-throughput sequencing technologies
Dr. Yuriy Orlov, Russia
19:00-20:00 WELCOME COCKTAIL
(Room 1)
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stOCTOBER 21
PROTEOMICS SESSION
08:30-09:30 Plenary Lecture:
Genomic-guided cancer therapy: selection from a
basket of options
Dr. Howard L McLeod, USA
Chairmans: Dr. Vladimir Besada / Dr. Akos Vertes
09:30-09:55 Targeted Proteomics-Driven Computational Modelin the Chemotaxis and Toll-like Receptor Signaling Pathways in MacrophagesDr. Aleksandra Nita-Lazar, USA
10:00-10:25 Collision Induced Unfolding: A New Paradigm in Protein Stability MeasurementsDr. Brandon Ruotolo, USA
10:30-10:55 Protein and peptide fractionation by electrophoresiswith online electroelution for proteomics studiesDr. Yassel Ramos, Cuba
11:00-10:25 Proteome-wide analysis of three species of LeishmaniaDr. Gabriel Padrón, Cuba
(Room 1)
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11:30-11:55
12:00-12:25 Beta-hairpin peptides as entry inhibitor of Dengue virus:relationship between structure and functionDr. Glay Chinea, Cuba
12:30-12:55 Does the Dengue virus interactome in human plasmadistinguish between viral serotypes?Dr. Vivian Huerta, Cuba
13:00-13:30 CIGB-814, a new therapeutic candidate for rheumatoidarthritis, from the bioinformatic design to the phase Iclinical trialDr. María del Carmen Domínguez, Cuba
13:30-15:00
15:00-17:00 POSTERS DISCUSSION
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stOCTOBER 21
GENOMICS SESSION
Chairmans: Dr. Julio Raúl Fernández / Dr. Yeang Chen-Hsiang
09:30-09:55 Genetics and Genomics in Obesity: Fatalism or Hope? Dr. Jose Fernández, USA
10:00-10:25 Integration of transcriptomic and proteomic data toelucidate the mechanism of action of novel compounds:the case of the antitumor peptide CIGB552.Dr. Julio Raúl Fernández, Cuba
10:30-10:55 A quantitative framework of integrating multi-modalcancer genomic dataDr. Yeang Chen-Hsiang,
11:00-10:25 Ecological Genomics: Microarray DNA sequencing andbioinformatics for massively parallel multi-speciesenvironmental studiesDr. Steven Car, Canada
11:30-11:55
12:00-12:25 Genomic approaches to improve the treatment oftumors with interferonsDr. Dania Vázquez-Blomquist, Cuba
(Room2)
Taiwan
12:30-12:55 Developing and applying network biology tools to differential gene expression data analysisDr. Ricardo Bringas, Cuba
13:00-15:00
15:00-17:00 POSTERS DISCUSSION
ndOCTOBER 22(Room 1)
08:30-09:30 Plenary Lecture: A population genomic health profilein Latin AmericaDr. King Jordan, USA
PROTEOMICS SESSION
Chairmans: Dr. Weston Struwe / Dr. Luis Javier González
09:30-09:55 Mapping HIV Viral Spike Glycosylation: a Critical Stepin Determining Neutralizing Antibody and LectinRecognitionDr. Weston Struwe, UK
(Room 1)
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10:00-10:25 Protein Content of the Hylesia metabus Eggnest Seta(Cramer [1775])(Lepidoptera: Saturniidae) and ItsAssociation With the Parental Investment for theReproductive Success and LepidopterismDr. Luis Javier González, Cuba
10:30-10:55 Structural characterization and biological implications of sulfated N-glycans in a serine protease from the
neotropical moth Hylesia metabus (Cramer [1775])(Lepidoptera: Saturniidae)Dr. Gleysin Cabrera Herrera, Cuba
11:00-10:25 A metabolic labeling approach for glycoproteomicanalysis reveals altered glycoprotein expression uponGALNT3 knockdown in ovarian cancer cells.Dr. Dimcho Bachvarov, Canada
11:30-11:55
12:00-12:25 Targeted LC-MS/MS quantification of glycopeptidesDr. Radoslav Goldman, USA
12:30-12:55 “Candidatus Liberibacter asiaticu”', Causal Agent ofCitrus Huanglongbing, Is Reduced by Treatment withBrassinosteroidsDr. Eduardo Canales, Cuba
13:00-13:30 Activity in vitro and in vivo of a novel growth hormonesecretagogue A233.Studies in fish and mammals.Dr. Rebeca Martínez, Cuba
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13:30-15:00
15:00-15:40 Universal and Digital NGS Solutions: From Sample toInsightJacobo Zuñiga Castillo, PhD. Regional Marketing Manager Mexico, Central America and Caribbean, QIAGEN Mexico
15:40-17:00 POSTERS DISCUSSION
stOCTOBER 23
(Room 1)
08:30-09:30 Plenary Lecture: High throughput metabolomics forthe analysis of tissue and cell phenotypesDr. Akos Vertes, USA
SYSTEM BIOLOGY SESSION (Room 1)
09:30-09:55 Mathematical models of the impact of IL2 modulationtherapies on T cell dynamics.Dr. Karina García, Cuba
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10:00-10:25 Dissection of programmed and externally instigatedageing in a fibroblast modelDr. Anna Baranova, USA
10:30-11:00 Analysis of nucleotide diversity to identify functionallyimportant regionsDr. Tatiana V. Tatarinova, USA
11:00-13:00 Break
13:00-14:00 CLOSING SESSION
3D genome structure analysis by high-throughput sequencing technologies
1,2 2Yuriy L. Orlov , Guoliang Li
1 2Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia. Novosibirsk State 3University, Novosibirsk, Russia. Huazhong Agricultural University, Wuhan, China
Key words: gene expression, chromatin, 3D chromosome structures, chromosome contacts, CTCF sites, ChIA-PET
Studying of 3D chromosome structure is important problem of molecular biology challenging sequencing technologies. Transcription regulation research challenges the sequencing technologies development and bioinformatics data analysis. ChIP-Seq detects interactions between DNA and proteins; ChIA-PET (Chromatin Interaction Analysis with Paired-End-Tag) technology allows detect interactions between pairs of DNA sites affecting gene regulation. Fullwood et al. [1] used ChIA-PET technology to construct chromatin interaction network bound by estrogen receptor alpha from human breast cancer cell line (MCF-7) and found long-range ER binding sites are mostly located at promoter regions. CTCF-mediated interactions found in mouse embryonic pluripotent stem cells and human cell lines [2]. Li et al. [2] detected promoter-centered distant interactions bound by RNA Polymerase II in cancer cells. The recent development of genome-wide proximity ligation assays such as Hi-C and its variant TCC has significantly facilitated the study of spatial genome organization. The Hi-C technology could capture all the interactions but with low resolution. The ChIA-PET technology greatly enhances the resolution; it can identify the interactions mediated by a known protein.
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AbstractsOral Presentations
We developed computer programs for 3D genomics data analysis. The data have been obtained experimentally by using Hi-C and ChIA-PET methods [3]. The programs were tested on CTCF binding sites, genes and spatial topological domains. These data have been obtained experimentally by using methods ChIP-seq, Hi-C, ChIA-PET. Five distinct chromatin domains revealed by CTCF ChIA-PET raised a new model of CTCF function for chromosome structure organization and linking enhancers to promoters for gene transcription regulation. Gene annotation was obtained from UCSC Genome Browser (http://genome.ucsc.edu). The result of the analysis is the distribution of CTCF transcription factor binding sites on domains on the human chromosomes and relative gene locations. The distributions of human genes relative CTCF binding sites and a randomly generated list of such sites as the program output were used to estimate statistical significance of the associations found. Distinct chromatin domains revealed by CTCF ChIA-PET raised a new model of CTCF function for chromosome structure organization and linking enhancers to promoters for gene transcription regulation. Chromatin interaction networks properties will be discussed.
References:
1. M.J.Fullwood et al. (2009) An oestrogen-receptor-alpha-bound human chromatin interactome, Nature, 462(7269):58-64.
2. G.Li et al. (2014) Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) sequencing technology and application, BMC Genomics, 15(Suppl 12):S11.
3. Z. Tang et al. (2015) CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription, Cell, 163(7):1611-27.
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Genomic-guided cancer therapy: selection from a basket of options
Howard L. McLeod
Medical Director, Personalized Medicine Institute, Moffitt Cancer Center, Tampa, FL USA
Precision medicine has become a focus of research and policy and has the opportunity to also change medical practice. The use of genomics in this context may impact many areas of medicine, but is becoming a real part of oncology, with implications for therapy selection, treatment avoidance, dosing, and risk prediction. However, there are many cancer treatments for which there are few tools for individualized selection based on patient-specific attributes. Selecting from amongst cytotoxic chemotherapy, antivascular agents, kinase inhibitors, immunotherapies is important for clinical management and for the use of very expensive medicines. Discovery strategies are needed to more thoroughly understand the pharmacological basis for drug toxicity, efficacy, and resistance in patients with cancer. The presence of clinically predictive germline variants has also opened the hope that objective predictors of patient toxicity will be in the future. This will allow for the development of robust risk/benefit models, whereby decisions between apparently equal treatment options can be made for an individual patient. These probabilistic strategies are important ways to make pharmacogenomic findings of relevance to modern cancer care. There is a need for personalized medicine approaches to also go beyond DNA, to include biomarkers that reflect the patients current situation. While this can include immunoproteomic or metabolomics strategies, blood level guided therapy remains an underexplored clinical tool. It is also clear that there are many barriers to clinical application. These include expanding the science to understanding the pathways of genes that regulate a drug's activity. There are also critical non-science issues, such as integration of new tests into health systems, changing old habits to allow application of new data, and the reality that the cost of both testing and the therapeutic options are a key driver in health care. As the scientific evidence matures, we must think beyond our favorite aspect of translational science if we are to overcome the many obstacles to delivering more careful selection of cancer therapy.
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Targeted Proteomics-Driven Computational Modeling of the Chemotaxis and TLR Signaling Pathways in Macrophages
1 1 1 1 1Manes, N. P. , Mann, J. M. , Angermann, B. R. , Koppenol-Raab, M. , An, E. , 1 1 2 1 1Sjoelund, V. H. , Sun, J. , Ishii, M. , Germain, R. N. ,Meier-Schellersheim,M. ,
1Nita-Lazar, A .
1 2Laboratory of Systems Biology, NIAID, NIH, Bethesda, MD, USA; Osaka University, Japan
Chemotaxis and toll-like receptor (TLR) signaling in macrophages are critical to the immune response. To model these signaling networks, we are using selected reaction monitoring (SRM) to measure the absolute abundance of pathway proteins, and the resulting values are being used as pathway model parameters.RNA-seq was performed to identify expressed transcripts, and shotgun mass spectrometry was used to identify proteotypic peptides. SRM using heavy-labeled internal peptide standards was used to quantify the chemotaxis pathway proteins. The transcript and protein abundance values correlated strongly, and estimated protein abundance values for the entire macrophage proteome were produced.Computational modeling of the chemotaxis pathway was performed using Simmune. Molecular reaction rates were not directly measured, but instead were constrained using a model training dataset consisting of multiple types of in vitro microscopy data. Subsequently, model testing used an orthogonal dataset from ELISA assays of RAC1-GTP and RHOA-GTP. The model produced in silico results consistent with both the training and testing datasets, and it was determined to be robust by assessing the accuracy of thousands of perturbed models. These findings demonstrate the feasibility and value of combining mass spectrometry-based measurements with pathway modeling for advancing biological insight.SRM assays for almost the entire TLR pathway have been successfully developed and are being used to accurately quantify the pathway proteins. In parallel, a preliminary model of the TLR pathway is being developed using the estimated protein abundance values.This research was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, NIH.
Collision Induced Unfolding: A New Paradigm in Protein Stability Measurements
Eschweiler JE, Tian Y, Ruotolo BT
University of Michigan, Department of Chemistry, 930 N. University Ave. Ann Arbor MI, 48109
A key factor in varied protein functionality is linked to their stabilities, enabling remarkable changes in 3D conformation in response to external stimuli and local environment. Because of their importance in biology, proteins are amongst the most important drug targets, but in many cases we lack the analytical tools necessary to characterize small molecule interactions within such protein assemblies, especially when those interactions require a degree of conformational-selectivity. Over the past several years, we have been building nano-electrospray ionization (nESI) coupled to ion mobility-mass spectrometry (IM-MS) into a tool capable of comprehensively characterizing multiprotein-ligand complexes from mixtures, using microgram amounts of protein and without requiring covalent labels or tagging. In our lab, we have pioneered an approach termed collision induced unfolding (CIU) which seeks to measure the stability of protein folds in the absence of solvent, along with pattern of intermediate unfolded states populated as a function of ion internal energy, in order to deduce subtle differences in protein tertiary structure and, in some cases, extract information directly related to the discovery and development of new therapeutics. In this presentation, our most recent CIU data will be shown in order to demonstrate the breadth and promise of such IM-MS technologies for applications in structural proteomics.
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Protein and peptide fractionation by electrophoresis coupled to electro-elution for proteomics studies.
Ramos Y., Carpio J., González A., Quiñones M., García D., García Y., González L. J. and Besada V.
Proteomics Group, Department of System Biology, Biomedical Research Division, Center for Genetic Engineering and Biotechnology, Havana, Cuba
Currently shotgun proteomics based on peptide fractionation via liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation via electrophoresis was the main tool. During the last decade, the applications of electrophoretic techniques for protein and peptide mixtures fractionation have emerged as an alternative to liquid chromatography systems. We recently proposed the combination of SDS-PAGE for protein fractionation and SDS-free PAGE for peptide separation as a novel procedure for proteomic studies. Due to the presence of SDS in the first step, the method named Dual Fractionation by Polyacrylamide Gel Electrophoresis (DF-PAGE) is suitable for very hydrophobic protein analysis. The second dimension of the method, the SDS-free PAGE technique is compared to Off-gel electrophoresis; a system commonly used for peptides fractionation previous to MS analysis. As the instrumental support of the method, a new device for electrophoresis coupled to electro-elution is also presented here. The device is used for SDS- or SDS free-PAGE to get the fractionated protein/peptides in solution increasing the number of identified proteins. Fractionated and on-line electro-eluted proteins could also be analyzed by MRM in order to detect low abundant protein.
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Proteome- wide analysis of three species of Leishmania
1 2 1Cuervo P. , Wisniewski J. and Padron G.
1 2Laboratory of Leishmaniosis, Institute Oswaldo Cruz, FIOCRUZ, Brazil; Max Planck
Leishmania species are responsible for a broad spectrum of diseases called Leishmaniasis affecting over 12 million people worldwide. Clinical forms range from self-healing localized cutaneous lesions to severe disseminated visceral forms that could be fatal. During the last decade, numerous proteome studies of Leishmania spp. have been reported. However, relative low number of proteins have been identified and quantified, and the Leishmania proteome remains mostly uncharacterized. Here, we report a comparative study of three Leishmania species: Leishmania braziliensis, L. panamensis and L. guyanensis. Parasite cells (five biological replicates) were lised in presence of 2% SDS and sample preparation was carried out by the FASP method. LC-MS-MS spectra were run in a QExactive HF MS coupled to a UPLC system using a 75 µm x 50 cm column packed with C18 1.8 µm diameter particles and 4h gradient. Data were analyzed with MaxQuant and Perseus. More than seven thousands proteins were identified and the absolute quantification of 6 500 were determined using label free quantification and total protein and proteomic ruler approaches, 2 054 proteins had statistically different concentrations in at least two of the species, and 53 in the three species. PCA analysis demonstrate very clear differentiation of the three species. Some of the most important biological processes having differences were identified. A group of proteins is proposed for selective identification of these species.
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Beta-hairpin peptides as entry inhibitor of Dengue virus: relationship between structure and function
Chinea G., Huerta V., Fleitas N., Toledo P., Martín A., Falcón V., Pupo D., Vidal Y., Guirola O., Sarría M., Reyes O., Garay H., Ramos Y., González L.J.
Centre for Genetic Engineering and Biotechnology, Havana, Cuba
Beta-Hairpins (BH) are common structural motifs of proteins often involved in protein-protein interactions. In this regard, several data indicate that the beta-hairpin “FG” of Domain III (DIII) of the envelope protein of Dengue virus (DV) plays an important role in the biological activity of the protein. Accordingly, we designed structurally constrained synthetic peptides based on the BH-FG of DIII which display antiviral activity and lead to the ultimate development of peptides showing potent antiviral activity. We further explored the impact of several structural properties of designed BH peptides on its biological activity, including BH sequence space, structural propensity of amino acids, secondary structure content, topology, loop length, turn type, linearity or cyclization, supramolecular structure, etc. BH peptide variants exhibiting high potency and avidity for receptor were designed by a combined approach aimed to optimize the putative receptor binding surface and peptide folding. We also show that antiviral activity of peptides correlates with putative receptor binding avidity and supramolecular structure. Docking simulations were performed between the peptides and the ligand binding domain of the receptor.
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Does the Dengue virus interactome in human plasma distinguish between viral serotypes?
1 1 1 1 1 1Huerta V. , Ramos Y. , Palomares S. , Guirola O. , Martin A.M. , Yero A. , Martín 1 1 1 2 2 1 1D. Pupo D. , Sarría M. , Gallien S. ,Domon B. , González L.J. and Chinea G.
1Department of System Biology, Center for Genetic Engineering and Biotechnology, Havana, 2Cuba. Luxembourg Clinical Proteomics Center, Luxembourg
Dengue virus complex consists of four serotypes (DENV1-4) of an arthropod-borne virus that affects tropical and sub-tropical areas of the world. Any of the four viral serotypes can cause a human disease that varies its clinical severity from a mild self-limited febrile syndrome to life-threatening manifestations. There is limited comprehension of host factors that can influence the efficiency of DENV infection and consequently the development of clinical manifestations. Envelope (E) protein is the major structural protein in the surface of the virion and is organized in three structural domains: domain I-III. Domain III of the E protein is target of neutralizing antibodies against the virus; is involved on the interaction with cellular receptors and amino acid changes between viral strains associated to differences in disease severity are also located in this region. We used affinity purification with recombinant preparations of domain III of the four virus serotypes: DIIIE1-4 as ligands to identify proteins from human plasma interacting in vivo with the virus. We identified 65 putative interacting proteins; some of them confirmed by Selected Reaction Monitoring and validated as DIIIE1-4 binders by ELISA. Structural and functional relation among the identified proteins suggest that domain III-mediated binding to plasma protein complexes could have a dual role for DENV2 infection in vivo: as a shield against the innate immune response but also to assist the virus in the interaction with cell surface receptors. Our studies disclose similarities and differences in the human plasma interactome of DENV serotypes and offer new data for the understanding of dengue pathogenesis.
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CIGB-814, a new therapeutic candidate for rheumatoid arthritis, from the bioinformatic design to the phase I clinical trial
2 2 1 1 1 2Prada D. , Gómez J. , Lorenzo N. , González E. , Corrales O. , Reyes Y. , Molinero 2 2 2 2 1C. , Mantecó A.M. , Torres A.M. , Hernández M.V. , Gonzalez L. , Domínguez
1M.C.
1Biomedical Research Department, Center for Genetic Engineering and Biotechnology, P.O.
2Box 6162, Havana, Cuba. Institute of Rheumatology, Havana, Cuba.
Induction of peripheral tolerance has long been considered a promising approach to the treatment of rheumatoid arthritis (RA). We aimed to evaluate the therapeutic potentialities of an altered peptide ligand (APL) derived from human heat-shock protein 60 (hHsp60) for the treatment of RA. A novel epitope of T cells located in the N terminal region of hHsp60, an autoantigen involved in the pathogenesis of autoimmune arthritis, was identified by bioinformatics tools and an APL (called CIGB-814) was designed starting from this epitope. We evaluated the therapeutic effect of this peptide in two animal models for RA (AA in Lewis rat and CIA in DBA1 mice) and in ex vivo assays using PBMC isolated from RA patients.CIGB-814 therapy reduced significantly the course of RA in both animal models and induced proliferation of regulatory T cells in ex vivo assays using PBMC from RA patients. In addition, we performed Phase I Clinical Trial with CIGB-814 in RA patients. This study was designed according three dosage levels of CIGB-814. The Schedule included 9 doses per patient in six months.Phase I Clinical Trial concluded showing safety of CIGB-814. Treatment reduced Il-17 and IFN-γ levels in patients. 17 patients achieved ACR 70, when they finished therapy. At 6 months, erosion and edema score by magnetic resonance imaging from baseline was significantly lower. These results reinforce the therapeutic possibilities of CIGB-814 as a novel candidate for treatment of RA.
Genetics and Genomics in Obesity: Fatalism or Hope?
The prevalence of overweight and obesity worldwide has more than doubled in the past 30 years. According to the World Health Organization, more than 1.9 billion worldwide adults were considered overweight in 2014. The etiology of obesity is complex and believed to be determined by action and interaction of genetic and environmental factors. Given the recent consideration of obesity as a disease, increased efforts have been directed toward the identification of strategies for prevention and treatment that can reduce the burden of this condition. Advances of genotyping methods have provided researchers with 'omics' data intended to translate bench sciences into clinical applications. However, limited success have been achieved in accounting for the estimated 50% of genetic contributions to body mass index, and no treatment strategy has been implemented based on genetic predisposition to obesity. Data from our laboratory and others supports the notion that the evaluation of gene-environment interactions provide greater explanation of phenotypic variance in obesity-related traits, promising to provide further translational insight into approaches to reduce obesity prevalence at the population level. This presentation will provide a general overview of the contributions of genetics and genomic research in obesity, will provide examples of how these findings can be applied to clinical outcomes, and will propose a framework for further research in the area of obesity.
Jose R. Fernandez
UAB Department of Nutrition Sciences, WEBB 522, 1675 University Boulevard,
Birmingham, AL 35294-3360
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Integration of transcriptomic and proteomic data to elucidate the mechanism of action of novel compounds: the case of the antitumor peptide CIGB552.
1 1 1 2Fernández Massó J.R. , Núñez de Villavicencio-Díaz T. , Ramos Y. , Oliva B. , 1 3 1 1 1 4Rodríguez A , Cruz Y , Guirola O. , Perez-Riverol Y. , González L.J. , TiscorniaI I. ,
4 4 1 5 1 1Victoria S. Bollati M. , Besada V. , Delgado L. , Palenzuela D. , Guillén I. , 1 1 2 2Vázquez-Blomquist D. , Novoa L.I. , Gomez Y. , Guerra-Vallespi M.
1 2Division of System Biology, CIGB, Havana, Cuba; Division of Pharmaceuticals, CIGB, 3Havana, Cuba; Department of Preclinical Studies, National Institute of Oncology and
4 5Radiobiology,Cuba, Cell Biology Unit, Institut Pasteur of Montevideo, Uruguay, Center of
Studies for Research and Biological Evaluations, Pharmacy and Food Sciences College,
University of Havana, 19250 Havana, Cuba.
Key words: antitumoral peptide, transcriptomics, proteomics, oxidative stress, network analysis
Motivation and Aim: CIGB-552 is a novel molecule with antineoplastic and cell-penetrating capacity in several tumor cell lines. Systemic injection of immunocompetent and nude mice with established solid tumor resulted in regression of tumor mass and apoptosis. The molecular target and the mechanism of action were unknown 1. Methods: Microarray experiments and proteomic approaches were used to identify the molecular target and the set of pathways regulated in tumor cells following treatment with the CIGB-522. Data integration was conducted using Bisogenet plugin 2. Results: We identified a set of 349 genes differentially expressed when compared treated versus untreated cells using oligonucleotide microarray. In addition the nuclear sub-proteome of HT-29 colon adenocarcinoma cells treated with CIGB-552 peptide was identified and analyzed. Pathway analysis enrichment using bioinformatic tools reveals NF-kB signaling and oxidative stress as relevant pathways in the antitumor activity of CIGB-552. In addition, using proteomic and network analysis tools COMMD1 protein was identified as a target of CIGB-552 peptide. The relevance of COMMD1 as the molecular target of the CIGB-552 was validated using shRNAi.
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Conclusion: COMMD1 protein was identified as the molecular target of the antitumoral peptide CIGB-552. NF-kB signaling and oxidative stress modulation by CIGB-552 contributes with its cytotoxic effect.
References:
1.Vallespi, M.G. et al. Identification of a novel antitumor peptide based on the screening of an
Ala-library derived from the LALF(32-51) region. Journal of peptide science : an official
publication of the European Peptide Society 16, 40-47 (2010).2.Martin, A. et al. BisoGenet: a
new tool for gene network building, visualization and analysis. BMC bioinformatics 11, 91
(2010).
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A quantitative framework of integrating multi-modal cancer genomic data
Chen-Hsiang Yeang
Institute of Statistical Science, Academia Sinica, Taipei, Taiwan
Cancer cells harbor simultaneous alterations at genomic, transcriptomic, proteomic, and epigenomic levels. To gain a comprehensive picture of cancer, multi-modal measurements have been applied to tumors. We developed a quantitative method to integrate those types of cancer molecular alteration data in the same modeling framework. The goal is to identify the statistical/causal associations that link molecular alterations on DNA (sequence mutations or variations, copy number variations, DNA methylations) to variations of gene (mRNA, microRNA and protein) expressions. The outputs are “association modules” consisting of the upstream molecular alterations (effectors), downstream gene expressions (targets), and regulators that mediate the associations from effectors to targets. We applied the method to the integrated datasets of NCI-60 cell lines and TCGA GBM and successfully identified and verified the putative causal links that have strong prognostic effects. Furthermore, we reconstructed the association modules from the lung adenocarsinoma dataset of a cohort of Taiwanese non-smoking females, and verified those modules with external datasets of East Asian and Western patients. Strikingly, the prognostic effects of the inferred modules are pronounced in East Asian or East Asian female datasets alone. The results echo ethnic specificity of molecular mechanisms in cancer and question the validity of Caucasian-dominated datasets (such as TCGA) in other ethnic groups.
Ecological Genomics: Microarray DNA sequencing and bioinformatics for massively parallel multi-species environmental studies
Carr, SM
Dept of Biology, Memorial University of Newfoundland, & Terra Nova Genomics, Inc., St John's NL, CANADA
A major goal of NextGen sequencing is to obtain the complete genome sequence of any single individual as a matter of routine. Geneticists interested in population variation face a different challenge, which is to harness this capacity to retrieve sequences from 100s ~1,000s of individuals from multiple species. Our Affymetrix microarray tiles a reference sequence of n bases as series of overlapping oligonucleotide quartets. Each quartet variant is specific to one of the four possible ACGTs at positions 1►n.The sequence is read as the succession of strongest signals from each quartet. Use of an “ArkChip” microarray with multiple reference sequences from species in different orders and classes prevents cross-hybridization, and ensures accurate recovery of species-specific data. Contig assembly and SNP identification algorithms are automated. Novel bioinformatic tools distinguish structured versus random phylogeography, and older from younger species diversity. Analysis of mtDNA genomes of species affected by Nearctic glacial cycles shows diverse patterns consistent with persistence in long-term refugia, trans-Atlantic movement, post-glacial recolonization, and (or) bottlenecks. The ArkChip technology is applicable to biomedical, agricultural, or other investigations that require parallel sequence data within and across species, e.g., typing of fungal pathogens, precautionary approach to species impacted by oil spills, or identification of origins of suspect commercial products.
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Genomic approaches to improve the treatment of tumors with interferons.
1 2 4 1 4Vázquez-Blomquist D , Miranda J , Leenstra S , Fernández JR , van der Kaaj M , 4 1 3 2 2 1Venkatesan S , Bello C , García Y , Bringas R , Fernández de Cossío J , Guillén I ,
1 2 2 5 5 5Pedroso S , Martín A , Ochagavia ME , Silva JA , Estrada R , Gell O , Palenzuela 1 1 3D , Novoa LI , Bello I .
1 2 3Pharmacogenomic Department, Bioinformatic Department, Clinical Assay Division, 5Oligonucleotide Department. Center for Genetic Engineering and Biotechnology (CIGB),
4Havana, Cuba, Department of Neurosurgery, Erasmus MC, Rotterdam, the Netherlands.
CIGB produces recombinant Interferons (IFNs) and combinations. Combinations have shown bigger impact on non-melanoma skin carcinoma than individual IFNs. Recent results in Glioblastoma Multiforme (GBM) compassionate study are encouraging. IFNs combinations have revealed higher antiproliferative effect on cervical- and GBM-derived cell lines HEp-2&U87MG. We wanted to investigate the mechanism of action of Interferons alpha, gamma and combinations on solid tumors using genomic approaches for the future improvement of treatments. By subtractive suppressive hybridization (SSH) & quantitative PCR (qPCR), we examined the mechanism of their actions alone and combined in HEp-2. In U87MG we performed qPCR and Illumina microarray studies. We tested the growth inhibitory response of 34 patient-derived GBM clones, with different molecular profiles, to three doses of an IFN combination. We found the inhibitory response was independent of TCGA molecular classification. Molecular data from untreated clones is available and RNA&DNA from treated and untreated clones was also purified for further molecular profiling. Interesting biomarkers validation by qPCR could give clues into molecular determinants of IFN combination response. The association between molecular profiles and the responses to IFNs in HEp-2, U87MG and GBM clones will help to identify mechanism-related biomarkers and biomarkers to predict the response to IFNs combination in different contexts for clinical applications.
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A population genomic health profile in Latin America
Irving K. JordanSchool of Biology at Georgia Tech and co-director of the PanAmerican Bioinformatics
Institute
Latin American populations are characterized by three-way genetic admixture with ancestry contributions from Europe, the Americas and Africa. The process of genetic admixture between these ancestral populations has resulted in the creation of Latin American genomes that can be considered to be novel in the sense that they carry combinations of ancestry-enriched alleles that had not previously existed together in the same genomic background. We are interested in understanding the implications of the creation of such evolutionarily novel, admixed Latin American genomes for human health and fitness in the Americas. To address this broad question, we are exploring the relationship between: (1) patterns of genetic admixture and ancestry in Latin American populations, and (2) genetic associations with a variety of health-related human phenotypes. Our initial analysis efforts are focused on a Colombian population, from the city of Medellin in the state of Antioquia, that has a particularly admixed population. By characterizing the genetic ancestry and admixture patterns for this population, we were able to identify genomic regions and individual SNPs that show anomalously high levels of ancestry from one of the three ancestral source populations. Interrogation of these 'ancestry enriched' regions revealed a number of associations with genetic determinants of human health. Genes of the immune system in particular appear to evolve via enrichment for specific ancestral contributions. For example, HLA (human leukocyte antigen) genes show both an excess of African ancestry and an excess of ancestry heterozygosity in the Colombian population. I will explore this case along with a number of other examples of ancestry enriched genes and SNPs that are associated with human health. Our results are consistent with an evolutionary scenario whereby specific alleles evolved separately in different ancestral human populations based on their regional-specific utility, i.e. their relationship to health and fitness, and then these pre-evolved ancestry specific-alleles were selected in modern, admixed populations based on their utility in the new environment. In this way, genetic admixture among previously isolated human populations appears to have enabled extremely rapid, adaptive evolution in the selective crucible of the New World.
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Mapping HIV Viral Spike Glycosylation: a Critical Step in Determining Neutralizing Antibody and Lectin Recognition
Weston B. StruweDepartment of Chemistry, University of Oxford, Chemistry Research Laboratory, 12
Mansfield Road, Oxford OX1 3TA, UK
An estimated 37 million people worldwide are HIV positive and efforts towards global eradication focus both on vaccine development as well parallel interventions to limit viral transmission, including physical barrier methods via anti-viral lectins. The surface of HIV is decorated by roughly twelve heavily glycosylated envelope glycoprotein (Env) spikes, comprised of gp120/gp41 proteins, which are 50% carbohydrate by mass. Crucially broadly neutralizing antibodies (bnAbs), which are elicited in up to 30% of infected individuals, target Env and the recent development of soluble Env mimics holds tremendous promise for an HIV vaccine. The abundance of Env glycans effectively shields the underlying peptides from innate immune responses but paradoxically many bnAbs target the viral spike via contact with conserved N-glycans. Lectins that have anti-viral properties have recently emerged as an alternative treatment to prevent HIV transmission, acting as microbiocides applied topically or even produced in situ by live engineered commensal microbes. Both bnAbs and microbiocide lectins (griffithsin and banlec) target the HIV “glycan shield” yet changes in Env glycosylation can shape bnAb and lectin epitope recognition. Therefore detailed glycoproteomic analysis of Env glycosylation is critical and will increase our knowledge of bNAb/lectin epitope recognition. Here we show through various mass spectrometry techniques, the glycosylation of HIV-1 clade A & B clades and also reveal the complex mechanisms by which HIV glycans are targeted.
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Protein Content of the Hylesia metabus Eggnest Setae and Its Association with defense mechanisms and lepidopterism.
1 2 1 3 1Cabrera G. , Lundberg U. , Rodríguez-Ulloa A. , Herrera M. , Machado W. , 6 4 1 1 6 1Portela M. , Palomares S. , Espinosa L.A. , Ramos Y. , Durán R. , Besada V. ,
5 1Vonasek E. , González L.J.
1
2Genetic Engineering and Biotechnology. PO. Box 6162. Havana. Cuba. Unit for Invertebrate
Toxins. Venezuelan Institute for Scientific Research. PO. Box 20632, Caracas 1020A. 3
Venezuela. Coordinación de Vigilancia Entomológica, Gerencia de Saneamiento Ambiental 4
y Control de Endemias, FUNDASALUD, Carúpano, Estado Sucre, Venezuela. Bioinformatic
Department. Center for Genetic Engineering and Biotechnology. PO. Box 6162. Havana. 5
Cuba. Proteomics Unit. Center of Structural Biology. Venezuelan Institute for Scientific 6
Research. PO Box 20632, Caracas 1020A. Venezuela. IIBCE y Unidad de Bioquímica y
Proteómica Analíticas. Institute Pasteur de Montevideo. Mataojo 2020. Montevideo.
Uruguay.
Hylesia metabus is a neotropical moth possessing toxic bristle-like hairs known as setae, which once in contact with the skin causes a severe dermatitis known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg hatchings stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28-45 kDa) showing sequence homology to other S1A serine proteases. The evaluation of a protease in an animal model of lepidopterism demonstrates that proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. A chitinase and a 30kDa lipoprotein with a glucan affinity are probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins by the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, able to recognize pathogen-associated patterns, suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal
Mass Spectrometry Laboratory and GlycoLab. Department of Proteomics. Center for
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reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.
Structural characterization and biological implications of sulfated N-glycans in serine proteases from the neotropical moth Hylesia metabus (Cramer [1775]) (Lepidoptera: Saturniidae)
1 2 5 6 7 3Cabrera G. , Salazar V. , Montesino R. , Tambara Y. , Struwe W.B. , Leon E. , 8 9 3 9 3 10Harvey D.J. , Lesur A. , Rincón M. , Domon B. , Méndez M. , Portela M. ,
1 1 11 4 3González-Hernández A. , Triguero A. , Durán R. , Lundberg U. , Vonasek E. , 1,González L.J.
1
2 36162, Havana, Cuba, Centro de Biofísica y Bioquímica, Proteomics Unit, Center of
4Structural Biology, Unit for Invertebrate Toxins, Venezuelan Institute for Scientific Research
5(IVIC), PO Box 20632, Caracas 1020A, Venezuela, School of Biological Sciences,
Universidad de Concepción, Víctor Lamas 1290, PO Box 160C, Concepción, Chile, 6Department of Proteomics, Center for Genetic Engineering and Biotechnology, PO Box
76162, Havana, Cuba, Department of Chemistry, University of Oxford, Chemistry Research
8Laboratory, 12 Mansfield Road, Oxford OX1 3TA, UK, Glycobiology Institute,Department of
9Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK, Luxembourg
Clinical Proteomics Center, 1A-B, rue Thomas Edison, L-1445 Strassen, Luxembourg, 10
Unidad de Bioquímica y Proteómica Analíticas, Institut Pasteur de Montevideo, Mataojo 11
2020, Montevideo, Uruguay, Unidad de Bioquímica y Proteómica Analíticas, Institut
Pasteur de Montevideo and IIBCE, Mataojo 2020, Montevideo, Uruguay,
Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with
18PNGase F in the presence of a buffer prepared with 40% H O allowed the 2
assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of whichwere exclusively α(1–6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1–6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological
Department of Carbohydrates, Center for Genetic Engineering and Biotechnology, PO Box
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reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.
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A glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells
1,2 3 4 4 4 1,4Sheta R. , Woo C. , Roux-Dalvai F. , Fournier F. , Bourassa S. , Droit A. , Bertozzi 3,5 1,2C. , Bachvarov D.
1 2Department of Molecular Medicine, Laval University, Québec PQ, Canada Centre de 3recherche du CHU de Québec, L'Hôtel-Dieu de Québec, Québec PQ, Canada. Department
4of Chemistry, Stanford University, Stanford, CA, USA. Centre de recherche du CHU de 5Québec, CHUL, Québec PQ, Canada. Howard Hughes Medical Institute, Stanford
University, Stanford, CA USA.
We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in advanced EOC. Our data also suggested for a role of GALNT3 in aberrant EOC O-glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we applied a metabolic labeling approach using the azido-sugar Ac4GalNAz, followed by affinity-purification with a bio-orthogonal chemical probe and consecutive mass spectrometry-based characterization of glycoproteins. This approach led to the identification of large number (589) of glycoproteins, found to be differentially regulated (≥ 2 fold, p-value ≤ 0.05) between control and GALNT3 knockdown (KD) A2780s EOC cells. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. Our glycoproteomics data are indicative of a potential role of GALNT3 in modulating post-translational modifications and metabolism pathways in EOC cells, which could significantly impact disease development. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach may be pursued as novel EOC biomarkers and/or therapeutic targets.
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Targeted LC-MS/MS quantification of glycopepides
1,2 1 2 1Goldman R. , Benicky J. , Edwards N.J. , Sanda N.
1 2Department of Oncology, Department of Biochemistry and Molecular & Cell Biology Georgetown University, Washington, DC, [email protected]
Objective: Our objective is to describe the selection of appropriate workflows adjusted for the quantification of glycopeptides. We discuss LC-MS/MS-MRM and MS3 methods as well as data independent (DIA) mass spectrometric workflows. Methods: Glycopeptide fragmentation was optimized for maximum recovery of informative ions using isolated glycoprotein standards. Proteins were reduced, alkylated, and digested with appropriate proteases under standard conditions. Protein digests were separated by C18 reversed phase chromatography and analyzed on a 6500 QTRAP or 5600 TripleToF mass analyzers (Sciex). Conditions for the CID fragmentation of glycopeptides and the quantitative workflows were adjusted to maximize sensitivity and specificity of detection. For further analyses, human serum was diluted in ammonium bicarbonate buffer for quantification of the protein glycoforms in complex background. Results: Optimized fragmentation conditions specific to different classes of glycoconjugates generate informative B- and Y- ions in high yield in addition to the peptide backbone fragments. Analyte-specific selection of the appropriate ions and workflows allows reliable quantification which substantially improves coverage of the glycoforms, including low abundant glycoforms. The optimized workflows allow simultaneous quantification of approximately 50 glycoforms derived from multiple proteins directly in the complex background without glycopeptide enrichment.Conclusion: Targeted quantification of glycopeptides by LC-MS/MS is feasible under optimized CID conditions, including DIA quantification in complex samples. Analyte-specific adjustments maximize the sensitivity and specificity of quantification.
“Candidatus Liberibacter asiaticu”', Causal Agent of Citrus Huanglongbing, Is Reduced by Treatment with Brassinosteroids Eduardo Canales Department of Plant Science, Center for Genetic Engineering and Biotechnology, Havana,
Cuba
Huanglongbing (HLB) constitutes the most destructive disease of citrus worldwide, yet no established efficient management measures exist for it. Brassinosteroids, a family of plant steroidal compounds, are essential for plant growth, development and stress tolerance. As a possible control strategy for HLB, epibrassinolide was applied to as a foliar spray to citrus plants infected with the causal agent of HLB, 'Candidatus Liberibacter asiaticus'. The bacterial titers were reduced after treatment with epibrassinolide under both greenhouse and field conditions but were stronger in the greenhouse. Known defense genes were induced inleaves by epibrassinolide. With the SuperSAGE technology combined with next generation sequencing, induction of genes known to be associated with defense response to bacteria and hormone transduction pathways were identified. The results demonstrate that epibrassinolide may provide a useful tool for the management of HLB.
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Activity in vitro and in vivo of a novel growth hormone secretagogue A233.Studies in fish and mammals
1 2 1 1 1 4 Martínez R. ,Gil L. ,Ubieta K. , Herrera F. , Morales A. Ramos Y. , Gonzalez 4 4 1 1 1 4L. ,Garay H. , Oliva A. , Rodriguez E , Estrada M.P ., Besada V.
1 2 Biotecnology Animal División. CIGB PO Box 6162. Cuba. Vaccine Division . CIGB PO Box 36162. Habana. Cuba. Department of Systems Biology, Center for Genetic Engineering and
Biotechnology; Havana, Cuba
Growht hormone(GH) synthetic secretagogues (GHS) are a family of compounds that includes non-peptide and peptide molecules, able to enhance GH releasing in vitro and in vivo. GHS bind to a receptor inducing calcium mobilization, a GHS receptor (GHSR). We inform here a peptide structure designed by molecular modeling ( the decapeptide A233 ) able to function as a GHS in teleosts. Their biological activity in vivo and in vitro was evaluated on growth and immune system of fish larvae. It was demonstrated their capacity to GH releasing in tilapia pituitary gland cell culture and enhance the superoxide anion production, which is related to phagocytic activity in head kidney leucocytes . The in vivo biological action of the peptides was also demonstrated for growth stimulation in fish larvae .Superoxide dismutase levels, antiprotease activity, and lectin titer was enhanced in tilapia larvae treated with this molecule. A comparative proteomic analysis was performed using a mammalian macrophage cell line J774.2 with peptide stimulation. In mice it was evidenced interferon gamma releasing and ability to decrease viral load, employing to the molecule in an experiment of challenge under controlled conditions with a viral pathogen, due to its immunomodulatory action.
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35
Universal and Digital NGS Solutions: From Sample to Insight
Jacobo Zuñiga Castillo PhD,
Regional Marketing Manager Mexico, Central América and Caribbean, QIAGEN México
Next Generation Sequencing (NGS) has become a powerful tool to get insight of different kind of samples with limited amount of it (FFPE, Microbiome, Liquid Biopsy, Single Cell and more). QIAGEN as the leader company for molecular biology solutions has launched the QIAseq portfolio which enables to every customer of different segments to get every detail of either DNA or RNA samples. In addition to, the bioinformatics knowledge of CLCbio Workbench and Ingenuity IPA report complete the customer needs for a better understating of genomic projects.
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Metabolomics: From Single Cell Analysis to Molecular Tissue Imaging
Akos Vertes
Department of Chemistry, The George Washington University, Washington, DC 20052, USA
Functioning biological systems continuously produce, consume, import, and export small metabolites and lipids to maintain homeostasis. There is growing interest in new metabolomic methods to rapidly characterize thousands of these species in tissues, cells and subcellular structures. Spatial distributions of metabolites and lipids in biological tissues have been studied by matrix-assisted laser desorption ionization mass spectrometry and secondary ion mass spectrometry. Despite the success of these techniques characterized by high mass capabilities and excellent spatial resolution, respectively, new ionization sources are being sought to provide better quantitation and fewer spectral interferences in the low mass region. Laser ablation electrospray ionization (LAESI) in combination with mass spectrometry (MS) enables the two- and three-dimensional molecular imaging of tissues and bacterial colonies under ambient conditions. Thin tissue sections and subcellular structures (e.g., lamellipodia) on silicon nanopost array (NAPA) platforms can be analyzed and imaged by laser desorption ionization MS. In this presentation, selected applications of these emerging high-throughput techniques will be discussed. They include following changes in the energy storage pathways of green algae due to genetic modifications, three-dimensional molecular imaging of microbial colonies by LAESI-MS to accelerate antibiotic susceptibility testing, imaging of lipid distributions in mouse brain and kidney tissue sections by NAPA-MS, and analyzing metabolic changes in hepatocytes during the stages of mitosis in the cell cycle using capillary microsampling electrospray ionization MS.
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Mathematical models of the impact of IL2 modulation therapies on T cell dynamics.
García-Martínez K., León K.
Molecular System Biology, Center of Molecular Immunology (CIM), Havana 11600, Cuba
Several reports in the literature have drawn a complex picture of the effect of treatments aiming to modulate IL2 activity in vivo. They seem to promote either immunity or tolerance, probably depending on the specific context, dose, and timing of their application. Such complexity might derive from the pleiotropic role of IL2 in T cell dynamics. To theoretically address the latter possibility, our group has developed several mathematical models for Helper, Regulatory, and Memory T cell population dynamics, which account for most well-known facts concerning their relationship with IL2. We have simulated the effect of several types of therapies, including the injection of: IL2; antibodies anti-IL2; IL2/anti-IL2 immune-complexes; and mutant variants of IL2. We studied the qualitative and quantitative conditions of dose and timing for these treatments which allow them to enhance either immunity or tolerance. Our results provide reasonable explanations for the existent pre-clinical and clinical data, predict some novel treatments, and further provide interesting practical guidelines to optimize the future application of these types of treatments.
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Dissection of programmed and externally instigated ageing in a fibroblast model
1 2 2 1,3,4Kural K.C. , Tandon N. , Kel-Margoulis O.V. , Baranova A.
1 2School of Systems Biology, George Mason University, Fairfax, VA USA. geneXplain, 3Wolfenbüttel Germany. Federal State Budgetary Institution "Research Centre for Medical
4Genetics," Moscow, Russia. ATLAS Biomed Group, Moscow, Russia;
Key words: senescence, ageing, expression landscapes
In cultured normal diploid cells, senescence may either happen naturally, in the form of replicative senescence, or it may be a consequence of external challenges such as oxidative stress. Here we present a comparative analysis aimed at reconstruction of molecular cascades specific for replicative (RS) and stress-induced senescence (SIPS) in human fibroblasts. An involvement of caspase-3/keratin-18 pathway and serine/threonine kinase Aurora A/ MDM2 pathway was shared between RS and SIPS. Moreover, stromelysin/MMP3 and N-acetylglucosaminyltransferase enzyme MGAT1, which initiates the synthesis of hybrid and complex N-glycans, were identified as key orchestrating components in RS and SIPS, respectively. In RS only, Aurora-B driven cell cycle signaling was deregulated in concert with the suppression of anabolic branches of the fatty acids and estrogen metabolism. In SIPS, Aurora-B signaling is deprioritized, and the synthetic branches of cholesterol metabolism are upregulated, rather than downregulated. Moreover, in SIPS, proteasome/ubiquitin ligase pathways of protein degradation dominate the regulatory landscape. This picture indicates that SIPS proceeds in cells that are actively fighting stress which facilitates premature senescence while failing to completely activate the orderly program of RS. The promoters of genes differentially expressed in either RS or SIPS are unusually enriched by the binding sites for homeobox family proteins, with particular emphasis on HMX1, IRX2, HDX and HOXC13. Additionally, we identified Iroquois Homeobox 2 (IRX2) as a master regulator for the secretion of SPP1-encoded osteopontin, a stromal driver for tumor growth that
39
is overexpressed by both RS and SIPS fibroblasts. The latter supports the hypothesis that senescence-specific de-repression of SPP1 aids in SIPS-dependent stromal activation.
Analysis of nucleotide diversity to identify functionally important
regions
Tatiana V. Tatarinova
University of Southern California, Center for Personalized Medicine, Children's Hospital
Los Angeles, Los Angeles, CA, USA
We analyzed genome-wide distributions of SNPs in rice, medicago, Arabidopsis, human, and other species. In this work we demonstrate existence of pronounced SNP density trends distinguishing various genomic regions. We also show that these trends are conserved between species and can be used to assess quality of genome annotation and identify positions of functionally important genomic regions. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions.In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3′ UTRs, the area rich with regulatory regions.
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Gen-01 Pharmacogenomics study of the effect of Dextran Sulfate Sodium in generating a biomodel of rats with acute ulcerative colitis.Guillén I.A., Camacho H., Roca J., Aguilera A., Palenzuela D.O., Silva J.A., Estrada R., Gell O., Novoa, L.I.
Gen-02 Pharmacogenomic study of the effect of Pellet-EGF in the colon of rat biomodel with ulcerative colitis.Camacho C., Guillén I.A., Roca J., Aguilera A., Palenzuela D.O., Novoa L.I.
Gen-03 Differential gene expression in mouse animal model of EAE treated with phycocyanobilin (PCB) by oral route.Camacho H., Piniella B., Cervantes M., Villareal A., Fernández-De Cossio M.E., Cintado A., Ale M., Fernández J.R., Palenzuela D.O., Pentón G.
Gen-04 Identification of predictive genetic markers for slow atorvastatin metabolizers in Mexican populationHerrera-González S., Martínez-Treviño D.A., Moreno-Treviño M.G., Aguirre-Garza M., León-Cachón R.B.R.
Gen-05 HLA DQ and DRB1 in Cuban patients with Multiple SclerosisFernández-de-Cossío M.E., Cintado A., Nazabal M., Camacho H., Díaz T., Villarreal A., Ale M., Grass D., Cervantes-Llanos M., Pavon-Fuentes N., Benitez J.V.1 Cabrera-Gomez J.A., Diaz de la Fe A., Pentón-Rol G.
Scientific program Poster Presentations
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Gen-07 De novo transcriptome assembly and gene annotation of the spotted rose snapper (Lutjanus guttatus)Escalante-Rojas M., García-Gasca A., Llera-Herrera R.
Gen-08 Deep transcriptome sequencing and ex vivo assays to study muscle growth in marine fish (Lutjanus guttatus)Torres-Velarde J., Escalante-Rojas M., Llera-Herrera R., Sifuentes-Romero I., García-Gasca A.
Prot-01 Protein fractionation by SDS-PAGE for detection by SRM of administered proteins at low concentrationQuiñones M., Ramos Y., Carpio J., Besada V., González L.J.
Prot-02 Approaches for the analysis of N-glycosylation sites on recombinant proteinsEspinosa L.A., Musacchio A., Bolaños A., Maxwell M., Rodríguez Y., Besada V.
Prot-03 Microbiome characterization of escamoles: a novel source of
microorganisms with biotechnological potentialGonzalez-Escobar J., Grajales-Lagunes A., Barba de la Rosa A.P.
Prot-04 Analysis of Fab and Fc N-glycans profile from human immunoglobulin G isolate from patients with rheumatoid arthritis.González-Hernandez A., Pousa S., Cabrera G.
Prot-05 Effect of silencing cmah and adaptation to serum free medium in the glycan profile of 14F7 MAb produced by NS0 cell line.Morales O., Rabasa Y., Dorvignit D., R. de La Luz Hernández K.
Prot-06 Mass spectrometry based methods for pharmacokinetic studies of three therapeutic peptide in phase I clinical trialsCabrales A., Ramos Y., Gil J., Garay H.E., Gómez J.A., Audain E., Berlanga J., Perera Y., Perea S.E., Domínguez M.C., Besada V., and González L.J.
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Prot-07 Characterization of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh7.5 cellsYero A., Ramos Y., Pupo D., Martín D., Márquez G., Martín A., Sarría M., Gallien S., Domon B., Chinea G., Huerta V.
Prot-08 Inter alpha Inhibitor complex components mediating the binding to the DIII of the Envelope protein of Dengue VirusMartín D., Ramos Y., Martín A., Sarría M., Chinea G., Huerta, V.
Prot-09 Synthetic peptides based on the domain III inhibit Dengue virus infectionPupo D., Fleitas N., Huerta V., Garay H., Chinea G.
Prot-10 Mining B23/NPM1 human genomic data from publicly available repositories: Is this protein a suitable cancer target beyond Acute myeloid leukemiaPerera Y., Fernandez-de-Cossío J., Perea S.E.
Prot-11 A comparative molecular dynamics study of thermophilic and mesophilic β-fructosidase enzymes.Musacchio A., Mazola Y., Guirola O., Palomares S., Chinea G., Menéndez C., Hernández L.
Prot-12 Potential inhibitory effects of the action of the CIGB 300 on substrates of CK2 identified in human plasmaPalomares S., Hardy A., Quiñones M., Ramos Y., Perera Y., Besada V., Guirola O., Perea S.E.
Prot-13 A web based application to manage Mass Spectrometry experiments.Vázquez Y., Guirola O., Palomares S., Espinoza L.A., Besada V.
Prot-14 CmPI-II, a non-classical Kazal inhibitor with exceptional characteristics : Structural characterization by NMRCabrera-Muñoz A., Rojas L., Alonso-del-Rivero M., Pires J.R.
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Prot-15 New family of β-defensin-like peptides on invertebratesMontero-Alejo V., Corzo C., Perera E., Rodríguez-Viera L., Sánchez-Díaz G., Hernández-Rodríguez E.W., Álvarez C., Perdomo-Morales R.
Prot-16 The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argusPerdomo-Morales R., Montero-Alejo V., Corzo G., Besada V., Vega-Hurtado Y., González-González Y., Perera E., and Porto-Verdecia M.
Prot-17 Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in the Spiny Lobster Panulirus argusRodríguez-Viera L., Perera E., Martos-Sitcha J.A., Perdomo-Morales R., Casuso A., Montero-Alejo V., García-Galano T., Martínez-Rodríguez G., Mancera J.M.
Prot-18 Proteomic analysis reveals the molecular basis on CIGB845 pharmacological interventionSubirós N., Palomares S., Quiñonez M., Pérez-Saad H., Ramos, Y., Guirola, O., González L.J., Besada V., García del Barco D.
Bio-01 In silico analysis of GMB and AML transcriptomesMiranda J., Bringas R.
Bio-02 Impact of ignoring germline susceptibility variants by restricting the analysis to somatic mutation data in cancer genetic studyFernandez-de-Cossio J., Perera Y.
Bio-03 QSAR and molecular docking studies. A rational approach to drug designÁlvarez-Ginarte Y.M.
Bio-01 In silico analysis of GMB and AML transcriptomesMiranda J., Bringas R.
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Bio-02 Impact of ignoring germline susceptibility variants by restricting the analysis to somatic mutation data in cancer genetic studyFernandez-de-Cossio J., Perera Y.
Bio-03 QSAR and molecular docking studies. A rational approach to drug designÁlvarez-Ginarte Y.M.
SB-01 BPb: Fast inference of ill-posed problems within a convex space. Applications in metabolic networksFernández-de-Cossío-Díaz J., Mulet R.
SB-02 In silico prediction of the Transforming Growth Factor beta and Activin Receptor-like Kinase 3 complexFernández-de-Cossío-Díaz J., Corría J., León K.
SB-03 Common gamma chain: relevant for the IL2-IL2R affinity? A mathematical approachPonce L.F., García-Martínez K., León K.
Gen-01
Pharmacogenomics study of the effect of Dextran Sulfate Sodium in generating a biomodel of rats with acute ulcerative colitis.
Guillén I.A., Camacho H., Roca J., Aguilera A., Palenzuela D.O., Silva J.A., Estrada R., Gell O., Novoa, L.I.
Pharmacogenomics group. Center for Genetic Engineering and Biotechnology CIGB. La Habana. Cuba.
Introduction: Animal models of ulcerative colitis are valuable because they provide information on the pathogenesis of the disease and its collateral effects in other organs and tissues. The most widely used chemical, which reproduces better the symptoms of the disease in humans, is dextran sulfate sodium (DSS). DSS produces damage to the intestinal epithelium, causing an immune response that promotes an inflammatory process in the colon and damage. We proposed to know the changes in the gene expression profile that help to justify the process of acute colitis occurring in the colon of rats treated for 5 days with 8% of DSS. Materials and methods: The workflow consisted of biopsy collection from distal colon, total RNA extraction and analysis of gene expression by qPCR of 28 genes related to inflammation, epithelial damage and oxidative stress. Results: 43% of the genes varied its expression, 6 genes was upregulates and 6 genes downregulate. The downregulates genes are involved in the defense mechanisms of the epithelial barrier, differentiation of goblet cells and epithelial renovation. The upregulates genes are involved in inflammation mechanisms. Conclusions: These results support the use of this animal model in the development of new drugs against ulcerative colitis.
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AbstractsPoster Presentations
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Gen-02
Pharmacogenomic study of the effect of Pellet-EGF in the colon of rat biomodel with ulcerative colitis.
Camacho H., Guillén I.A., Roca J., Aguilera A., Palenzuela D.O., Novoa L.I.
Pharmacogenomics group. Centre of Genetic Engineering and Biotechnology. CIGB. La
Havana, Cuba.
Introduction: Ulcerative colitis (UC) is an inflammatory bowel disease, causing inflammation and ulcers in intestinal epithelium. The diagnosis of the disease and the possible prediction of the development of colon cancer from prolonged illness are still unclear. We use an animal model of colitis based on the use of dextran sodium sulfate (DSS) to evaluate the effect of the application of EGF-Pellet in the expression of genes involved in inflammation, oxidative stress and restoration of the intestinal epithelium.Materials and methods: The gene expression analysis was conducted with distal colon biopsies of male Wistar rats with UC. For this, 5 animals were treated with Pellet-placebo for 7 days and 8 animals were treated with Pellet-EGF for 7 days. Total RNA extraction was performed using the QIAcube platform. Quality control of RNA was performed with the Nanodrop and Bioanalyzer. The RNA was employed in quantitative real time polymerase chain reaction (qPCR) to measure the differential expression of 36 genes.Results: A positive effect to treatment with Pellet-EGF was observed by decreased expression of proinflammatory cytokines such as IL1, IL6 and TNF-alpha. Other genes such as INOS, GPX1 and SOD2, related with oxidative stress, also decrease its expression levels. Furthermore, an increase of expression of genes involved in restoring the intestinal epithelium (MUC2, KLF and TFF3) was observed.Conclusions: Pellet-EGF promotes differential gene expression that may promote inhibition of inflammation, oxidative stress and damage to epithelial tissue.
48
Gen-03
Differential gene expression in mouse animal model of EAE treated with phycocyanobilin (PCB) by oral route.
Camacho H., Piniella B., Cervantes M., Villareal A., Fernández-De Cossio M.E., Cintado A., Ale M., Fernández J.R., Palenzuela D.O., Pentón G.
Biomedical research group. Centre of Genetic Engineering and Biotechnology. CIGB. La Havana, Cuba.
Multiple sclerosis (MS) is an autoimmune, inflammatory and demyelinating disease of the central nervous system. There are few drugs targeting axonal and myelin loss, which are prominent pathological features of MS, from those approved by FDA. Phycocyanobilin (PCB) is a tetrapirrolic ring from C-Phycocyanin with different pharmacological properties reported. We evaluated the effect of PCB on the modulation of demyelinating/remyelinating genes in Experimental Autoimmune Encephalomyelitis (EAE), animal model of MS. Materials and methods: C57BL6 EAE mice treated with three doses (0.2, 1and 5 mg/kg/day) of PCB by oral route were assessed. Brains were collected into RNAlater® and stored at -20°C. Total RNA extraction was performed using the Quiacube platform. The RNA quality controls were conducted with Nanodrop and Bioanalyser instruments. The differential expression of genes CXCL12, LINGO, MAL, NOTCH1, FOXP3, IL6, CYBA, CYBB, HMOX1, NCF1, NQO1 and NRF2.were evaluated by Real-time-quantitative-PCR (qPCR).Results: The CXCL12, FOXP3, IL6, LINGO, MAL, NCF1 and NOTCH1, genes changed expression levels, compared to animals with EAE, regardless of the dose of PCB used. The CYBA and NQO1 genes were expressed similarly in animals with EAE. The CYBB, HMOX1 and NRF2 genes changed their expression, depending on the PCB dose used.Conclusions: Our results show the effectiveness of treatment with PCB by oral route in modulating demyelinating, remyelinating, immunoregulatory and oxidative stress gene expression.
Gen-04
Identification of predictive genetic markers for slow atorvastatin metabolizers in Mexican population
Herrera-González S., Martínez-Treviño D.A., Moreno-Treviño M.G., Aguirre-Garza M., León-Cachón R.B.R.
Centro de Diagnóstico Molecular y Medicina Personalizada, Departamento de Ciencias Básicas, División Ciencias de la Salud, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México
The therapeutic response to atorvastatin (ATV) treatment is variable, mainly due to polymorphisms located in genes that encode for enzymes involved in metabolism and transport. These polymorphisms can also affect drug therapeutic target proteins. In our previous study using microarrays, we found polymorphisms in ABCG2 (rs2231142), AGT (rs699), AGTR1 (rs5186) and BDKBRB2 (rs1799722) genes that tend to be associated with metabolizer phenotypes. Our objective was to re-evaluate the effect of polymorphisms on the ATV pharmacokinetic parameters and the association of these polymorphisms with metabolizer phenotypes. Sixty healthy male volunteers were re-genotyped using real-time PCR and TaqMan® probes. Statistical analysis was done by Student's t, Mann Whitney U, ANOVA, Kruskal-Wallis H, Chi-square and Fisher´s exact tests. Two polymorphisms were found to have a significant effect on ATV pharmacokinetics: AGTR1 (rs5186) and BDKBRB2 (rs1799722). However, only BDKBRB2-rs1799722 was associated with a slow metabolizer phenotype. To our knowledge, this study is the first reporting an effect of AGTR1-rs5186 and BDKBRB2-rs1799722 polymorphisms on ATV pharmacokinetics. Further studies using a genetic screening method and a larger population are needed to confirm these polymorphisms as predictive biomarkers for ATV slow metabolizers.
49
Gen-05
HLA DQ and DRB1 association with Multiple Sclerosis in a sample of the admixed Cuban population
1 1 1 1 1 Fernández-de-Cossío M.E. , Cintado A. , Nazabal M. , Camacho H. , Díaz T. ,1 1 2 1 2Villarreal A. , Ale M. , Grass D. , Cervantes-Llanos M. , Pavon-Fuentes N. , Benitez
1 2 2 1J.V. , Cabrera-Gomez J.A. , Diaz de la Fe A. , Pentón-Rol G.
1Pharmacogenomics Department. Biomedical Research Division. Center for Genetic 2Engineering and Biotechnology, 31 entre 158 & 190, Cubanacán, Playa, Cuba. International
Center of Neurological Restoration. Ave 25 no 15805 entre 158 & 160 Cubanacan Playa.
Multiple sclerosis (MS) is a neuro-inflammatory autoimmune disease. The MHC class II region produces the strongest effect on MS genetic susceptibility. The purpose of this study is to evaluate DRB1*, DQA1*, and DQB1* alleles among MS Cuban patients. Disease–HLA associations were controlled by population stratification. 100 MS patients and 200 unrelated healthy controls (matched by age, gender, and ethnicity) were included. Ancestry informative markers were used. MS associated HLA susceptibility/protection alleles were ascertained by PCR using specific primers. Statistical analyses were conducted using STRUCTURE 2.1, ADMIXMAP 3.7, SPSS 16.0 packages. Logistic regression analysis showed odds ratio (OR) of 5.7 (95% CI 1.2–36) for MS associated with a unit change in European admixture proportion. Evidence for susceptibility to MS was observed for the presence of HLA-DRB1*15, DRB1*14, DQA1*01 and DQB1*06 alleles, with OR 3.40, 4.96, 2.53, and 2.77 respectively compared to healthy controls. A protective effect of HLA DRB1*01, *07, *10 was found among MS patients (OR 0.35, 0.2, 0.4 respectively). After correcting for admixture, a new association to HLA-DRB1*1101 was identified. Conclusion: Alleles HLA-DRB1*, DQ were associated with MS in Cuban patients. HLA-DRB1*1101-1104 was associated to MS protection in this study sample, where the European ancestry proportion was identified.
50
51
Gen-06
Myostatin regulates cardiac beat rate in zebrafish embryos
1 2 3Lizárraga-Lizárraga D. , Martínez-Torres A. , Llera-Herrera R. , 1García-Gasca A.
1Laboratorio de Biología Molecular, Centro de Investigación en Alimentación y Desarrollo
2(CIAD). Mazatlán, Mexico. Departamento de Neurobiología Celular y Molecular, Laboratorio de Neurobiología Molecular y Celular, Instituto de Neurobiología, Universidad Nacional
3Autónoma de México. Querétaro, Mexico. CONACYT research fellow - Centro de Investigación en Alimentación y Desarrollo (CIAD). Mazatlán, Mexico.
Myostatin is a negative regulator of skeletal muscle growth. In zebrafish, mstn-1 is highly expressed in skeletal and cardiac muscle, however, its function in the latter is not known. In this study, the CRISPR/Cas9 system was used to inactivate mstn-1 in zebrafish. Microinjection was performed in zygotes. Genomic DNA from F0 offspring was analyzed. The region containing the target sequence was amplified, and nested PCR reactions were performed using a set of four different M13 forward primers. Final PCR reactions were carried out using IonXpress primers containing different barcode sequences. The barcode libraries were sequenced using a 316 chip and the Hi-Q sequencing chemistry on the Ion Torrent PGM. The sequencing reads were retrieved as FASTQ and demultiplexed based on the combinatorial barcodes in both IonXpress and M13 primers used in the third and second PCR, respectively. A strict quality-filter in any position was then applied, obtaining an average of 341 clean reads per individual. Sequences from each individual were transformed to FASTA, aligned and compared to the wild type sequence. From the 27 mutant fish, a total of 9024 sequences were analysed, with an average of 334 sequences per individual. From these sequences, a high degree of mosaicism was evident, since one individual showed several genotypes. Mutations were classified into different types, depending on the number of inserted or deleted nucleotides. Mutant fish presented the expected phenotype of skeletal muscle growth. Interestingly, the inactivation of the gene produced a significant decrease in the cardiac rate, indicating that mstn-1 plays a role in development and function of the cardiac muscle.
Espino-Saldaña 2A.E. ,
52
Gen-07
De novo transcriptome assembly and gene annotation of the spotted rose snapper (Lutjanus guttatus)
1 1 1,2Escalante-Rojas M. , García-Gasca A. , Llera-Herrera R.
1Laboratory of Molecular Biology and Tissue Culture of Aquatic Organisms, Centro de 2Investigación en Alimentación y Desarrollo (CIAD), Mazatlán, Sinaloa, Mexico. CONACYT
research fellow - Centro de Investigación en Alimentación y Desarrollo (CIAD). Mazatlán, Mexico.
Fish farming is an important activity in Mexico. The spotted rose snapper (Lutjanus guttatus) is one of the species of interest because it has proved to be amenable for aquaculture in terms of reproduction and development; nevertheless, efforts are carried out to increase the growth rate. Nowadays, genomic information is necessary to understand gene function and to elucidate and manipulate signaling pathways in order to improve production. Since genomic and transcriptomic information was not available for this species, the aim of this study was to generate a comprehensive transcriptome for the future development of technologies and tools to boost production. To achieve this, cDNA barcoded libraries were sequenced from 11 different organs (spleen, gills, brain, heart, testis, liver, gut, muscle, white skin, dark skin, and visceral fat) from a single fish maintained under rearing conditions and fed with a fish meal and a fish oil-based diet. Sequencing was performed in the Illumina HiSeq2000 platform, yielding a total of 222856278 paired end raw reads. From them, 207493630 reads were de novo assembled with Trinity RNA-Seq Assembler. A total of 267601 contigs were assembled with an average contig length of 845 base pairs. From the assembled contigs, 85677 open reading frames were identified and annotated using the Trinotate pipeline. Homology search with BLASTp algorithm against the SwissProt database resulted in 129072 hits. Also, an expression analysis was done for each library, being the brain the most complex of all the tissues. These results provide important information for further studies in this and other related species.
53
Gen-08
Deep transcriptome sequencing and ex vivo assays to study muscle growth in marine fish (Lutjanus guttatus)
1 1 1,2 1Torres-Velarde J. , Escalante-Rojas M. , Llera-Herrera R. , Sifuentes-Romero I. , 1García-Gasca A.
1Laboratory of Molecular Biology and Tissue Culture of Aquatic Organisms, Centro de 2Investigación en Alimentación y Desarrollo (CIAD), Mazatlán, Sinaloa, Mexico. CONACYT
research fellow - Centro de Investigación en Alimentación y Desarrollo (CIAD). Mazatlán, Mexico.
The spotted rose snapper, Lutjanus guttatus, is considered an important commercial species in the Pacific coast of Mexico and Central America. In adult fish, the skeletal muscle covers over 60% of the total body and is used for human consumption. Understanding the mechanisms governing muscle growth will allow the development of technologies aimed to improve growth in captivity. Using Illumina HiSeq2000 as a sequencing platform, we assembled the skeletal muscle transcriptome of L. guttatus and identified important genes involved in proliferation, differentiation and atrophy of muscle cells. Different paralogues were identified for MyoD, Mrf4, Foxo1, Foxo3a, Murf1 and myostatin. Due to the importance of myostatin as a negative regulator of myocyte proliferation and differentiation, we selected this gene to conduct RNAi-silencing experiments, first using an immortal cell line (NHI-3T3-L1) expressing myostatin from L. guttatus, and then an ex vivo system consisting of fish skeletal muscle tissue; in this system, tissue integrity is conserved, cells express endogenous myostatin and/or myogenic factors for several days, and gene silencing by RNAi can be performed by simple transfection. We tested two blocking strategies, dsiRNA (dicer substrate RNAi) and shRNA (small hairpin RNA). So far, our results (although preliminary) are promising. The tissue culture system proved to be a good approach to perform short duration gene silencing assays in fish muscle. Our strategy (deep sequencing plus functional ex vivo experiments) could be especially useful when working with marine fish, since no genomic information is available for most species, and in vivo assays could be expensive due to fish size and availability.
54
Prot-01
Protein fractionation by SDS-PAGE for detection by SRM of administered proteins at low concentration.
Quiñones M., Ramos Y., Carpio J., Besada V., González L.J.
1Proteomics Group, Department of Systems Biology, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
The identification of low abundant proteins is one of the most important challenges of current proteomic. The approaches developed to mitigate this limitation are based on the combination of protein fractionation methods in order to decrease the sample complexity. Because of its extraordinary resolving power, gel electrophoresis has been used successfully in these strategies. However, the protein recovery from a gel and the sample processing substantially compromises the ability to link this technique with downstream mass spectrometry analysis. In this work we established the protein fractionation using a new gel electrophoretic system coupled to electro-elution that allows on-line in-solution collection of separated proteins. This technic was combined with mass spectrometric analysis via Selective Reaction Monitoring for the detection of the antitumor therapeutic candidate CIGB-370. Proteotypic peptides and their transitions were experimentally stablished for this protein. Using this strategy, we successfully detected CIGB-370 at concentrations up to 0.0012 mg / mL in human plasma.
Prot-02
Approaches for the analysis of N-glycosylation sites on recombinant proteins
1 2 3 1 1 1Espinosa L.A. , Musacchio A. , Bolaños A. , Maxwell M. , Rodríguez Y. , Besada V.
1 2Proteomics Group, Chrystalography and Modelling Group. Department of Systems Biology, 3Physico-Chemistry Department. Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Protein glycosylation is one of the most common and important pos-translational modifications (PTMs) in eukaryotic cells and the attachment of glycans affect both structural and functional roles of target proteins. N-glycosylation analysis is of increasing interest in biomedical and biological research, the pharmaceutical and healthcare industry and biotechnology. The aim of the work was to compare several methodologies that allow the identification of N-glycosylation sites by (1) combination of enzymatic digestions and deglycosylation with Endo H, (2) lectin affinity enrichment, (3) glycopeptide separation on reverse-phase HPLC column
18and (4) enzymatic deglycosylation with PNGase F in buffer containing 40% H O. 2
For this purpose we used a recombinant protein expressed in the methylotrophic yeast P. pastoris as model, which present five possible glycosylation sites with consensus sequence N-X-S/T. Analysis of N-glycosylation sites was carried out by
TMESI-MS and MS/MS in a hybrid mass spectrometer QTOF-2 . Best results were obtained from deglycosylation experiments with Endo H of several enzymatic
355 389 404digestions which provided clear evidence of asparagine residues N , N , N and 523N representing N-glycosylation sites.
55
Prot-03
Microbiome characterization of escamoles: a novel source of microorganisms with biotechnological potential
1 2 1Gonzalez-Escobar J. , Grajales-Lagunes A. , Barba de la Rosa A.P.
1Department of Molecular Biology of Instituto Potosino de Investigación Científica y 2Tecnológica A.C., S.L.P., México. Department of Bioprocesos of Facultad de Ciencias
Químicas, Universidad Autónoma de San Luis, S.L.P., México.
There are about one million of known species of insects in the world, from them, about 1900 species are edible. Insects are ecologically sustainable because do not harm the environment, use less natural resources and produce animal protein more efficiently. It has been reported, particularly in the ants, that multiple associations with microorganisms that live in their guts, help them to produce enzymes, vitamins, amino acids, and antimicrobial compounds. This has led to recognize the microbiota of ants as major resources for biotechnology applications. In Mexico, ant larvae of Liometopum apiculatum better known as Escamoles or “Mexican Caviar” are considered a food with high nutritive value due to their high-protein content. In addition, adult castes have many interactions with other insects, plants and microorganisms that allow diversify their microbiome. Therefore, the aim of this work was to carried out the characterization of the adult ants and their larvae microbiomes through metagenomic analysis of 16S rRNA. So far we have identified the microbial profile of the instars of the ants. The resulting data will be integrated to a bioinformatics analysis to know the organizational, functional and metabolic activities of microbial system. The workflow will allow us identify bacteria with specific metabolic characteristics with high biotechnological potential.
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57
Prot-04
Analysis of Fab and Fc N-glycans from human immunoglobulin G isolate from patients with Rheumatoid Arthritis
González-Hernandez A., Pousa S., Cabrera G.
Center for Genetic Engineering and Biotechnology, System Biology Department,
Proteomics. PO Box 6162, Havana, Cuba.
Glycosylation studies associated to rheumatoid arthritis (RA) are mainly focused to analyzing only the neutral glycans profile in total IgG. Thus, the information about charged N-glycans, located mostly in Fab fragment, is lost. The aim of this work is to establish the methodology for Fab and Fc N-glycans analysis of IgG in RA. Pepsin and papain digestions were performed to obtain Fab and Fc fragments. The isolation of fragments from digestion contaminants was realized by molecular exclusion chromatography. Additionaly, for IgG fragments derived to papain digestion was necessary one step by protein A affinity purification to separate Fab and Fc fragments. N-glycans linked to IgG fragments were obtained by PNGase F deglycosylation and labeled with 2-aminobenzamide. The N-glycosylation profiles were obtained by NP-HPLC and quantitation of structures was performed from relative area under curve (AUC). In Fab2 regions, sialylated N-glycans were more abundant than neutral one. Also sialylated structures are less represented in patients comparing to healthy individual. We found a more abundance of neutral N-glycans on Fc region (obtained from papain digestion) than on Fab regions. Also, we analyzed the content of bisecting and fucosylated structures linked to both IgG fragments of patient and healthy individual. Optimization steps in the procedure will be necessary to obtain larger quantities of fragment, and its N-glycans for subsequent analysis. This research is a preliminary approach to study several IgG samples and correlate the N-glycosylation changes in these fragments with total profile of IgG N-glycans.
58
Prot-05
Effect of silencing cmah and adaptation to serum free medium in the glycan profile of 14F7 MAb produced by NS0 cell line.
Morales O., Rabasa Y., Dorvignit D., R. de La Luz Hernández K.
Center of Molecular Immunology, Havana, Cuba
14F7 is a monoclonal antibody that targets NGcGM3 ganglioside: a tumor specific antigen in humans. Adapting the antibody producing NS0 cell line to grow in serum free conditions induced loss of culture viability because these cells express NGcGM3 in the cellular membrane. This problem was solved by silencing monophosphoryl cytidine N-acetyl-sialic acid hydroxylase enzyme (cmah) on the producing cell line.In this study we evaluate by glycomics techniques the impact that silencing cmah and adapting the producing cells to grow in protein-free medium had on the glycosylation profile of the recombinant protein obtained. We also demonstrate that this changes did not affect the physical and chemical attributes of 14F7 (molecular size, primary sequence, cyclization of N-terminal glutamine in the heavy chains, processing of the C-terminal lysines, deamidation, isoelectric point and aggregation pattern) or its biological properties (NGcGM3 ganglioside recognition and ability to induce lysis of tumor cell line P3-X63).
59
Prot-06
Mass spectrometry based methods for pharmacokinetic studies of three therapeutic peptide in phase I clinical trials
1 2 2 1 3 3Cabrales A. , Ramos Y. , Gil J. , Garay H.E. , Gómez J.A . , Audain E. , Berlanga 4 4 4 4 2 2J. , Perera Y. , Perea S.E. , Domínguez M.C. , Besada V. , and González L.J.
2
1 Physics and Chemistry Department, Center for Genetic Engineering and Biotechnology, 2Havana, Cuba, Systems Biology Department, Center for Genetic Engineering and
3Bioechnology, Havana, Cuba. Systems Biology Department, Center for Molecular 4Immunology, Havana, Cuba. Pharmaceutical Department, Center for Genetic
Engineering and Biotechnology, Havana, Cuba
Mass spectrometry is a powerful analytical tool in drug discovery and development due to its impressive capabilities in terms of structural elucidation, resolution, and mass accuracy. However, a quantitative determination of therapeutic peptides in biological samples still remains as an analytical challenge, even for mass spectrometry. The availability of the proper internal standards as well as the choice of a suitable method for detection and quantitation, are key elements to a successful and reliable outcome. Peptides are molecules with a wide diversity of physicochemical properties, so the development of methodologies cannot be implemented automatically as generic approaches, they need to be tailored. We present here our experiences in the development and validation of mass spectrometry based methods for peptide absolute quantitation in human plasma and their applications to pharmacokinetic studies in phase I clinical trials. Three novel therapeutic peptide candidates were analyzed in this context: CIGB-814 (Rheumatoid Arthritis, patients); CIGB-500 (Cardiovascular diseases, healthy volunteers) and CIGB-300 (Cancer, patients). It had been used Signal-Ion-Monitoring in combination with MALDI-TOF (CIGB-300) and QTOF (CIGB-500) mass spectrometers for the absolute quantification of two peptides administered by intravenous route. Also, we used Single Reaction Monitoring in a triple quadrupole tandem mass spectrometer to quantify a peptide administered subcutaneously (CIGB-814).
60
Prot-07
Characterization of a human plasma sub-proteome able to modulate the Dengue 2 virus infection in Huh7.5 cells
1 1 1 1 1 1 1Yero A. , Ramos Y. , Pupo D. , Martín D. , Márquez G. , Martín A. , Sarría,M. , 2 2 1 1Gallien S. , Domon B. , Chinea G. , Huerta V.
1 2Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Luxembourg
Clinical Proteomics Center, Luxembourg
Introduction: The outcome of Dengue virus (DENV) infections, which produce a wide range of symptoms going from mild febrile disease to hypovolemic shock, depends on interactions between viral and host factors whose interplay, despite ongoing research, remains poorly understood. Methods: Anion exchange chromatography was used to obtain a plasma derived preparation enriched in proteins capable of binding viral proteins and inhibiting DENV2 infection: ELUDE52.
Components proteins were identified by LC-MS/MS shotgun proteomics approach using a linear trap quadrupole (LTQ)-Orbitrap mass spectrometer. Protein identification was performed by searching the Swissprot database with the Mascot ver.2.3 engine. The influence of ELU on DENV2 infection was studied by time-DE52
of-addition experiments conducted in the human hepatoma cell line Huh-7.5. Results and discussion: A total of 239 proteins were identified in ELU . Results DE52
of function enrichment analysis emphasize the association between DENV2 proteins and proteins from the complement and coagulation cascades. The experiments show that ELU inhibits DENV2 infection in vitro during adhesion DE52
and internalization, and in the intracellular stages of viral replication. Conclusions: This work describes conditions of chromatographic fractionation of human plasma resulting in a protein sample able to modulate DENV infection in mammalian cells, provides the identity of proteins contained in the different chromatography fractions and presents data on the infection of Huh-7.5 cells with DENV2 in presence of abovementioned human plasma sample. These results will further contribute to understand virus-host factors interaction during DENV infection.
61
Prot-08
Inter alpha Inhibitor complex components mediating the binding to the DIII of the Envelope protein of Dengue Virus
Martín D., Ramos Y., Martín A., Sarría M., Chinea G., Huerta, V.
Department of System Biology, Center for Genetic Engineering and Biotechnology. Havana. Cuba
Dengue disease can be caused by any of the four virus serotypes of Dengue virus complex i.e. DV1-4. Symptomatic Dengue disease can vary from very mild symptoms to a life threatening syndrome with hemorrhagic manifestations. The interplay between viral and host factors that determine virus pathogenesis is not well understood. In previous work, a Proteomic approach was used to study DV interactome in human plasma. Results pointed to Inter alpha trypsin inhibitor complexes (IαIc) as putative interaction partners of DV. IαIc consist of Protein-glycosaminoglycan-protein structures that participate in the regulation of acute inflammatory response, a process typically altered in severe cases of Dengue disease. Plasma levels of IαIc are lower in children infected with DV and the magnitude of the decrease correlate with disease severity. In this work, IαIc were highly purified from human plasma and identified by mass spectrometry. IαIc components were separated by chemical treatment and used to further validate and characterize the direct interaction with recombinant proteins comprising the domain III of the envelope protein of DV1-4: DIIIE1-4. Two types of independent interactions were identified; one of low affinity with the light chain Bikunin presumably via the chondroitin sulfate chain of this protein and a stronger interaction was found IαIc heavy chains 1 and 2. Our results suggest the direct participation of IαIc in the pathogenesis of the infection from DV1-4.
Prot-09
Synthetic peptides based on the domain III inhibit Dengue virus infection
1 2 1 1 1Pupo D. , Fleitas N. , Huerta V. , Garay H. , Chinea G.
1 2Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Current address: Department of Physics, University of Sonora. 83000 Hermosillo, Sonora, México
Mosquito-borne flaviviruses are a group of viruses belonging to the genus Flavivirus. Dengue Virus (DENV), Yellow Fever Virus, West Nile Virus and Zika Virus are some examples of important flaviviruses. Dengue is caused by four serologically and genetically distinct viruses termed DENV1, DENV2, DENV3 and DENV4. Secondary infections with DENV heterologous serotypes are frequently associated with the most severe symptoms of disease because of antibody immune enhancement effect. Even though 2.5 billion people worldwide are currently at risk of infection with DENV, a safe and effective dengue vaccine is still not available. Nevertheless, development of antiviral drugs is also considered an attractive strategy for treatment of dengue infections, mainly due to the high correlation observed between viremia and illness severity. It is generally accepted that an effective antiviral treatment against DENV should be efficient against the four DENV serotypes. Based on the structure of domain III of viral envelope protein we have initially designed and synthesized a number of structurally constrained peptides which display antiviral activity. One of these peptides inhibits the viral infection of DENV1-4 in vitro at 15-50 M concentration with selectivity index of 20-67. It also shows significant protection in the mice model of encephalitis caused by intra-cranial inoculation of the virus. This peptide proves valuable for further development of antiviral candidates for therapeutic and prophylactic treatments against DENV and perhaps other flavivirus-associated diseases.
62
Prot-10
Mining B23/NPM1 human genomic data from publicly available repositories: Is this protein a suitable cancer target beyond Acute myeloid leukemia?
1 2 1Perera Y. , Fernandez-de-Cossío J. , Perea S.E.
1Laboratory of Molecular Oncology, Division of Pharmaceuticals, Center for Genetic 2Engineering and Biotechnology (CIGB), Bioinformatics Department, CIGB, P.O. Box 6162,
CP 10600, Havana, Cuba.
Background: B23/NPM1 is a multifunctional protein frequently overexpressed and eventually associated with tumor stage and malignancy in solid tumors. Paradoxically, only in blood tumors where the B23/NPM1 gene harbors specific somatic mutations, a clear cancer-driver effect for this protein has been demonstrated. Otherwise, not a driver or cooperative effect for the commonly upregulated-wildtype B23/NPM1 has been unambiguously assessed in cancer cells; therefore raising questions concerning its actual relevance in the malignant transformation. Aim: We sought to explore publicly available multiomics data to identify B23/NPM1 alterations which per se or in combinations with other mutational events, may promote or support the malignant transformation. Methods: For selected human cancers we analyzed genome-level Sequencing, Copy Number Variation and Expression data using integrated multi-dimensional analyses platforms and bioinformatics tools. Results: Although B23/NPM1 gene harbors nonsynonymous somatic mutations in three of the selected tumors (AML, LUAD, CESC), a driver effect was only predicted in AML. Otherwise, CNV analysis indicated low frequent gene copy number gain and losses with more prevalence of copy number gain overall. Global expression analysis indicated B23/NPM1 mRNA is upregulated in several tumors, but heterogeneity was seen among patients. Interestingly, co-occurrence or mutually-exclusive patterns between mutations in driver genes and B23/NPM mRNA overexpression were observed in some tumor localizations, impacting the overall survival of patients. Conclusion: Our findings suggest that although B23/NPM is not widely mutated in cancer, once overexpressed it might cooperate with established driver's genes to maintain a neoplastic state in some tumor localizations.
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64
Prot-11
A comparative molecular dynamics study of thermophilic and mesophilic β-fructosidase enzymes.
1 1 1 1 1Musacchio A. , Mazola Y. , Guirola O. , Palomares S. , Chinea G. , 2 2Menéndez C. , Hernández L.
1 2System Biology Department, Biomedical Research Division, Department of Plant-
Microbe Interactions, Center for Genetic Engineering and Biotechnology (CIGB), Ave. 31
e/ 158 and 190, Playa, P.O. Box 6162, Havana 10600, Cuba
Arabidopsis thaliana cell wall invertase 1 (AtcwINV1) and Thermotoga maritima β-fructosidase (BfrA) are among the best structurally studied members of the glycoside hydrolase family 32. Both enzymes hydrolyze sucrose as the main substrate but differ strongly in their thermal stability. Mesophilic AtcwINV1 and thermophilic BfrA have divergent sequence similarities in the N-terminal five bladed β-propeller catalytic domain (31 %) and the Cterminal β-sandwich domain (15 %) of unknown function. The two enzymes were subjected to 200 ns molecular dynamics simulations at 300 K (27 °C) and 353 K (80 °C). Regular secondary structure regions, but not loops, in AtcwINV1 and BfrA showed no significant fluctuation differences at both temperatures. BfrA was more rigid than AtcwINV1 at 300 K. The simulation at 353 K did not alter the structural stability of BfrA, but did increase the overall flexibility of AtcwINV1 exhibiting the most fluctuating regions in the β-propeller domain. The simulated heat treatment also increased the gyration radius and hydrophobic solvent accessible surface area of the plant enzyme, consistent with the initial steps of an unfolding process. The preservation of the conformational rigidity of BfrA at 353 K is linked to the shorter size of the protein loops. Shortening of BfrA loops appears to be a key mechanism for thermostability.
65
Prot-12
Proteomic and bioinformatics analysis of the CIGB-300 interacting proteins in plasma.
1 2 1 1 3 1Palomares S. , Hardy A. , Quiñones M. , Ramos Y. , Perera Y. , Besada V. , 1 3Guirola O. , Perea S.E.
1Department of System Biology, Biomedical Research Division, Center for Genetic
2Engineering and Biotechnology, Havana, Cuba. University of Havana, Havana, Cuba. 3Molecular Oncology Laboratory, Pharmaceutical Division, Center for Genetic Engineering
and Biotechnology, Havana, Cuba
CIGB-300 is a synthetic peptide-based drug that targets the CK2 phospho-acceptor domain, inhibiting it's interaction with substrates. This therapeutic candidate has been suggested for the treatment of acute myloid leukemia and lung cancer by intravenous application. Therefore, proteomics experiments have been made for the identification of CIGB-300 interacting proteins in plasma. Interestingly, three CK2 enzyme substrates were found among the identified proteins: fibrinogen alpha chain, vitronectin, and C3 complement component. Finally, using appropriated bioinformatics tools, in this work we also analyzed the biological meaning of such interactions in terms of the mechanism of action of CIGB-300.
66
Prot-13
A web based application to manage Mass Spectrometry experiments.
Vázquez Y., Guirola O., Palomares S., Espinoza L.A., Besada V.
Department of System Biology, Center for Genetic Engineering and Biotechnology, Havana, Cuba
ProteoLims (Laboratory Information Management System for Proteomics) is a web application to manage and retrieve information about proteomic experiments design by Bioinformatics group of the System Biology department at the CIGB. The Spectrometry framework contains four main modules to capture the information from project level to results. The application let researchers to efficiently document their work, to describe materials and methods and to collect and analyze even the unprocessed data obtained by mass spectrometry. The system have a user-friendly web interface where can be added, edited, browsed and searched data. Additionally the system can also trace the projects and sub-projects of investigation that were processed on the lab. The web application is based on the Minimal Information About a Proteomics Experiment (MIAPE-MS). ProteoLims aims to increases the operational efficiency of the Mass Spectrometry lab trough the delivering of reliable results in a shorter amount of time.
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Prot-14
CmPI-II, a non-classical Kazal inhibitor with exceptional characteristics : Structural characterization by NMR
1 1 1 2Cabrera-Muñoz A. , Rojas L. , Alonso-del-Rivero M. , Pires J.R.
1 2Centro de Estudios de Proteínas, Universidad de La Habana, Cuba, Instituto de Bioquímica Medica, Universidade Fedelar do Rio de Janeiro, RJ, Brazil
Proteases and its inhibitors are subject of intense research for studying the protein-protein interactions and the development of new drugs. A protease inhibitor (CmPI-II) (UNIPROT: IPK2_CENMR) was isolated and characterized from the marine mollusk Cenchritis muricatus. It is a non-classical Kazal tight-binding inhibitor of serine proteases: trypsin, human neutrophil elastase, subtilisin A and pancreatic elastase. This specificity is exceptional in the members of Kazal-type inhibitor family. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In this work, we determined for first time the solution structure of CmPI-II and studied its backbone dynamics using NMR techniques. CmPI-II has the typical scaffold described for Kazal-type inhibitors: a central a-helix and a antiparallel three-stranded ß-sheet, but has some significant differences in the N-terminal moiety and in the reactive loop conformation. In addition, analysis of local mobility showed that the N-terminal region (2-10 aa) is flexible in solution, with the three first residues as the most flexible zone in the structures and also showed that residues V5, G22, Y25 and N27 have internal motion in the ms-ms timescale. Finally, the global correlation time diminished with the dropping of the protein concentration, indicating that CmPI-II is able to dimerize reversibly. All these structural features could explain the unusal specificity of CmPI-II.
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Prot-15
New family of β-defensin-like peptides on invertebrates
1 2 3 4Montero-Alejo V. , Corzo C. , Perera E. , Rodríguez-Viera L. , Sánchez-Díaz G. 5, 6 5, 6 7 1, Hernández-Rodríguez E.W. , Álvarez C. , Perdomo-Morales R.
1
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7
Beta_defensin have been solely found in vertebrates until β-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered, as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/β motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of β-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of β-defensins from vertebrates.
Biochemistry Department, Center for Pharmaceuticals Research and Development, Havana, Cuba. Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico. Department of Fish Physiology and Biotechnology, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellon, Spain. Center for Marine Research, University of Havana, Havana, Cuba. Department for Basic and Biomedical Sciences, Medicine Faculty, Artemisa, Cuba. Laboratory of Computational and Theoretical Chemistry, University of Havana, Havana,
Cuba. Center for Protein Studies, Faculty of Biology, University of Havana, Havana, [email protected]
Prot-16
The Trypsin Inhibitor Panulirin Regulates the Prophenoloxidase-activating System in the Spiny Lobster Panulirus argus.
1 1 2 3Perdomo-Morales R. , Montero-Alejo V. , Corzo G. , Besada V. , Vega-Hurtado 1 4 5 1Y. , González-González Y. , Perera E. , and Porto-Verdecia M.
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The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.
. Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico. Proteomics, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, CP 10600 Havana, Cuba. Biochemistry Department, Federal University of Sao Paulo, Rua 3 de Maio 100, CEP 04044-020 Sao Paulo, Brazil Department of Fish Physiology and Biotechnology, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellon, [email protected]
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Prot-17
Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in the Spiny Lobster Panulirus argus
1,3,4 2a 2b 3Rodríguez-Viera L. , Perera E. , Martos-Sitcha JA , Perdomo-Morales R. , 1 3 1 2Casuso A. , Montero-Alejo V. , García-Galano T , Martínez-Rodríguez G. ,
4Mancera JM.
1 2Center for Marine Research, University of Havana, Havana, Cuba. Instituto de Ciencias 3Marinas de Andalucía, ICMAN-CSIC, Puerto Real, Cadiz, Spain. Department of
Biochemistry, Center for Pharmaceuticals Research and Development, Havana, Cuba. 4 aDepartment of Biology, University of Cadiz, Puerto Real, Cadiz, Spain. Current address: Control of Food Intake Group, Department of Fish Physiology and Biotechnology,Institute of
bAquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain. Current address: Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain.
Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. The physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work, we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favored trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
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Prot-18
Proteomic analysis reveals the molecular basis on CIGB845 pharmacological intervention
Subirós N. , Palomares S. , Quiñonez M. , Pérez-Saad H. , Ramos, Y. , Guirola, O. , González L.J. , Besada V. , García del Barco D.
Pharmaceutical Department, Department of Systems Biology, Center for Genetic Engineering and Biotechnology, Biomedical Research Direction, Havana, Cuba
Neuroprotective effect of CIGB845 has been previously assessed in experimental models of stroke. The objective of this study was to identify differentially expressed proteins in the cerebral cortex of vehicle- and CIGB845-treated animals after ischemic injury. Focal cerebral ischemia was induced by Endothelin-1 intracerebral injection in rats, and two hours later vehicle or CIGB845 were administered. Cerebral cortexes were collected 3, 5 and 24 hours later. Cerebral cortex protein extracts were label-free analyzed by liquid chromatography mass spectrometry.More than 900 proteins were identified from the rat brain with <5% false discovery rate. Among them, 296 proteins significantly changed in response to CIGB845 treatment; 43 (14 %) at 3 hours, 61 (21 %) at 5 hours; and 220 (74%) at 24 hours. Comparison between vehicle and CIGB845 treated groups reveals that CIGB845 treatment reduced the expression of 97 (44%) proteins and enhanced the expression of 123 (55 %) proteins. Functional categorization analysis of altered proteins revealed that CIGB845 is influencing relevant biological process such as inflammation, biogenesis, and neuroregeneration, thus explaining from a molecular point of view, the salutary effects of CIGB845. This proteomic data may provide new insights into the protective mechanisms of this pharmacological intervention which was intended to preserve the ischemic penumbra area after stroke.
1 2 2 1 2
2 2 2 1
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72
Bio-01
In silico analysis of AML and GBM transcriptomes.
Miranda J., Bringas R.
Bioinformatics Group, Center for Genetic Engineering and Biotechnology (CIGB), Havana,
Cuba.
DNA microarrays and massive sequencing have become the most widely used technologies to generate expression profiles on a genomic scale, allowing the simultaneous measure of mRNA levels of thousands of genes from samples corresponding to different experimental conditions. Thus, it has been applied to the understanding of the molecular basis of diseases such as leukemia and malign gliomas. Functional analysis of differential expressed genes may lead to the discovery of new drug targets candidates. Moreover, the identification and validation of therapeutic targets in complex diseases such as cancer remains one of the key challenges in the biomedical sciences. In this work, using the data published by The Cancer Genome Atlas (TCGA) on AML (200 samples) and GBM (529 samples) we studied the relevance of most highly expressed genes in these tumors of very poor prognosis. Others repositories of data were used for validation of results. Selection of the most expressed genes in each cancer type was done in two ways: (1) applying one-group t-test and (2) choosing the top 200 higher expressed genes. This was done on all samples and by subtype in the case of AML. A combination of functional (Toppgene) and networks (BisoGenet) analysis of these lists was then performed. In AML we identified significant genes for each subtype and also those common to all of them, some already known as markers like SRGN. In GBM, additionally we analyzed the behavior of integrin's expression profiles.
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Bio-02
Impact of ignoring germline susceptibility variants by restricting the analysis to somatic mutation data in cancer genetic study
Fernandez-de-Cossio J., Perera Y.
‡Bioinformatics Department and Pharmaceutical Division of the Center for Genetic Engineering and Biotechnology, Havana Cuba
Cancer is a genetic disease with a substantial contribution of acquired (somatic) alteration. Though it is believed that most inherited (germline) susceptibility variants are not clinically actionable, germline mutations underlying advanced cancers is turning more frequent than anticipated . However, reporting germline changes usually require patient consents and only filtered somatic alteration are directly accessible . Nonetheless, both somatic and germline alterations ascertained from cancer genomes data can be evaluated to infer a more integral picture of the genetic contribution to disease . Here we assess the impact of ignoring germline susceptibility variants by restricting the analysis to somatic mutation data in the study of the genetic contribution to cancer.
Bio-03
QSAR and molecular docking studies. A rational approach to drug design
Álvarez-Ginarte Y.M.
Laboratory of Theoretical and Computational Chemistry, Faculty of Chemistry, University
of Havana, 10400, La Habana, Cuba.
Keywords: virtual screening, quantum and physicochemical molecular descriptor, QSAR and docking studies.
Parallel quantitative structure-activity relationship (QSAR) and computational simulations of a candidate ligand binding to a receptor study (a “docking” procedure) of 269 steroids with anabolic activity evaluated in vivo were performed. The QSAR model expressed by selected descriptors as the octanol-water partition coefficient, the molar volume and the quantum mechanical calculated charge values on atoms C1, C2, C5, C9, C10, C14 and C17 of the steroid skeleton, express structural features of anabolic steroids contributing to the transport and steroid-receptor interaction. On the other hand, a docking procedure predicts the association of these steroids with the human androgen receptor. Fourteen compounds were identified as lead; the most potent was the 7α-Methylestr-4-en-3, 17-dione. It was concluded that a good anabolic activity requires hydrogen bonding interactions between either Arg752 and Gln711 residues (in the vicinity of cycles A) with the O3 atom of the steroid and either Asn705 and Thr877 residues (in the vicinity of cycles D of steroid) with O17 atom.
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SB-01
Bpb: Fast inference of ill-posed problems within a convex space. Applications in metabolic networks
1 2Fernández-de-Cossío-Díaz J. , Mulet R.
1Molecular System Biology, Center of Molecular Immunology (CIM), Havana 11600, Cuba. 2 University of Havana, Havana
11600, Cuba.
One of the most intensively investigated biological systems is cellular metabolism. While the topological nature of individual reactions and pathways is well understood there is still a lack of comprehension regarding the global functional behavior of the system. Flux-balance analysis (FBA) has been the most succesful technique for studying metabolism. Relying on the strong hypothesis that the organism maximizes an objective function, this method produces a single optimal metabolic state. Unfortunately, only under very specific biological conditions the cell obeys such an optimization law. An alternative strategy pursued by researchers is to explore the set of all possible metabolic states, but computational limitations hinder this approach. Our work proposes a novel algorithmic strategy, BPb, that provides an efficient characterization of the whole set of stable fluxes compatible with metabolic constraints. We redesigned a message-passing algorithm derived from the field of statistical physics. We parameterized the standard Belief Propagation message equations with Beta distributions, significantly improving the speed of previous implementations. The algorithm, based on the well known Bethe approximation, can be used to estimate the volume of a sub-dimensional convex polytope in high dimensions. We compared the results of our algorithm with an exact solver in small networks, and with those of hit and run Monte Carlo (MC) on larger systems, demonstrating that both methods agree but BPb significantly outperforms MC on real networks. In this work we present applications of BPb to metabolic networks. To verify the validity of our algorithm in this arena we compare the flux distributions obtained by BPb and MC on the standard red blood cell
Theoretical Physics Department, Faculty of Physics,
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network. Then we assess the reduction in the space of possible metabolic states by simulated enzymopathies in the E. coli central metabolism, and compare with the genome scale version of the network, unraveling non-trivial redundancies.
76
SB-02
In silico prediction of the Transforming Growth Factor beta and Activin Receptor-like Kinase 3 complex
Fernández-de-Cossío-Díaz J., Corría J., León K.
Molecular System Biology, Center of Molecular Immunology (CIM), Havana 11600, Cuba
Transforming growth factors (TGF)b1, b2, b3 are homodimeric polypeptides that play crucial non-overlapping roles in embryogenesis, tissue development, carcinogenesis, and immune regulation. They signal through the formation of complexes involving two pairs of receptor serine/threonine kinases known as the type I and type II receptors, respectively, which in turn propagate the signal by phosphorylating Smad2/3 transcription factors, inhibiting cell proliferation and migration. For some time now it has been known that TGFb can induce an opposite cellular response through the recruitment of the activin receptor-like kinases (ALK) 3 or 1 to the receptor complex, which can stimulate cell proliferation and migration through phosphorylation of Smad1/5. Even though the three isoforms of TGFb share a significant similarity in sequence and structure, they can have very different effects in certain contexts. The structural explanation of these functional differences is still incomplete. The crystal structures of TGFb1 and TGFb3 in complex with the type I and type II receptors have been resolved, but the structures of the complexes involving ALK3 or ALK1 have not been resolved at present. In this work we employed the ROSETTA modelling suite to dock ALK3 to the TGFb1 and b3 receptor complexes. The resulting model structures are compared with the experimental data available in the literature. Based on these models, we suggest a plausible explanation for the different effects TGFb1 and b3.
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SB-03
Common gamma chain: relevant for the IL2-IL2R affinity? A mathematical approach
Ponce L.F., García-Martínez K., León K.
Molecular System Biology, Center of Molecular Immunology (CIM), Havana 11600, Cuba
Interleukin-2 (IL-2) mediates its function in target cells through a multimeric receptor (IL-2R), which is constituted by three different chains: IL-2Rα, IL-2Rβ and IL-2Rγc. The mechanism for assembling the different subunits to yield IL-2/IL-2Rαβγ complexes remains to be elucidated. We demonstrate, through mathematical modeling, that the affinity conversion model, is compatible with experimental data of IL2 binding to the cells. The mathematical model developed here, allow us to study how γc contributes to the apparent affinity of IL2:IL2R complex. The model predicts that the apparent affinity of IL2:IL2R complex is directly proportional to the γc expression, indicating a relevant role of this chain in the IL2R assembly. Furthermore, we simulate the effect of an antagonist IL2 mutein, that have affected the interaction with γc (IL2 no-γ). We studied the effect of competition between IL2 and IL2 no-γ for binding to IL2R. We obtain that, at high concentration of IL2 no-γ, is possible to displace IL2 from the formation of signaling complexes IL2/IL-2Rβγc and IL2/IL-2Rαβγc. The mutein efficacy can be improved, by enhancing its interaction with IL-2Rα or IL-2Rβ. Overall, our results predict a relevant role for the γc expression, in the capacity of cells to capture IL2, despite being unable to bind IL2 along. This affects the capacity of IL2 no-γ muteins to compite with IL2 in a real scenario, which can be solved by increasing its affinity for IL-2Rα or IL-2Rβ.
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PARTICIPANTS LIST
Abel Paredes Biotechnology, Havana, Cuba.Center for Genetic Engineering and Biotechnology, Havana, Cuba
Anna BaranovaSchool of Systems Biology, George
Akos Vertes Mason University, Fairfax, VA USA. Department of Chemistry, The George Federal State Budgetary Institution Washington University, Washington, "Research Centre for Medical DC 20052, USA Genetics," Moscow, Russia. ATLAS
Biomed Group, Moscow, Russia ;
Aleksandra Nita-Lazar Laboratory of Systems Biology, NIAID, NIH, Bethesda, MD, USA Annia L. González
Center for Genetic Engineering and Biotechnology, System Biology
Alexis Musacchio Department, Proteomics. PO Box System Biology Department, 6162, Havana, Cuba.Biomedical Research Division, 2 Department of Plant-Microbe Interactions, Center for Genetic Aymara Cabrera MuñozEngineering and Biotechnology Centro de Estudios de Proteínas,
Universidad de La Habana, Cuba
Alexis Yero DíazDepartment of System Biology, Center Brandon Ruotolo for Genetic Engineering and University of Michigan, Department of Biotechnology (CIGB), Havana, Cuba Chemistry, 930 N. University Ave. Ann
Arbor MI, 48109
Ania Cabrales RicoPhysics and Chemistry Department, Chen-Hsiang YeangCenter for Genetic Engineering and Institute of Statistical Science,
[email protected]@[email protected]
[email protected]@fbio.uh.cu
[email protected]@umich.edu
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Academia Sinica, Taipei, Taiwan Eduardo CanalesDepartment of Plant Science, Center for Genetic Engineering and
Dania Vázquez Blomquist Biotechnology, Havana, CubaPharmacogenomic Department, Center for Genetic Engineering and Biotechnology (CIGB), Elda Dennisse Lizarraga LizarragaHavana, Cuba. Laboratorio de Biología Molecular,
Centro de Investigación en Alimentación y Desarrollo (CIAD).
Dayron Martín Mazatlán, Mexico. Department of System Biology, Center for Genetic Engineering and Biotechnology. Havana. Cuba Gabriel Padrón
Laboratory of Leishmaniosis, Institute Oswaldo Cruz, FIOCRUZ, Brazil
Diana García del Barco
Gleysin Cabrera HerreraMass Spectrometry Laboratory and GlycoLab. Department of Proteomics. Center for Genetic Engineering and Biotechnology. PO. Box 6162.
Dianne Pupo Havana. Cuba.Department of System Biology, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. Hamlet Camacho
Pharmacogenomics group. Centre of Genetic Engineering and
Dimtcho.Batchvarov Biotechnology. CIGB. La Havana, Department of Molecular Medicine, Cuba. Laval University, Québec PQ, [email protected] Canada. Centre de recherche du CHU de Québec, L'Hôtel-Dieu de Québec, Howard L. McLeodQuébec PQ, Canada. Medical Director, Personalized
Medicine Institute, Moffitt Cancer
Pharmaceutical Department, Center for Genetic Engineering and Biotechnology, Biomedical Research Direction, Havana, Cuba
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Center, Tampa, FL USA Jorge Fernández de Cossío Díaz Molecular System Biology, Center of
Molecular Immunology (CIM), Irving King Jordan Havana 11600, CubaSchool of Biology at Georgia Tech and co-director of the PanAmerican Bioinformatics Institute Jorge Luis Gonzalez Escobar
Department of Molecular Biology of Instituto Potosino de Investigación
Isabel Guillén Científica y Tecnológica A.C., S.L.P., Pharmacogenomics group. Center for México. Genetic Engineering and Biotechnology CIGB. La Habana. Cuba. Jose R. Fernandez
UAB Department of Nutrition Sciences, WEBB 522, 1675
Jacobo Zuñiga Castillo University Boulevard,Regional Marketing Manager Mexico, Birmingham, AL 35294-3360Central América and Caribbean, QIAGEN México
Julia M. Torres VelardeLaboratory of Molecular Biology and
Jamilet Miranda Tissue Culture of Aquatic Organisms, Bioinformatics Group, Center for Centro de Investigación en Genetic Engineering and Alimentación y Desarrollo (CIAD), Biotechnology (CIGB), Havana, Mazatlán, Sinaloa, MexicoCuba. [email protected]
Julio Raúl Fernández
Jorge Fernández de Cossío Division of System Biology, Center for Bioinformatics Department, Center for Genetic Engineering and Genetic Engineering and Biotechnology, Havana,Biotechnology, Havana Cuba Cuba.
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Karina García Martínez María del Carmen DomínguezMolecular System Biology, Center of Biomedical Research Department, Molecular Immunology (CIM), Havana Center for Genetic Engineering and 11600, Cuba Biotechnology, P.O. Box 6162,
Havana, Cuba
Leandro Rodríguez VieraCenter for Marine Research, María Elena Fernández de CossíoUniversity of Havana, Havana, Cuba. Pharmacogenomics Department. Department of Biochemistry, Center Biomedical Research Division. Center for Pharmaceuticals Research and for Genetic Engineering and Development, Havana, Cuba. Biotechnology, 31 entre 158 & 190, Department of Biology, University of Cubanacán, Playa, Cuba. Cadiz, Puerto Real, Cadiz, Spain.
Mario Valdés
Luis Ariel Espinosa Centro de Estudios de Proteínas, Proteomics Group, Center for Genetic Universidad de La Habana, CubaEngineering and Biotechnology, Havana, Cuba.
Mauricio E. Escalante RojasLaboratory of Molecular Biology and
Luis Felipe Ponce Tissue Culture of Aquatic Organisms, Molecular System Biology, Center of Centro de Investigación en Molecular Immunology (CIM), Havana Alimentación y Desarrollo (CIAD), 11600, Cuba Mazatlán, Sinaloa, Mexico.
Luis Javier GonzálezMass Spectrometry Laboratory and Mauricio QuiñonesGlycoLab. Department of Proteomics. Proteomics Group, Department of Center for Genetic Engineering and Systems Biology, Center for Genetic Biotechnology. PO. Box 6162. Engineering and Biotechnology, Havana. Cuba Havana, Cuba.
[email protected]@cigb.edu.cu
[email protected]@cim.uh.cu
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Odalys Maestre Biotechnology (CIGB), Havana, Center for Genetic Engineering and Cuba.Biotechnology, Havana, Cuba
Rolando Perdomo Morales
Orlando Morales Biochemistry Department, Center for Center of Molecular Immunology, Pharmaceuticals Research and Havana, Cuba Development, Havana, Cuba
Radoslav Goldman Roberto Mulet Department of Oncology, Department Group of Complex Systems and of Biochemistry and Molecular & Cell Statistical Physics and Department ofBiology Georgetown University, Theoretical Physics, Physics Faculty. Washington, DC, USA. University of Havana
Rafael B. Reyes León Cachón Steven M. CarrCentro de Diagnóstico Molecular y Dept of Biology, Memorial University Medicina Personalizada, of Newfoundland, & Terra Nova Departamento de Ciencias Básicas, Genomics,División Ciencias de la Salud, Inc., St John's NL, CanadaUniversidad de Monterrey, San Pedro Garza García, Nuevo León, México
Sucel PalomaresDepartment of System Biology,
Rebeca Martínez Biomedical Research Division, Biotecnology Animal División. Center Center for Genetic Engineering and for Genetic Engineering and Biotechnology, Havana, Cuba. Biotechnology; Havana, Cuba
Tatiana V. Tatarinova
Ricardo Bringas University of Southern California, Bioinformatics Group, Center for Center for Personalized Medicine, Genetic Engineering and Children's Hospital Los Angeles, Los
ricardo,[email protected]@cigb.edu.cu
[email protected] [email protected]
[email protected] [email protected]
[email protected]@cigb.edu.cu
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Angeles, CA, USA Biotechnology, Havana, Cuba
Vivian Huerta Yasser PereraDepartment of System Biology, Laboratory of Molecular Oncology, Center for Genetic Engineering and Division of Pharmaceuticals, Center Biotechnology, Havana, for Genetic Engineering and Cuba. Biotechnology
Vivian Montero Alejo Yeny Mercedes CascaretBiochemistry Department, Center for Center for Genetic Engineering and Pharmaceuticals Research and Biotechnology, Havana,Development, Havana, Cuba. Cuba.
Weston B. Struwe Yoanna M. Álvarez-GinarteDepartment of Chemistry, University Laboratory of Theoretical and of Oxford, Chemistry Research Computational Chemistry, Faculty of Laboratory, 12 Chemistry, University of Havana, Mansfield Road, Oxford OX1 3TA, 10400, La Habana, Cuba.UK
Yuriy L. Orlov
Yamilet Vázquez Institute of Cytology and Genetics SB Department of System Biology, RAS, Novosibirsk, Russia.Center for Genetic Engineering and Biotechnology, Havana, Cuba
Yassel RamosProteomics Group, Department of System Biology, Biomedical Research Division, Center forGenetic Engineering and
[email protected] [email protected]
[email protected] [email protected]
[email protected] [email protected]
[email protected]@bioch.ox.ac.uk
NOTEBOOK
