9th International Symposium on OMICS

and Bioinformatics

October 24 to 27, 2019

Melia Las Dunas Hotel, Cuba

Index OPENING SESSION ...................................................................................................... 1

Thursday October, 24th ......................................................................................... 1 ORAL PRESENTATIONS ............................................................................................... 2

Friday October, 25th ............................................................................................... 2 Saturday October, 26th ........................................................................................... 6 Sunday October, 27th ........................................................................................... 10

ORAL SESSION ABSTRACTS ...................................................................................... 13 Extending the small molecule similarity principle to all levels of biology ............ 13 Biomarker Discovery in Cancer: Building a bridge between preclinical and clinical research ............................................................................................................... 14 It takes two to tango: TIMS and PASEF ................................................................ 15 İ Viva la adquisición! MaxQuant.Live revolutionizes instrument control............. 16 Absolute quantitative proteomics of blood ......................................................... 17 Electrophoretic fractionation previous to MS...................................................... 18 Proteoforms. The new proteomics challenge. Application to the study of protamines in the human sperm nucleus ............................................................ 19 Epidermal growth factor and growth hormone-releasing peptide. A combined therapeutic approach for brain damage .............................................................. 20 Heberferon, combination of ifns for cancer treatment. The omics opportunities. ............................................................................................................................. 21 Omics approaches in glioblastoma multiforme under interferon co-formulation treatment ............................................................................................................. 22 Phosphoproteomic study of the antitumorals CIGB300 and CX-4945. ................ 23 OMICS-credentialing of patient-derived glioblastoma models for pre-clinical studies .................................................................................................................. 24 Novel gene targets in head-and-neck cancer revealed by co-expression networks using multi-omic data integration ........................................................................ 25 Personalized Cancer Therapy Prioritization Based on Driver Alteration Co-occurrence Patterns ............................................................................................. 26 Investigation of epithelial-to-mesenchymal transition mechanisms implicated in ovarian cancer dissemination .............................................................................. 27 Molecular taxonomy of metastatic renal cell carcinoma and its correlation with outcome to immunotherapy ................................................................................ 28 Steroidogenic Factor-1 is a Goldilocks transcription factor regulating key genes implicated in tumorigenesis ................................................................................. 29 Serum microRNAs in osteoarthritis: novel biomarkers and therapeutic targets . 30 Quantitative MEMS Molecular Diagnostics ......................................................... 31 Nanooncological platform as a tool for proteomic, lipidomic, glycomic, epigenetic studies. ................................................................................................................. 32 Pregnancy specific glycoprotein (PSG9) as a potential biomarker in Preeclampsia (PE) ....................................................................................................................... 33 Application of tensor decomposition based unsupervised feature extraction to multi-omics data set ............................................................................................ 34

Detection of low abundant viral CD8+ T cell epitopes and tumor neoepitopes by targeted mass spectrometry ................................................................................ 35 Enabling proteomic scrutiny in recalcitrant tissues of tropical fruits ................... 36 Top-down sequencing of frog skin peptides, antibiotics of the next generation . 37 Bioinformatics as a tool for treatment plant diseases. ........................................ 38 Skeletal muscle-specific methyltransferase METTL21C trimethylates p97 and regulates autophagy-associated protein breakdown .......................................... 39 Early proteome movements depicting a GHRP6-mediated plausible mechanism towards aesthetic wound healing ........................................................................ 40 Multi-omics analysis of in vivo circadian data reveals mechanisms of enzyme activity regulation ................................................................................................ 41 Biological and mass spectrometric characterization of four peptide-protein conjugated vaccine candidates against ticks. ...................................................... 42 Bufadienolides isolated from peltophryne fustiger (amphibia: bufonidae) are potent and selective inhibitors of the membrane-bound neutral ectoaminopeptidase n: preliminary effects on the melanoma mewo cell line. ... 43 Artificial Neural Network Algorithms for Biomarker Identification, Pathway Modelling and Drug Target Discovery. ................................................................. 44 Bioinformatics approaches for sing cell and big data analysis ............................. 45 Drug taxonomy based on large pharmacogenomic data ..................................... 46 Advances and Challenges in Single Cell RNA-Seq Analysis ................................... 47 Resolution of the concept of interaction ............................................................. 48 Functional Analysis and Network approaches elucidate the mechanism of action of Heberferon in the U87MG Glioma cell line...................................................... 49 CE-MS in clinical proteomics: application in oncology, kidney and cardiovascular diseases ................................................................................................................ 50 Heberprovac, a GnRH based vaccine directed to prostate Cancer. Clinical trials results and new challenges. ................................................................................. 51 Overpowering multiple inhibitory immune checkpoints with a single peptide inhibitor. .............................................................................................................. 52 CRISPR/Cas9-mediated gene knockout of COMMD1 decreases sensitivity of H460 and HCT-116 cell lines to the antitumoral peptide CIGB-552. ............................. 53 Integrated ‘Omics Strategies to Advance Alzheimer’s Disease Disparities Research .............................................................................................................. 54 Modulation of genes for the combination phycocyanobilin and ifn beta promises greater expectations for a new therapy for multiple sclerosis ............................ 55 Effects of phycocyanobilin on genes expression reveal neuroprotective mechanisms in a rat model of ischemic penumbra ............................................. 56

POSTER SESSION ...................................................................................................... 57 POSTER SESSION ABSTRACTS ................................................................................... 61

Association of rs4278932 polymorphism of MERKT gene with susceptibility to multiple sclerosis in Cuban population. ............................................................... 61 Glycosylation pattern of the Human Epidermal Growth Factor Receptor obtained in Human Embryonic Kidney tissue derived cell line (HEK293). ........................... 62

Analysis of a library of compounds with possible inhibitory properties in the MyD88-mediated signaling pathway. .................................................................. 63 Comparative genomics with Neisseria Meningitidis serogroup B: insights into cross reactivity of VA-MENGOC-BC. ..................................................................... 64 Proinflammatory cytokines in alzheimer’s disease patients ................................ 65 Structural insights into High Affinity PD-1 (HACPD1) and PD-L2 interaction. An in silico approach ..................................................................................................... 66 Rational basis for MyD88 inhibitors development: an in silico approach ............ 67 Challenges of Pharmacoeconomics towards Precision Medicine ........................ 68 The impact of flow regime over dengue virus shadow effect of propeller shapes proteins ................................................................................................................ 69 Molecular Dynamics Simulations of the cyclic and antiparallel dimer analogues of the antifungal peptide Cmp5 in a fungal membrane model ................................ 70 Strategy based on pan-genomics for rational design of vaccines against Neisseria Meningitidis serogroup B. .................................................................................... 71 Mass spectrometric characterization of Bm86-Cys1pP0: A bivalent vaccine candidate to control the cattle-tick population. .................................................. 72 Expression profile of TLR pathway genes in a colitis biomodel. ........................... 73 CGWork: a tool for the search of the common genome with applications in the rational design of vaccines. .................................................................................. 74 Mass spectrometric characterization of low‐abundance species in the active pharmaceutical ingredient of synthetic peptides ................................................ 75 Phycocyanobilin molecular mechanisms on a damage model induced with glutamate in shsy5y cells ..................................................................................... 76 An integrative analysis of evolutionary pressures over 3C and VP1 segments in Coxsackievirus A24v ............................................................................................. 77 Characterization by mass spectrometry of the KLH1-Cys1pP0 and KLH2-Cys1pP0 conjugates: a vaccine candidate against ticks. ..................................................... 78 Integrative model of rhodanine derivatives as tau aggregation inhibitors in alzheimer’s disease .............................................................................................. 79 BitClust: Fast conformational clustering of long Molecular Dynamics simulations. ............................................................................................................................. 80 Prediction of molecular interactions and physicochemical properties relevant for vasopressin v2 receptor antagonism ................................................................... 81 Metabolome Analysis of Breast Tumors for Discovery of Oncometabolites ....... 82 Toward the uses of antimicrobial peptides against multidrug resistant bacteria 83 Proteotranscriptomics Reveals Associations of Protein-mRNA Concordance with Breast Cancer Subtypes and Survival ................................................................... 84 LC-MS/MS characterization of two conjugated vaccine candidates against ticks obtained by different crosslinking strategies between the carrier protein p64K from N. meningitidis and a peptide derived from the acidic ribosomal P0 protein from R. sanguineus. ............................................................................................. 85 Integrated Genomics to Uncover Clinically-relevant Liver Cancer Drivers........... 86 Epidermal Growth Factor regulates LPS-induced inflammatory scenario in fibroblasts derived from diabetic foot ulcers ....................................................... 87

Network interactions and signaling pathways analysis for a phosphoproteomics experiment with heberferon in glioblastoma cells .............................................. 88 Functional analysis of a phosphoproteomics experiment with heberferon in glioblastoma cells ................................................................................................. 89 Oxidative stress parameters as biomarkers for new multiple sclerosis therapies90

1

OPENING SESSION

Thursday October, 24th (Room I)

20:00-20:15 Welcome remarks:

Dr. Luis Javier González (Center for Genetic

Engineering and Biotechnology, Havana, Cuba)

20:15-21:00 Opening lecture:

Dr. Gerardo Guillén (Director of Biomedical

Research at CIGB, Cuba)

21:00-21:45 Plenary lecture:

Dr. Patrick Aloy (Principal Investigator of the

Structural Bioinformatics Lab in the Institute for

Research in Biomedicine (IRB Barcelona), Spain)

“Extending the small molecule similarity principle to all

levels of biology”

21:45-22:45 “GET-TOGETHER WELLCOME COCKTAIL

2

ORAL PRESENTATIONS

Friday October, 25th

08:30-9:15 Plenary Lecture (Room I):

Benjamin Haibe-Kains, PhD (Canada)

“Biomarker Discovery in Cancer: Building a bridge between

preclinical and clinical research.”

“Novel instrumental approaches, Sample preparation” (Room I)

Chairpersons: Dr. Florian Meier, Dr. Yassel Ramos

09:20-09:45 Florian Meier, PhD (Germany)

“It takes two to tango: TIMS and PASEF”

09:50-10:15 Christoph Wichmann, PhD (Germany)

“Viva la adquisición! MaxQuant.Live revolutionizes

instrument control”

10:20-10:45 Prof. Jacek Wisniewski (Germany)

“Absolute quantitative proteomics of blood”

10:50-11:15 Dr. Yassel Ramos (Cuba)

“Electrophoretic fractionation previous to MS”

11:15-11:35

3

“PTM enrichment, top-down, quantitative approaches” (Room I)

Chairpersons: Dr. Marta Vilaseca, Dr. Vladimir Besada

11:35-12:00 Dr. Marta Vilaseca (Spain)

“Proteoforms. The new proteomics challenge. Application to

the study of protamines in the human sperm nucleus”

12:05-12:30 Dr. Diana García del Barco (Cuba)

“Epidermal growth factor and growth hormone-releasing

peptide. A combined therapeutic approach for brain

damage”

12:35-13:00 Dr. Iraldo Bello (Cuba)

“HeberFERON, combination of IFNs for cancer treatment.

The OMICS opportunities.”

13:05-13:30 Dr. Dania Marcia Vazquez-Blomquist (Cuba)

“OMICs Approaches In Glioblastoma Multiforme under

INTERFERON co-formulation Treatment”

13:35-14:00 Dr. Vladimir Besada (Cuba)

“Phosphoproteomic study of the antitumorals

CIGB300/CX4945”

14:00-15:00

15:00-17:00 Bilateral meetings

4

“Integration of OMICs in Biomarker discovery and Personalized

Medicines” (Room II)

Chairpersons: Benjamin Haibe-Kains, PhD (Canada), Ana C. deCarvalho,

PhD (USA)

09:20-09:45 Ana C. de Carvalho, PhD (USA)

“OMICS-credentialing of patient-derived glioblastoma

models for pre-clinical studies.”

09:50-10:15 Prof. Marcelo A. Soares (Brazil)

“Novel gene targets in head-and-neck cancer revealed by co-

expression networks using multi-omic data integration.”

10:20-10:45 Prof. Patrick Aloy (Spain)

“Personalized cancer therapy prioritization based on driver

co-occurrence patterns.”

10:50-11:15 Prof. Dimcho Bachvarov (Bulgaria)

“Investigation of epithelial-mesenchymal transition

mechanisms implicated in epithelial ovarian cancer

dissemination by modulation of the LY75 gene expression.”

11:15-11:35

11:35-12:00 Alejandra Bernardini, PhD (Spain)

“Molecular taxonomy of metastatic renal cell carcinoma and

its correlation with outcome to immunotherapy.”

12:05-12:30 Enzo Lalli, M.D. (France)

“Steroidogenic Factor-1 is a Goldilocks transcription factor

regulating key genes implicated in tumorigenesis.”

5

12:35-13:00 Prof. Aspasia Tsezou (Grecia)

“Serum microRNAs in osteoarthritis: novel biomarkers and

therapeutic targets.”

13:05-13:30 Prof. Halina Abramczyk (Poland)

“Pregnancy specific glycoprotein (PSG9) as a potential

biomarker in Preeclampsia (PE).”

13:30-15:00

15:00-15:25 Prof. Martin Hegner (Ireland)

“Quantitative MEMS Molecular Diagnostics.”

15:30-16:15 Dr. Zsuzsanna Izsvak (Germany)

“Pregnancy specific glycoprotein (PSG9) as a potential

biomarker in Preeclampsia (PE).”

6

Saturday October, 26th

08:30-9:15 Plenary Lecture (Room I):

Prof. Y-h. Taguchi (Japan)

“Application of tensor decomposition based unsupervised

feature extraction to multi-omics data set.”

“Human diseases, clinical applications, biomarkers discovery,

plants proteomics” (Room I)

Chairpersons: Prof. J. Wisniewski, Prof. Albert Lebedev

09:20-09:45 Renata Blatnik, PhD (Germany)

“Detection of low abundant viral CD8+ T cell epitopes and

tumor neoepitopes by targeted mass spectrometry”

09:50-10:15 Dr. Eliel Ruiz-May (Mexico)

“Phosphopeptide enrichment for deep phosphoproteomics

in recalcitrant tissues such as fruit peels”

10:20-10:45 Prof. Albert Lebedev (Russia)

“Sequencing of natural frog peptides as possible

pharmaceuticals of future generation”

10:50-11:15 Dr. Meylin Rodriguez (Cuba)

“Bioinformatics as a tool for treatment plant diseases.”

11:15-11:35

7

“System biology, interactomics” (Room I)

Chairpersons: Prof. Marcus Krueger, Dr. Luis Javier González

11:35-12:00 Prof. Marcus Krueger (Germany)

“Skeletal muscle-specific methyltransferase METTL21C

trimethylates p97 and regulates autophagy-associated

protein breakdown”

12:05-12:30 Dr. Maday Fernández-Mayola (Cuba)

“Early proteome movements depicting a GHRP6-mediated

plausible mechanism towards aesthetic wound healing”

12:35-13:00 Hamid Hamzeiy, PhD (Germany)

“Multi-omics analysis of in vivo circadian data reveals

mechanisms of enzyme activity regulation”

13:05-13:30 Dr. Luis Javier González (Cuba)

“Biological and mass spectrometric characterization of

peptide-protein conjugated vaccine candidates against ticks”

13:35-14:00 Dr. Isel Pascual (Cuba)

“Bufadienolides isolated from Peltophryne fustiger

(amphibia: bufonidae) are potent and selective inhibitors of

the membrane-bound neutral Ectoaminopeptidase N:

preliminary effects on the melanoma MEWO cell line.”

14:00-15:00

15:00-16:00 Posters discussion

16:00-17:00 Bilateral meetings

8

“Bioinformatics resources in omics integration and drug

discovery” (Room II)

Chairpersons: Prof. Y-h. Taguchi (Japan) Ph.D Ricardo Bringas Perez

(Cuba)

09:20-09:45 Prof. Graham Ball (UK)

“Artificial Neural Network Algorithms for Biomarker

Identification, Pathway Modelling and Drug Target

Discovery.”

09:50-10:15 Dr. Ming Chen (China)

“Bioinformatics approaches for sing cell and big data

analysis”

10:20-10:45 Benjamin Haibe-Kains, PhD (Canada)

“Drug taxonomy based on big pharmacogenomic data”

10:50-11:15 Dr. Susmita Datta (USA)

“Advances and Challenges of Single Cell RNA Sequencing

Data Analysis.”

11:15-11:35

11:35-12:00 Dr. Jorge Fernández de Cossio (Cuba)

“Resolution of the concept of interaction.”

12:05-12:30 Msc. Jamilet Miranda (Cuba)

“Functional analysis and network approaches elucidate the

mechanism of action of Heberferon in the U87MG glioma cell

line.”

9

12:35-13:35 Benjamin Haibe-Kains, PhD (Canada)

Workshop: “Biomarker discovery in large

pharmacogenomics datasets using R.”

13:35-15:00

15:00-16:00 Posters discussion

16:00-17:00 Bilateral meetings

10

Sunday October, 27th

08:30-9:15 Plenary Lecture (Room I):

Prof. Harald Mischack (Germany)

“CE-MS complementarity to LC-MS: clinical proteomics on

oncology, kidney and cardiovascular diseases.”

“Original tumor targeting strategies (Room I)

Chairpersons: Prof. Ranjan J. Perera (USA), Dr Iraldo Bello Ph.D (CUBA)

09:20-09:45 Ana Campal (CUBA)

“Heberprovac, a GnRH based vaccine directed to prostate

Cancer. Clinical trials results and new challenges.”

09:50-10:15 Prof. Michael Olin (USA)

“Overpowering multiple inhibitory immune checkpoints with

a single peptide inhibitor.”

10:20-10:45 Dr. Julio Raul Fernández (CUBA)

“CRISPR/Cas9-mediated gene knockout of COMMD1

decreases sensitivity of H460 and HCT-116 cell lines to the

antitumoral peptide CIGB-552”

10:45-11:15 Closing remark, closing toast and official group

pictures

“Biomarkers and Drugs in Neurodegenerative Diseases”

(Room II)

Chairpersons: Renã A.S. Robinson, Ph.D Dr. Giselle Pentón-Rol

09:20-09:45 Renã A.S. Robinson, Ph.D. (USA)

“Integrated ‘Omics Strategies to Advance Alzheimer’s

Disease Disparities Research.”

11

09:50-10:15 Dr. Giselle Pentón-Rol (Cuba)-

“Molecular mechanisms involved on the effect of

phycocyanobilin in animal models of multiple sclerosis and

cerebral ischemia.”

10:20-10:45 Javier Marin- Prida, PhD (Cuba)

“Effects of phycocyanobilin on genes expression reveal

neuroprotective mechanisms in a rat model of ischemic

penumbra.”

10:45-11:15 Closing remark, closing toast and official group

pictures

12

13

ORAL SESSION ABSTRACTS Extending the small molecule similarity principle to all levels of

biology Patrick Aloy

Principal Investigator of the Structural Bioinformatics Lab in the Institute for Research in Biomedicine (IRB Barcelona), Spain

paloy@irbbarcelona.org

Large scale small molecule bioactivity data are not routinely integrated in daily biological research to the extent of other ‘omics’ information. Compound data are scattered and diverse, making them inaccessible to most researchers and not suited to standard statistical analyses. In this talk, I will present the Chemical Checker (CC), a resource that provides processed, harmonized and integrated bioactivity data on small molecules. The CC divides data into five levels of increasing complexity, ranging from the chemical properties of compounds to their clinical outcomes. In between, it considers targets, off-targets, perturbed biological networks and several cell-based assays such as gene expression, growth inhibition and morphological profiles. In the CC, bioactivity data are expressed in a vector format, which naturally extends the notion of chemical similarity between compounds to similarities between bioactivity signatures of different kinds. I will show how CC signatures can boost the performance of drug discovery tasks that typically capitalize on chemical descriptors, including compound library optimization, target identification and anticipation of failures in clinical trials. Moreover, we demonstrated and experimentally validated that CC signatures can be used to reverse and mimic biological signatures of disease models and genetic perturbations, options that are otherwise impossible using chemical information alone.

14

Biomarker Discovery in Cancer: Building a bridge between

preclinical and clinical research Benjamin Haibe-Kains1,2,3,4

1Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada 2Department of Medical Biophysics, University of Toronto, Toronto,

Ontario, Canada 3Department of Computer Science, University of Toronto, Toronto, Ontario, Canada 4Ontario Institute for Cancer Research, Toronto, Ontario, Canada

5Vector Institute for Artificial Intelligence, Toronto, Ontario, Canada One of the main challenges in precision medicine consists of developing predictors of drug response to select the most beneficial therapy for each individual patient. In this context, preclinical models are crucial to study the association between molecular features of tumor cells and response to chemical perturbations. However, only few predictors have been successfully translated to clinical settings. Such a low success rate is due not only to the complexity of the mechanisms underlying anticancer drug response, but also to the lack of robustness of the predictors developed in preclinical settings. To address this issue we developed PharmacoGx, a computational platform enabling meta-analysis of large-scale drug screenings of in vitro and in vivo model systems, and PharmacoDB (pharmacodb.ca), a web-application enabling quick access to a large compendium of pharmacogenomics datasets. In this presentation I will show how we used our new platforms to develop univariate and multivariate predictors of drug response that can be further tested in clinical trial data.

15

It takes two to tango: TIMS and PASEF Meier F, Brunner A, Vasilopoulou C, Mann M

Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany

fmeier@biochem.mpg.de

Ion mobility spectrometry separates ions based on their shape and size on a time scale that can be readily nested with liquid chromatography and time-of-flight mass spectrometry. Building on trapped ion mobility spectrometry (TIMS), we have recently introduced parallel accumulation – serial fragmentation (PASEF) which multiplies peptide sequencing speed without any loss in sensitivity by synchronizing ion mobility separation and precursor selection (Meier et al., JPR 2015 and MCP 2018). In data-dependent acquisition mode, more than 100 fragmentation scans per second yield over 6,000 protein identifications from a whole-cell digest of a human cancer cell line in single runs and the sensitivity extends to the lower nanogram range. Transferring the PASEF principle to data-independent acquisition has great potential to further improve coverage and data completeness, while highly reproducible ion mobility measurements provide the basis for deep learning technologies (Meier et al., bioRxiv 2019). Here, we further demonstrate how PASEF extends to other ‘omics’ areas, such as lipidomics (Vasilopoulou et al., bioRxiv 2019). We conclude that PASEF is a valuable addition to the mass spectrometry toolbox, with a number of unique opportunities that are only beginning to be explored.

16

İ Viva la adquisición! MaxQuant.Live revolutionizes instrument

control Christoph Wichmann, Florian Meier, Sebastian Virreira Winter, Andreas-David

Max-Planck Institute of Biochemistry, Martinsried, Germany

Mass spectrometry (MS)-based proteomics is often performed in a shotgun format, in which as many peptide precursors as possible are selected from full or MS1 scans so that their fragment spectra can be recorded in MS2 scans. Although achieving great proteome depths, shotgun proteomics cannot guarantee that each precursor will be fragmented in each run. In contrast, targeted proteomics aims to reproducibly and sensitively record a restricted number of precursor/fragment combinations in each run, based on prescheduled mass-to-charge and retention time windows. Here, we set out to unify these two concepts by a global targeting approach in which an arbitrary number of precursors of interest are detected in real-time, followed by standard fragmentation or advanced peptide-specific analyses. Global Targeting combines the advantages of two classical approaches in MS-based proteomics, whereas greatly expanding the analytical toolbox. Our software tool MaxQuant.Live has a graphical user interface to specify and apply many predefined data acquisition strategies like Global Targeting and BoxCar for example.

17

Absolute quantitative proteomics of blood Jacek R Wisniewski

Max Planck Institute of Biochemistry, Martinsried, Germany Determination of protein concentrations by mass spectrometry-based proteomic

typically requires isotopically labeled standards, making any proteome wide

quantification of proteins difficult and expensive. In contrast, the ‘Total Protein

Approach’ (TPA) allows absolute protein quantitation without any biochemical input.

In the TPA method, calculation of protein abundances is based on spectral intensities

acquired in the large-scale proteomic analyses. Blood tests are often used in health

care to determine physiological and biochemical states, such as disease and organ

function. Many tests include measuring of titers in plasma; however, the number of

monitored proteins is limited. In contrast, proteomics has the capacity to provide

quantitative information on hundreds of plasma proteins. Combination of the TPA

method combined with MED-FASP-consecutive sample digestion strategy allows

accurate quantification of plasma proteins. Analyses of unfractionated plasma cover

protein concentrations spanning six orders of magnitude of protein abundance. In

addition, this technology has been proven as a powerful tool for investigation of

blood cells and vesicles.

18

Electrophoretic fractionation previous to MS

Ramos Y.1, &, Almeida A.1, &, Carpio J.1, Wisniewski J. R.2, González L. J.1 and Besada V1.

1 Department of Proteomics, Center for Genetic Engineering and Biotechnology. Ave 31 e/ 158 y 190. Cubanacán, Playa. La Habana. Cuba. 2 Biochemical Proteomics

Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

yassel.ramos@cigb.edu.cu Protein fractionation is still an important issue in proteomics. The existing

fractionation methods disperse the same protein in few fractions. This aspect is

critical when samples with very high dynamic concentration range are analyzed. The

most remarkable exception is the SDS-PAGE based protein fractionation that due to

its extraordinary resolution and the effectiveness of the SDS for protein

disaggregation and as solubilizing agent, separate proteins more evenly distributed.

Poor recovery of the gel trapped proteins is the main concern of this technique while

the protein electro-elution from the gel is the most successful approach to overcome

this drawback. We developed an equipment for the separation of complex mixture

of proteins and peptides based on the continuous gel electrophoresis/electro-elution

of these molecules. In a user unattended process, complex mixtures of proteins or

peptides are fractionated into the gel while separated fractions are simultaneously

and sequentially electro-eluted to solution containing wells. The performance of the

equipment was studied for SDS-PAGE based protein fractionation in terms of

reproducibility, protein recovery and loading capacity. In a SDS-free PAGE setup,

complex peptide mixture can also be fractionated. The equipment was successfully

applied to bottom-up proteomics. More than 11,000 proteins were identified in the

human cell line CaSki by using the gel electrophoresis/electro-elution sorting

equipment combined with the Filter Aid Sample Preparation method (FASP) and LC-

MS/MS analysis.

19

Proteoforms. The new proteomics challenge. Application to the

study of protamines in the human sperm nucleus Marina Gay2, Ada Soler-Ventura1, Mar Vilanova2, Laura Villarreal2, Gianluca Arauz-Garofalo2,

Josep Lluís Ballescà3, Judit Castillo1, Meritxell Jodar1, Rafael Oliva1, Marta Vilaseca2 1Molecular biology of Reproduction and Development Research Group, Institut

d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Faculty of Medicine, University of Barcelona, Barcelona 08036, Spain, and Biochemistry and Molecular Genetics Service, Hospital

Clinic, Barcelona 08036, Spain 2Mass Spectrometry and Proteomics Core Facility, Institute for Research in Biomedicine (IRB

Barcelona), BIST (The Barcelona Institute of Science and Technology), Barcelona 08028, Spain, 3Clinic Institute of Gynecology, Obstetrics and Neonatology, Hospital Clínic, Barcelona, Spain.

"Top-down proteomics is the only mass spectrometry (MS) technology capable of directly identifying and quantifying proteins with full sequence coverage, without the need for enzymes that digest them into peptides. This technology allows the identification of truncations and mutations, as well as the combinatorial effects of multiple post-translational modifications (PTMs) that occur on the same polypeptide chain. Its ability to provide direct access to the identification and quantification of the huge variety of proteoforms present in dynamic biological systems is indispensable to gain a complete picture of functional regulation at the protein level. Through the detection of precise proteoforms, top-down proteomics holds great promise for the identification of key diagnostic and prognostic markers and therapeutic targets in certain human diseases, and it is expected to make a significant contribution to precision medicine. Developing new proteome-wide strategies to measure proteins with complete molecular specificity presents a formidable but not insurmountable technological challenge. However, once available, such strategies will greatly benefit the biomedical community. I will talk about the developments our group has made in top-down methods focused on the characterization of basic proteins (protamines) in the human sperm nucleus, a clinical study led by Dr. Rafael Oliva. The protamine 1 (P1) and protamine 2 (P2) family comprises the most abundant basic proteins in human spermatozoa and packs 85-95% of the paternal genome. P1 is synthesized as a mature form, whereas P2 components (HP2, HP3, and HP4) are generated from the proteolysis of the precursor. The particular physical-chemical properties of protamines hinder their identification by the standard bottom-up MS strategy. Importantly, the top-down MS approach allowed the characterization of the protamine PTM profile at the intact level. The establishment of the normal protamine PTM profile in fertile individuals and the identification of alteration patterns in different types of infertile patients will provide insight into the role of protamine PTM codes in male fertility and their potential as epigenetic markers during early stages of preimplantation embryogenesis."

20

Epidermal growth factor and growth hormone-releasing peptide. A

combined therapeutic approach for brain damage

Professor Diana Garcia del Barco M.D., Ph.D Center for Genetic Engineering and Biotechnology, Cuba

diana.garcia@cigb.edu.cu Considering the multiple pathophysiological mechanisms involved in

neurodegenerative diseases, combined therapy might be a convenient approach to

induce brain protection. In this regard, some of the pathophysiological phenomena

produced during brain damage are targeted by the citoprotective effects of CIGB845

- the combination of Growth Hormone Releasing Peptide-6 (GHRP6) and Epidermal

Growth Factor (EGF)-, both of which simultaneously target several pathophysiologic

key points involving not only neurons, but also glial cells and vascular endothelium.

The most relevant of the above mentioned citoprotective effects occur on oxidative

stress-induced damage, on mitochondrial dysfunction and on glutamate-induced

excitotoxicity. The latter can explain the salutary therapeutic effects of the

EGF+GHRP6 co-administration observed in experimental models of amyotrophic

lateral sclerosis, multiple sclerosis and stroke during our preclinical evaluations. In

particular, the combined therapy with EGF and GHRP-6 in stroke models has shown

striking evidence of neuroprotective effects. Further proteomic analysis and

functional categorization contributed to explain the positive effects of CIGB845 at a

molecular level, since it was revealed that proteins altered by the effect of CIGB845

act upon relevant biological processes such as inflammation, biogenesis, and

neuroregeneration. The results of our preclinical research have been recently

translated into the clinical arena, wherein “Courage” , a phase I/II clinical trial has

demonstrated that CIGB845 shows a highly safe profile, together with a great impact

on both survival and reduction of disability (assessed by the Rankin scale) at three

and six months after stroke.

21

Heberferon, combination of ifns for cancer treatment. The omics

opportunities. Bello-Rivero I1, Garcia-Vega Y2, Collazo-Caballero S3, Anasagasti-Angulo L4,

Valenzuela C2, Duncan-Roberts1 Y, Santana-Milian H5, Roben-Aguiar Y6, Martinez-Suárez C1, Acosta-Reyes O7, Nodarse-Cuni H1, Muzio-Gonzales V1.

1) Clinical Investigation Department, Center for Genetic Engineering and Biotechnology (CIGB); 2) Clinical Trial Department, Center for Molecular

Immunology, 3) Dermatology Department, Hermanos Ameijeiras Hospital; 4)Peripheral Tumor Department, National Institute of Oncology and Radiobiology;

5) Formulation Department, 6) Production Direction, 7) Business and Project Development, CIGB.

iraldo.bello@cigb.edu.cu HeberFERON is pharmaceutical formulation that combines in synergic proportions

IFNs α-g. The formulation reproduces several proprieties described for IFNS.

Pharmacological distinguishing property is the improved pharmacodynamics with

respect to separated IFNs, even PEGylated variants. Antitumor effectivity has been

proven in phase IV and real world treatment of patients with high risk and advanced

basal cell carcinoma with more that 60% of complete responses. However there is a

need for personalized use due to differences in clinical response rate depending on

tumor subtypes and likely patient characteristics. Patients with renal cell carcinoma

grade III or IV after nephrectomy greatly benefited from the parenteral

administration of HeberFERON with estimated overall survival of more than 50

months. The influence of tumor subtype and immunological patient’s environment

could dictate the final clinical outcome. While the origins of oncologic disease are

genetically encoded, the disease process is largely mediated through altered protein

function. Recent investigations suggest that each individual patient’s tumor

possesses unique kinase-driven cell signaling derangements, and that these

derangements derive, in part, from the tumor’s relationship with its host

microenvironment. Samples from more than 2000 patient with BCC treated with

HeberFERON, is an excellent and valuable opportunity for monitoring treatment

efficacy and toxicity and for predicting treatment outcome using OMICS approaches.

22

Omics approaches in glioblastoma multiforme under interferon co-

formulation treatment Vázquez-Blomquist D1, Besada V4, Miranda J2, Palomares CS5, Hardy A5, Baez S5, Ramos Y4, Guirola O5, Bringas R2, Leenstra S6, Wisniewski J7, Vonasek E8, Gil Y8,

Fernández de Cossío J2, Quiñones M4, Novoa LI1, Palenzuela D1, González LJ4, Bello I3 1Pharmacogenomic Group/System Biology Department, 2Bioinformatic Department,

3Clinical Assay Division, 4Proteomic Group/System Biology Department, 5 Bioinformatic Group/System Biology Department. Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba, 6Department of Neurosurgery, Erasmus MC, Rotterdam, the Netherlands, 7Protein Signaling and Proteomics, Max-Planck

Institute for Biochemistry, Munich, Germany. 8Instituto Venezolano de Investigaciones Científicas (IVIC), Venezuela.

dania.vazquez@cigb.edu.cu CIGB produces recombinant Interferons (IFNs) α and g and the co-formulation HeberFERON, with proved impact on non-melanoma skin carcinoma. In the last years, IFN combinations have also shown promising results in the aggressive and invasive brian tumor, Glioblastoma Multiforme (GBM). As HeberFERON could be used as a therapeutic for this tumor it would be interesting and necessary to understand the way it works in GBM and to obtain some predictive biomarkers for future clinical applications. To accomplish these objectives we used high throughput OMICs methodologies in two different models. We selected the very well-known U87MG cell line (TCGA molecularly as “classical like”) to perform Microarray (Illumina) and Proteomic (LC-MS/MS) experiments, using the same design, with HeberFERON treatment for 72h in comparison to treatments with IFNα or IFNg. We also carried out a Phosphoproteome experiment in this cell line treated with HeberFERON for 72h. A second approach took 34 GBM patient-derived clones, representing the four TCGA molecular classifications (15 classical, 4 proneural, 4 mesenchymal, 3 neural and 8 unclassified) which showed different proliferative responses to HeberFERON, independently of that classification. A Clariom S microarray experiment of these clones treated vs untreated with the co-formulation for 72h was also included as part of the data analysis. Additional validation by quantitative PCR was also included in the experimentation. It is our purpose to share the main OMICs results in both models in an individual but also integrative point of views. This analysis will guide us to describe a general mechanism of action for HeberFERON in GBM. The application of this product could be more precise based on that knowledge.

23

Phosphoproteomic study of the antitumorals CIGB300 and CX-

4945.

Besada, V.1, Perera, Y.2, Ramos, Y.1, Perea, S.2, Guirola, O.3, Wisniewski, J.4, González, L.J.1

1Mass Spectrometry Laboratory, Department of Proteomics; 2Department of Pharmaceuticals, 3Group of Bioinformatics, CIGB, Havana; 4 Proteomics and Signal

Transduction, Max-Planck Institute for Biochemistry, Munich vladimir.besada@cigb.edu.cu

Casein kinase protein (CK2) is a ubiquitous enzyme that catalyze phosphorylation of

hundreds of substrates within cells. The enzyme is involved in cell growth,

proliferation and apoptosis, among others. CX-4945 is a small organic molecule that

binds to the catalytic cleft of casein kinase 2 alpha subunit impairing downstream

phosphorylation of hundreds of substrates. On the other side CIGB300 is a peptide

raised from a phage display library purposed to bind the phosphoaceptor substrate

target of the CK2. Both molecules are facing clinical trials as antitumoral drugs. A

phosphoproteomic study of both molecules was evaluated in two similar acute

myeloid leukemia cell lines: HL-60 and OCI-AML3. Here we present a preliminar

quantitative analysis.

24

OMICS-credentialing of patient-derived glioblastoma models for

pre-clinical studies

deCarvalho AC1,2, Datta I3, Kim H4, Berezovsky A1,2, Nuga O1,2, Malta T1, Verhaak R4, Noushmehr H1, Poisson LM3

1) Dept. of Neurosurgery, Henry Ford Hospital, Detroit, MI USA; 2) School of Medicine, Wayne State University, Detroit MI USA; 3) Dept. of Public Health

Sciences, Henry Ford Hospital, Detroit, MI USA; 4) The Jackson Laboratory for Genomic Medicine, Farmington CT USA.

adecarv1@hfhs.org Glioblastoma is treated with maximal surgical removal, followed by radiation therapy

(RT) and DNA-alkylating agent temozolomide (TMZ). Despite this aggressive multi-

modality treatment, most patients relapse within months, with a dismal 2-year

survival rate of 15.2%. Small molecules and biologicals targeting multiple genomic

abnormalities and oncogenic signaling pathways in glioblastoma have largely failed

interventional clinical trials due to the lack of efficacy. The clinical translational value

of the use of patient-derived models in studies to uncover bona fide vulnerabilities

of cancer cells for targeted therapy depends on recapitulation of the genomic,

epigenomic and phenotypic heterogeneity of the original tumors. We will present

data credentialing a multiple model approach to studying therapeutic response in

glioblastoma through integrated multi-omics analysis. For a panel of molecularly

diverse glioblastoma tumors, whole genome copy number variation, exome

sequencing, DNA methylation, RNA sequencing and targeted proteomics were used

to compare multiple models with the original tumor, to investigate patterns of

molecular adaptive changes in the neoplastic cells to different selective pressures.

Taking advantage of the host uniformity of patient derived orthotopic mouse

xenograft models, we have identified tumor cell intrinsic transcriptional signatures of

fitness and response to DNA-targeting therapy. We further show that oncogene

amplifications in extra-chromosomal DNA is represented in the models and play a key

role in glioblastoma evolution and in therapeutic resistance.

25

Novel gene targets in head-and-neck cancer revealed by co-

expression networks using multi-omic data integration

Costa, RL1; Boroni, M2; Soares, MA1* 1Oncovirology Program, 2Bioinformatics Laboratory, Instituto Nacional do Câncer,

Rio de Janeiro, Brazil masoares@inca.gov.br

Human papillomavirus (HPV) is present in a significant fraction of head-and-neck squamous cell cancer (HNSCC). The main goal of this study was to identify distinct co-expression patterns between HPV+ and HPV− HNSCC and to provide insights into potential regulatory mechanisms/effects within the analyzed networks. We selected cases deposited in The Cancer Genome Atlas database comprising data of gene expression, methylation profiles and mutational patterns, in addition to clinical information. The intersection among differentially expressed and differentially methylated genes showed the negative correlations between the levels of methylation and expression, suggesting that these genes have their expression levels regulated by methylation alteration patterns in their promoter. Weighted correlation network analysis was used to identify co-expression modules and a systematic approach was applied to refine them and identify key regulatory elements integrating results from the other omics. Three distinct co-expression modules were associated with HPV status and molecular signatures. Validation using independent studies reporting biological experimental data converged for the most significant genes in all modules. This study provides insights into complex genetic and epigenetic particularities in the development and progression of HNSCC according to HPV status, and contribute to unveiling specific genes/pathways as novel therapeutic targets in HNSCC.

26

Personalized Cancer Therapy Prioritization Based on Driver

Alteration Co-occurrence Patterns

Patrick Aloy Principal Investigator of the Structural Bioinformatics Lab in the Institute for

Research in Biomedicine (IRB Barcelona), Spain paloy@irbbarcelona.org

Molecular profiling of personal cancer genomes, and the identification of actionable

vulnerabilities and drug-response biomarkers, are the basis of precision oncology.

Tumors often present several driver alterations that might be connected by cross-

talk and feedback mechanisms, making it difficult to mark single oncogenic variations

as reliable predictors of therapeutic outcome. In the current work, we uncover and

exploit driver alteration co-occurrence patterns from a recently published in vivo

screening in patient-derived xenografts (PDXs), including 187 tumors and 53 drugs.

For each treatment, we compare the mutational profiles of sensitive and resistant

PDXs to statistically define Driver Co-Occurrence (DCO) networks, which capture both

genomic structure and putative oncogenic synergy. We then use the DCO networks

to train classifiers that can prioritize, among the available options, the best possible

treatment for each tumor based on its oncogenomic profile. In a cross-validation

setting, our drug-response models are able to correctly predict 66% of sensitive and

77% of resistant drug-tumor pairs, based on tumor growth variation. Perhaps more

interesting, our models are applicable to several tumor types and drug classes for

which no biomarker has yet been described. Additionally, we experimentally

validated the performance of our models on 15 new tumor samples engrafted in

mice, achieving an overall accuracy of 75%. Finally, we adapted our strategy to derive

drug-response models from continuous clinical outcome measures, such as

progression free survival, which better represent the data acquired during routine

clinical practice and in clinical trials. We believe that the computational framework

presented here could be incorporated into the design of adaptive clinical trials,

revealing unexpected connections between oncogenic alterations and increasing the

clinical impact of genomic profiling.

27

Investigation of epithelial-to-mesenchymal transition mechanisms

implicated in ovarian cancer dissemination

Mehdi, S1,2, Bachvarova, M2, Scott-Boye,r M.P2, Droit, A1,2, Mcdonald, E3, Vanderhyden B3, Bachvarov, D1,2

1Dept. molecular medicine, Université Laval, Québec (Québec), Canada, 2Centre de recherche du CHU de Québec, Québec (Québec), Canada, 3Dept. molecular and

cellular medicine, University of Ottawa, Ottawa, ON, Canada. dimtcho.batchvarov@crhdq.ulaval.ca

The mechanisms for the tumorigenesis, progression and dissemination of epithelial

ovarian cancer (EOC) have not been yet fully clarified. We have recently shown that

the mannose receptor LY75 modulates epithelial-to-mesenchymal transition (EMT)

in EOC cells, as LY75 knockout (KO) induced mesenchymal-to-epithelial transition

(MET) in EOC cell lines with mesenchymal morphology, accompanied by a reduction

of their migratory and invasive capacity in vitro and enhanced tumor cell colonization

and metastatic growth in vivo. We used the Ly75-KO model to investigate for DNA

methylation alterations during EMT in EOC by applying the Reduced Representation

Bisulfite Sequencing (RRBS) technology. Numerous genes showing alterations in DNA

methylation during EMT were identified, which could represent new EOC therapeutic

targets. Consecutive methylation data analyses were indicative for the strong

implication of the Wnt/β-catenin signaling pathway in EMT-induced DNA methylation

alterations in EOC cells. This was indirectly confirmed by the identification of Ly75-

interacting partners using an immunoprecipitation technique, followed by mass

spectrometry. Moreover, we are currently applying the LY75 mediated-modulation

of EMT in EOC cells as a model to study in vivo the mechanisms of EMT implication in

EOC metastasis. We are using LY75-KO and control EOC cells to clarify the role of EMT

in EOC progression and treatment response in vivo in an intrabursal orthotopic

xenograft mouse EOC model.

28

Molecular taxonomy of metastatic renal cell carcinoma and its

correlation with outcome to immunotherapy

Bernardini A, Carril L, Suarez-Cabrera C, Dueñas M, Rodriguez Peralto JL, De Velasco G, Paramio JM.

There are three main histolopathological types of renal cell carcinomas (RCC): clear cell RCC –the most common type-, cromophobe RCC and papilar RCC. Furthermore, other RCC subtypes have been decrypted by mutational and transcriptomic data. The metastatic disease (mRCC) is frequently lethal due to its refractoriness to conventional chemotherapeutic agents. The application of targeted therapies and immune checkpoints inhibitors into the clinical of mRCC has increased the overall survival of RCC patients. The mRCC molecular subtypes are different in terms of prognosis but it is not clear how are related to therapy outcome: a variety of treatment responses is observed in mRCC patients subjected to 12 approved drugs belonging to 6 molecularly different mechanisms. We aimed to establish associations between transcriptomic patterns of mRCC patient samples and the response to immunotherapy. We included in the study mRCC samples from 80 patients that have been through at least two lines of treatment. Samples were histologically analyzed and classified into the main RCC types. Tissue samples with at least 60% of tumor content were selected and included in the study. We performed whole gene expression analysis of 45 RCC tumor samples using microarrays. Quality control and gene expression analysis were performed and it was possible to classify the samples in groups accordingly to patterns of gene expression and by hierarchical unsupervised classification independently of their histological type. We did comparisons between histological RCC types in terms of gene expression, gene set enrichment analysis and gene ontology pathways, finding different predictive responses to immunotherapy and the closest relationship between papilar and cromophobe types. We compared tumor transcriptomic features of responders and non-responders patients to the main drugs used in the set of patients: tyrosine kinase inhibitors and PDL1/PD1 immune checkpoint inhibitors, in order to look for transcriptomic profiles that differentiate response groups. The results of this study insight the increasingly importance of describing the complex molecular classification of RCC. This categorization contributes to define predictive biomarkers for current treatments of mRCC, and consequently, to stratify patients in order to provide the best possible treatment plan.

29

Steroidogenic Factor-1 is a Goldilocks transcription factor

regulating key genes implicated in tumorigenesis

LALLI, E Institut de Pharmacologie Moléculaire et Cellulaire; EXPOGEN-CANCER CNRS

International Associated Laboratory; Université Côte d'Azur, Sophia Antipolis, 06560 Valbonne, France

ninino@ipmc.cnrs.fr The transcription factor Steroidogenic Factor-1 (SF-1; NR5A1) regulates tissue-

specific gene expression in steroidogenic cells and has an essential role in the

development of adrenal glands and gonads. Furthermore, it is implicated in the

pathogenesis of adrenocortical tumors and its overexpression correlates with a

severe prognosis in adrenocortical carcinoma (ACC). We have shown that SF-1

regulates distinct set of genes in ACC cells according to its dosage. Those genes are

involved in defining several aspects of the malignant phenotype in ACC cells. We have

performed detailed kinetic analyses of the expression of genes regulated by SF-1 in

adrenocortical cancer cells. Our results reveal that different thresholds of gene

activation or repression exist according to the precise levels of SF-1 expression. We

could also ascertain that SF-1 autoregulates the expression of its own transcript, a

property that can be relevant to modulate its levels in ACC. The scenario then

emerges of SF-1 working as a Goldilocks transcription factor, with distinct dosages of

the protein each regulating a specific set of target genes. These properties are likely

to have an important role for transcriptional regulation of gene expression by SF-1 in

steroidogenic tissue development, physiology and disease. Further studies are in

progress with the aim to correlate SF-1 target gene expression to local chromatin

modifications and to perform single-cell gene expression analysis in conditions of

basal and increased SF-1 dosage in tissue culture cells and in a new mouse model of

Sf-1 overexpression.

30

Serum microRNAs in osteoarthritis: novel biomarkers and

therapeutic targets

Papathanasiou I1., Mourmoura E1., Trachana V2., Tsezou A1,2* 1.Department of Cytogenetics and Medical Genetics, University of Thessaly, Faculty

of Medicine, Larissa, Greece 2. Department of Biology, University of Thessaly, Faculty of Medicine, Larissa, Greece

atsezou@med.uth.gr Osteoarthritis (OA) is a complex degenerative musculoskeletal disease and a leading

cause of pain and disability. Until now there are no disease-modifying drugs

(DMOAD) to slow down or reverse disease onset and progression, leaving costly

surgical interventions (joint arthroplasties) as the only end-point treatment.

MicroRNAs (miRNAs) play a pivotal role in normal musculoskeletal function and in

pathological processes of OA. We established a global serum miRNA signature in OA

patients using a high-resolution microarray technology interrogating 2,549 miRNAs.

Among the 279 differentially expressed miRNAs in the serum of OA patients and

healthy individuals, miR-140 and miR-146a were identified as potential OA

biomarkers and were selected for further investigation. Bioniformatics analysis

revealed common gene targets, pathways and networks for both miRNAs. Functional

analysis in chondrocytes derived from articular cartilage of OA patients that had

undergone knee arthroplasty revealed the synergistic role of miR-140 and miR-146a

in activating the TLR4/NFkB pathway and promoting inflammatory cytokines (IL6,

IL1b, TNFa) production. Using an animal model of osteoarthritis, we showed that

intra-articular injections of miR-140 and/or miR-146a resulted in downregulation of

TLR4, IL1b, IL6 and matrix degrading enzymes, as MMP-13 and in macroscopical and

histological restoration of articular cartilage and synovium integrity, suggesting their

use as potential OA therapeutic targets. In conclusion, miR-140 and miR-146a were

identified, utilizing high resolution microarray technolgoy, in vitro and in vivo

approaches, as potential biomarkers and therapeutic targets for osteoarthritis.

31

Quantitative MEMS Molecular Diagnostics

Duffy, J., Rotella, C., Brunetti, G., DePastina, A., Niosi, F., Certa, U.*, Padovani, F., Hegner, M.

Centre for Research on Adaptive Nanostructures and Nanodevices, School of Physics, Trinity College Dublin, Ireland; *

Martin Hegner@tcd.ie Efficient changes in prevention, diagnosis and therapy are coupled to innovation and

development of new medical tools, which allow a parallel analysis of multiple factors.

We developed an integrated and automatic biosensor platform that provides a non-

invasive, rapid and personalised diagnosis of various onsets of clinical disorders.

Quantitative labelfree nanomechanical bio-analytical measurements provide high-

sensitivity, fast and specific bioassays. Silicon based cantilever array sensors

functionalized with native biomolecules present excellent tools for genomic,

proteomic, microorganism applications and clinical micro-rheology measurements.

MEMS sensors change their mechanical properties (e.g. bend or change their

oscillation frequency) due to forces arising during the interaction of molecules, the

accumulation of interfacial stress, or an increase of mass loading. Real-time

measurements are evaluated using in-situ differential readout (important for specific

nanomechanical measurements). MicroRNAs are involved at the molecular scale and

are extractable biomarkers for a multitude of aberrant biological effects. We

detected specific expression patterns of miRNA relevant to cancer from malignant

cell lysates. miRNA expression profiles associated to adverse drug effects in

hepatocytes derived from necrotic liver tissue were monitored directly in serum using

few microliters within 20 minutes. These new platforms pave the way for portable

nanomechanical diagnostic devices.

32

Nanooncological platform as a tool for proteomic, lipidomic,

glycomic, epigenetic studies.

Abramczyk, H.*1, Kopec, M.1, Surmacki, J.1, Brozek-Pluska, B.1 1 Lodz University of Technology, Faculty of Chemistry, Laboratory of Laser Molecular

Spectroscopy, Lodz, Poland abramczy@mitr.p.lodz.pl

Raman-guided methods for monitoring ‘omic’ (proteomic, lipidomic, glycomic and

epigenetic) modifications will be presented. We developed a novel Raman based

alternative for currently existing epigenetics research approaches. The proposed

Raman approach can ‘upgrade’ cancer epigenetic tests and answer many questions

by monitoring the biochemistry of cancer cells. It will be demonstrated that the

results based on the use of multimode oncological platform: Raman-IR-Fluorescence-

SNOM-AFM imaging-femtosecond spectroscopy provide ultrasensitive, quick, non-

invasive, objective method for monitoring cancer development of the human brain1,

human breast, head and neck, and colon. The oncological platform monitors

metabolic alterations, cell polarity disruption, epigenetic changes that will increase

the sensitivity of diagnostic methods to detect markers of cancer–raising the

possibility of using Raman methods for screening and early detection of cancer and

development of immunotherapy and targeted drug therapy leading to enhanced

ability of patient response to therapy. The Raman–driven oncological platform will

help to analyze proposed biological explanations for the Warburg effect and

enhanced fatty acid synthesis de novo, emphasize their rationale, and discuss their

controversies.

33

Pregnancy specific glycoprotein (PSG9) as a potential biomarker in

Preeclampsia (PE)

Qu, Y; Zadora, J; Dechend, R; Izsvák, Z. All: Max Delbrück Center for Moleculare Medicine, Berlin, Germany

zizsvak@mdc-berlin.de Preeclampsia (PE) is a complex and common human-specific pregnancy syndrome

associated with placental pathology, affecting both mother and foetus. Pregnancy-

specific glycoproteins (PSGs) are the most abundant trophoblast-derived proteins in

maternal circulation during pregnancy. Human PSGs seem to play a role in

modulation of maternal immune responses during pregnancy, regulate trophoblast

cell invasion and vascular remodeling. PSGs are encoded by multigene family (PSG1

– PSG9, PSG11 in human) clustered on chromosome 19. Whether the expansion of

PSGs was to increase the gene dosage or for diversification of function, is not clear.

Although PSG genes share >93% sequence similarity, the expression levels between

different members varies significantly. PSGs are regulated by ancient retrovirus

derived enhancers, sensitive to epigenetic changes. The PSG locus exhibits a high

level of copy number variation and is highly polymorphic in the different ethnic

groups of the human population. Our data shows that PE is associated with an

elevated expression of PSG9. In our RNA-seq data we detected significant

upregulation of PSG9 in human trophoblast cells isolated from early-onset PE vs term

control placenta samples. PSG9 is of a particular interest as a biomarker, as its

elevated expression level is detectable at 15 weeks of gestation in plasma of women

who later developed early-onset PE.

34

Application of tensor decomposition based unsupervised feature

extraction to multi-omics data set

Taguchi, Y-h. Department of Physics, Chuo University, Tokyo 112-8551, Japan

tag@granular.com Although there have been numerous proposals on how to integrate multi-omics data

sets, most of them require the criteria on how to model the interaction between

individual omics data. In contrast to these so called “supervised methods”, the

proposed pethood is fully unsupervised and completely data driven method, which

is free from the bias introduced by human subjectivity. The proposed method can

achieve competitive performance with DIABLO proposed in mixOmics package in R.

In this lecture, In this lecture, I apply the proposed method to integrate, (A) mRNA

and miRNA expression of breast cancer [1], (B) multi-omics data taken from 26 non-

small cell lung cancer (NSCLC) cell lines [2], (C) gene expression and methylation taken

from brain of social insect [3], and (D) promoter methylation and miRNA expression

in TCGA data set [4]. The results are (A) treatment of multi classes (B) one-class

differential expression analysis (C) identification of genes that mediate social caste

development, (D) identification of correlation between regulation of genes by

promoter methylation and miRNA expression. Thus, the proposed method is

expected to have ability to integrate multi-omics data set successfully.

35

Detection of low abundant viral CD8+ T cell epitopes and tumor

neoepitopes by targeted mass spectrometry

Renata Blatnik Center for Molecular Biology of Heidelberg University (ZMBH), Germany

For development of immunotherapies for cancer treatment or treatment of persistent viral infections, detailed knowledge about CD8+ T cell epitopes truly presented on cell surface is fundamental. CD8+ T cell epitopes are 8- to 11-mer peptides that are endogenously produced by the antigen processing machinery and presented on the cell surface for immune recognition. Such peptides do not always contain charged amino acids, which result in poor peptide ionization efficiency. Furthermore, viral and tumor mutation derived epitopes are usually presented at low abundance due to immune evasion mechanisms. Both properties complicate the MS-based identification of epitopes for immunotherapies. We established a targeted LC-MS3 workflow to identify low abundant human leukocyte antigen A2 (HLA-A2) binding epitopes presented on the surface of human papillomavirus (HPV) transformed cells. To this end, epitopes were in silico predicted for their binding affinities and tested for actual binding to HLA-A2 in in vitro binding assays. Verified binders were used for MS reference spectra generation. HLA-A2-epitope complexes were immunoprecipitated (IP) from HPV16-transformed cells and analyzed by targeted LC-MS3. To confirm the presence of target peptides in IP samples, reference peptide spectra were compared to the ones of IP samples. We successfully detected the known HLA-A2-restricted epitope E711-19 and ten additional E7-derived peptides on the surface of HPV16-transformed cells. T-cell reactivity was shown for all 11 detected peptides in ELISpot assays. Interestingly, identified peptides exhibit high, moderate and weak binding characteristics. Thus, it is important that the discovery of epitopes for immunotherapies does not focus only on strong (predicted) binders. Moderate and weak binders should be also considered as immunotherapy targets. Our strategy was successfully applied to detection of low abundant epitopes from cell surface of HIV and CMV infected cells and tumor mutation-derived neoepitopes from tumor cells. This demonstrates that the developed strategy for direct identification of low abundant epitopes on the cell surface is broadly applicable to various major histocompatibility complex I (MHC I) types and target cells. In general, directly identified epitopes are a solid base of future immunotherapy design.

36

Enabling proteomic scrutiny in recalcitrant tissues of tropical fruits

Eliel Ruiz May Red de Estudios Moleculares Avanzados, Clúster Científico y Tecnológico

BioMimic®, Instituto de Ecología A.C. (INECOL), Carretera Antigua a Coatepec No. 351, Congregación el Haya, CP 91070, Xalapa, Veracruz, México.

Arabidopsis thaliana is the plant model for studying several biological processes,

including the application of proteomics approach tools and the comprehensive

profiling of posttranslational modifications. However, the extrapolation of these

proteomics protocols to other plant species is not a straight line. Tropical fruits,

having recalcitrant tissue, limited genomics information, and prevalence of hardened

structures including waxes, cuticle, and cell wall exponentially increase the

complexity of proteomics scrutiny. The molecular composition of the structures

mentioned above, including compounds with different chemical characteristics like

fatty acids, polyphenolics, and sugars challenge current proteomics pipelines.

Consequently, proteomics protocol should be optimized for each plant species and

tissue to starts to envisage the secretes of the biology of tropical fruits. In these five

years we were able to dig deep into the proteome and posttranslational modification

by combining different extraction approaches, fractionation, high-resolution mass

spectrometry approach and multiple search engine (MASCOT, AMANDA, SEQUEST,

ANDROMEDA, and BYONIC) in tropical fruits such as avocado to mention an example.

With our proteomic pipelines, we are paving the avenue for the understanding

biological processes associated with tropical fruits including zygotic and somatic

embryogenesis, postharvest shelf life and fruit-pathogen-plague interactions.

37

Top-down sequencing of frog skin peptides, antibiotics of the next generation

A.T. Lebedev, T.Yu. Samguina Organic Chemistry Department, M.V.Lomonosov Moscow State University,

Moscow, Russia, 119991 Modern mass spectrometry is the most powerful, sensitive, and informative tool for the identification and quantification of chemical compounds. It handles anything beginning from isotopes of chemical elements and finishing with the most complex biopolymers. Nowadays mass spectrometry has become a key-method of bio-medical studies including proteomics. Modern studies in the field of the diseases of the XXI Century make the investigators look at the living representatives of flora and fauna in order to understand their mechanisms of immune defense and protection from the deteriorating environment and pathogenic micro organisms. Amphibians as one of the leaders of immune resistance live on the Earth for hundreds millions of years. Their dorsal glands produce a cocktail of biologically active compounds, mainly peptides, which may successfully fight micro organisms and even predators. Skin secretion of amphibians contains wide spectrum antibiotic and neuro peptides, critical for the immune response and active at the levels of 10-9 М. They can also show antifungal and antiviral activities, stimulate insulin synthesis, inhibit NO synthesis, and be analgesics. The mechanism of action of antibiotic peptides is completely different in comparison to that of the existing pharmaceuticals: amphipatic α-helix destroys phospholipid bilayer, leading to the lysis of the pathogenic cells. Since this mechanism prevents development of the pathogens resistance, antimicrobial peptides are very perspective pharmaceuticals of future generations. Skin secretions of various frog species were obtained by mild electric stimulation. Their LC-ESI-MS/MS analysis was carried out with Thermo ICR and Orbitrap mass spectrometers (Thermo Scientific). CID, ECD, HCD, and ETD were applied in MS2 and MS3 modes to achieve the targeted sequence coverage. To sequence SS-containing peptides crude secretions were preliminary reduced (DTT+ iodoacetamide) or oxidized with performic acid. The developed de novo sequencing algorithm involves the analysis of three versions of original samples of the frogs’ skin secretion: intact, carboxamidomethylated and oxidized ones. The combined analysis allows achieving complete sequence coverage of all frog peptides including long (up to 50 aa) ones. It resolves the problems of S-S bonds, cyclization of short peptides, the presence of isobaric (e.g. lysine/glutamine) amino acid residues in the sequence. An efficient approach of easy and reliable differentiation between isomeric Leu/Ile involves production and isolation of primary z. ions, followed by radical site initiation of their fragmentation with formation of w-ions, characteristic of the isomeric amino acid residues. The resulting spectra are very selective with targeted w ions usually being the most abundant. Extracted ion plotting often applied in environmental tasks demonstrated its efficiency to detect all peptides related to a certain family. More than 200 new natural peptides were sequenced in terms of the present study. Their biological activity against microorganisms was studied. Thus activity of brevinin 1Tb measured with PMEU Spectrion® (Portable Microbe Enrichment Unit) technology appeared to be in the nanomole range, i.e. that of the modern antibiotics. Peptidome representation with 2D-maps based on the simple mass spectrometry parameters shows itself as a very convenient method to distinguish frogs of closely related species, and making mass spectrometry a powerful tool for taxonomy studies. Moreover the applicability of the proposed approach to differentiate the frogs of the same species but different populations was successfully demonstrated. It involves changes in the sequences of similar peptides due to diversity of natural habitat. The animals face different microbes and synthesize the most efficient peptides to fight them. Therefore interspecies and intraspecies biomarkers revealed by mass spectrometry may be very helpful for future taxonomy and biodiversity studies.

38

Bioinformatics as a tool for treatment plant diseases.

Canales, E1; Hernández, I1; Guirola, O2; González, LJ2; Borrás-Hidalgo, O1; Rodríguez, M1; López, Y1; Portieles, R1; Ubieta, R3; Pimentel, E1; Pujol, M1; González, S3 and

Rodríguez, M1 1 Plant Functional Genomics Group, Direction of Agricultural Biotechnology, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. 2 Bioinformatics

Group, Department of Systems Biology, Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba. 3 Patent Department, Center for Genetic

Engineering and Biotechnology (CIGB), Havana, Cuba. meilyn.rodriguez@cigb.edu.cu

Understanding plant responses to pathogen infection is essential to study the

mechanisms of plant-microbe interactions and develop novel strategies for therapy.

Methods and techniques developed in molecular biology in recent years allow the

creation of innovative bioinformatics platforms to structure and analyze genomic

databases that are applied in agricultural biotechnology. In this sense, this work

describes the use of Computer Assisted Drug Design (CADD) method for the selection

of novel synthetic compounds for treating diseases in plants, specifically to develop

a strategy for stimulating the defense and induction of the plant disease natural

resistance. Taking account, the experience of Plant Functional Genomics group in the

study of plant-pathogen interactions in different models, we decided to study the

relationship between brassinosteroid (BR) and immunity in plants. The results

obtained from this study allowed to select a group of synthetic compounds that

activate various plants signaling pathways related to immune response, such as,

salicylic acid, ethylene and jasmonic acid. Also, the novel synthetic compounds

stimulate the systemic acquired response and give the plant a higher level of

resistance against subsequent infection by pathogen. In summary, this work study

the role of new synthetic compounds capable of stimulating defense mechanisms in

plants and by consequently be used in the treatment of diseases.

39

Skeletal muscle-specific methyltransferase METTL21C

trimethylates p97 and regulates autophagy-associated protein

breakdown

Prof. Dr. Marcus Krüger Leader of Research Platform A, Institute for Genetics, Faculty of Math. Nat. Sciences

Skeletal muscle is a plastic tissue that rapidly responds and adapts to many different

patho-physiological conditions. However, the genetic and molecular mechanisms

regulating the protein turnover in skeletal muscles are still incompletely understood

and until now there are no therapeutic interventions available to cure aging-related

muscle loss and muscle-related diseases such as ALS, Duchenne muscle dystrophy,

and inclusion body myositis (IBM).We have characterized the muscle-specific lysine

protein methyltransferase METTL21C and gene ablation in mice results in reduced

muscle performance and elevated cellular residuals during aging. Our integrative

study provides a detailed view of the autophagy lysosomal pathway and highlights

the important function of METTL21C for protein degradation in muscle tissue. We

have carried out extensive proteomics measurements of single muscle fibers and

whole muscle tissue under various conditions, including sciatic nerve sections to

study the METTL21C function under enhanced protein break down conditions. Most

interestingly, we associated the function of METTL21C to the AAA ATPase p97 and

found a reduced trimethylation on p97 reducing its hexamer formation and activity.

Since p97 is an important molecule for protein quality control, we show that the

methyltransferase METTL21C is an essential modulator for protein degradation and

our study is a significant step forward to understand the mechanism underlying

muscle loss in human patients.

40

Early proteome movements depicting a GHRP6-mediated plausible

mechanism towards aesthetic wound healing

Maday Fernández-Mayola (1) | Sucel Palomares (2) | Lázaro Betancourt (2) | Vladimir Besada (2) | Luis J. González (2) | Yssel Mendoza-Marí (1) | Ariana García-

Ojalvo (1) | Jorge Berlanga-Acosta (1) 1. Wound Healing and Cytoprotection Group, Biomedical Research Direction, Center

for Genetic Engineering and Biotechnology, Havana, Cuba 2. Mass Spectrometry and Bioinformatics Group, Department of Proteomics. Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Havana, Cuba

maday.fernandez@cigb.edu.cu Skin fibrosis is a phenomenon characterized by the accumulation of excessive

extracellular matrix during wound healing. The clinical expressions of this pathology

are hypertrophic scars (HTS) and keloids, both affecting patient’s self-esteem and

quality of life. Current therapies still fall short and cause undesired effects. We have

thoroughly proved growth hormone releasing peptide 6 (GHRP6) to be able to

prevent skin fibrosis without untoward reactions in the HTS model in rabbits. We

therefore aimed to elucidate a GHRP6-mediated plausible mechanism towards

aesthetic wound healing by using early proteomics. The results enlightened

unexpected biological processes as to be involved in GHRP6 ability to strikingly

prevent skin over-scarring.

41

Multi-omics analysis of in vivo circadian data reveals mechanisms

of enzyme activity regulation

Hamid Hamzeiy1, Maria S. Robles2*, and Jürgen Cox1* 1Computational Systems Biochemistry, Max-Planck Institute of Biochemistry, Martinsried, Germany. 2Institute of Medical Psychology, Ludwig-Maximilians-

University Munich, Munich, Germany cox@biochem.mpg.de, charo.robles@med.uni-muenchen.de

A common way to study a complex system is to monitor and analyze the multivariate

temporal behavior of its degrees of freedom in order to infer properties of the rules

governing its dynamics. In case the system is a cell, the degrees of freedom are the

concentrations and dynamics of all the biomolecules making up the cell. Multiomics

technologies can monitor comprehensive subsets of these cellular degrees of

freedom, which can be done in a time-dependent manner. Here, we study circadian

rhythms which are endogenously regulated changes experienced by living organisms

at many different levels by combining publically available transcriptomics,

proteomics, phosphoproteomics, lipidomics and metabolomics data in combination

with large-scale metabolic network reconstruction and kinase activity prediction

based on phosphoproteomics data. Our particular interest focuses on enzymatic

control of metabolism via phosphorylation of key sites on proteins. Investigating the

cross-correlation between quantitative phosphorylation changes on a metabolic

enzyme and the abundance changes of the products and reactants of the reaction

being catalyzed can reveal information on the activation mechanism of the enzyme,

which is clearly indicated in our analysis. We also show that the time needed for both

the accumulation and depletion of products and reactants of enzymatic reactions

follows a bimodal pattern and successfully remodel previously known reactions along

with several novel reactions.

42

Biological and mass spectrometric characterization of four peptide-

protein conjugated vaccine candidates against ticks.

González LJ1, Rodríguez-Mallón A1, Encinosa P1, Pousa S1, Espinosa LA1, Cabrera G1, Machado W1, Guirola O1, Almeida F1, Garay H1, Alejandro Leyva2, Rosario Durán2,

Alexei Licea-Navarro3, Samanta Jiménez3, Cabrales A1, Besada V1, Wisniewski J4 and Estrada M1

1Center for Genetic Engineering and Biotechnology, Havana, Cuba. 2Pateur Institut de Montevideo, Uruguay. 3CICESE, Ensenada, México. 4Max Planck Institute for

Biochemistry, Munich, Germany. luis.javier@cigb.edu.cu

GAVAC vaccine has demonstrated to be efficient in the control of the cattle tick

population in field conditions. This vaccine is based on the recombinant Bm86

protein, a hidden antigen located in the intestine of ticks. GAVAC has as a drawback;

it is only efficient against B. microplus, a tick that affect cattle. The Cys1P0 peptide

derived from the acidic P0 protein in the 60S ribosomal subunit from Rhipicephalus

microplus contains B-epitopes for mammals and also is very well-conserved in

sequence alignments vs many others tick species. Taking into account the limitations

of peptides for vaccine development , Cys1P0 was chemically conjugated to three

highly immunogenic carrier proteins: Bm86, KLH (M. crenulata) and p64K(N.

meningitidis). The synthetic procedure uses the MPS chemistry and yields conjugates

highly heterogeneous in size once the peptide is randomly cross-linked to a

considerable number of exposed lysine residues in the carrier proteins. In a second

approach, the six free cysteine residues of p64K carrier protein were used as reactive

centers to obtain less heterogeneous conjugates using a N-terminal maleyl derivative

of bAla1P0 as a modifying reagent. The conjugates were reduced, S-alkylated,

digested with different proteases and analyzed by LC-MS/MS. The identification of

type 2 cross-linked peptides (carrier protein-P0) and the manual validation of the

data allowed the assignment of the conjugation sites in the carrier protein. SDS-PAGE

analysis determined of the average number of P0 peptide units linked to the carrier

proteins. Two conjugates conferred protection in biological evaluations. Challenges

faced in the structural characterization of these conjugates will be presented and

discussed.

43

Bufadienolides isolated from peltophryne fustiger (amphibia:

bufonidae) are potent and selective inhibitors of the membrane-

bound neutral ectoaminopeptidase n: preliminary effects on the

melanoma mewo cell line. Pascual Alonso I1, Rivera L1, Alonso Bosch R2, Valdes-Tresanco ME1, Perera-Córdova

W3, Sánchez B4, Bergado G4, Charli JL5 1Center for Protein Studies, Faculty of Biology, University of Havana, Cuba, 2Museo

de Historia Natural Felipe Poey, Faculty of Biology, University of Havana, Cuba, 3ORISE fellow-Agricultural Research Service, U.S. Department of Agriculture, Natural

Product Utilization Research Unit, University of Mississippi, MS, 38677, USA., 4Centro de Inmunología Molecular, Cuba, 5Instituto de Biotecnología, UNAM,

Mexico. l isel@fbio.uh.cu

Neutral aminopeptidase (APN) is a M1 family membrane ectopeptidase that plays pivotal roles in many physiological and pathophysiological processes; it is a current target for cancer chemotherapy. APN inhibitors from natural sources are scarce, being bestatin and betulinic acid the most widely used, with IC50 values in the µmol/L range. Recently, in Peltophryne fustiger venom, we identified compounds, belonging to the bufadienolide family, characterized by a high hydrophobicity and voluminous groups; both properties agreed with the substrate preference of APN. Considering the biomedical relevance of the development of new APN inhibitors, we validated (using biochemical, bioinformatics and in vitro cellular approaches) various bufadienolides as a new class of APN inhibitors. We studied the APN-Inhibitor interaction using a kinetic approach with a specific substrate. We made an in-depth analysis of their selectivity inside the M1 family. A bioinformatic approach was followed to study APN:inhibitor complex interactions. Additionally, we demonstrated for the first time the presence of APN on the surface of the HTB–65 (MEWO) human melanoma cell line using kinetic assays and evaluated the effect of the new inhibitors on cell viability, cell cycle and DNA degradation. The new molecules inhibited APN in the 10-6-10-7 mol/L range, with kinetic mechanisms that differed according to inhibitor structure. Among M1 family enzymes, aminopeptidase A was the only additional target of some of the inhibitors, with an inhibition mode differing from that of APN. All the molecules displayed strong effects on APN+ MEWO cell viability and DNA fragmentation, but did not induce cell cycle arrest. Bufadienolides are new inhibitors of APN with potential biomedical applications in cancer studies.

44

Artificial Neural Network Algorithms for Biomarker Identification,

Pathway Modelling and Drug Target Discovery.

Ball G.R. Nottingham Trent University, School of Science and Technology, Clifton Lane,

Nottingham. NG11 8NS. United Kingdom. Graham.ball@ntu.ac.uk

Cancer is a complex disease with a myriad of forms and prognoses occurring within

each type. For example, in breast cancer using genomic profiling in excess of 80 sub

types have been identified. The ability to characterise the disease for each patient

may offer the potential to assess the molecular sub-type of the disease and thus

accurately determine the patients’ prognostic outcome. Methodologies such as mass

spectrometry-based proteomics, RNASeq and gene expression arrays offer the

potential for characterisation of disease derived samples using a huge number of

proteins or genes. This depth of information while providing a comprehensive

overview of a disease state also proves problematic in its complexity. One has to

search through potentially hundreds of thousands of pieces of information for

consistent features that address a clinical question in the population. The human

mind is very good at finding patterns in a system but is not able to conduct the task

repetitively for large numbers of parameters. Conversely computers are very good

at searching for features in such a data space, but previously defined statistical

methods are not able to cope with the high complexity. Here we present the

application of Artificial Neural Networks (ANNs, a form of artificial intelligence having

the characteristics of both human pattern recognition and computer automated

searching) to finding genomic solutions to questions in cancer. Here we present the

use of a range of statistical and artificial intelligence-based machine learning

techniques to develop prognostic models for breast cancer. Here we present results

of use of ANN algorithms for biomarker discovery, whereby we have undertaken a

parallel analysis of multiple molecular databases for breast cancer and have

identified markers that drive proliferation and thus predict response to anthracycline.

45

Bioinformatics approaches for sing cell and big data analysis

Chen, M Department of Bioinformatics, College of Life Sciences, Zhejiang University,

Hangzhou 310058, China mchen@zju.edu.cn

With the rapid development of information technology and biological technology, we

are in the big data era. Multi-omics data is available, which brings us a challenge to

develop appropriate bioinformatics approaches to model complex biological systems

at spatial and temporal scales. We were motivated to characterize coding and non-

coding RNAs including microRNAs, siRNAs, lncRNAs, ceRNAs and cirRNAs. An

integrative interactome model of non-coding RNAs is built. Moreover, single-cell RNA

sequencing makes it possible for bioinformatics to reveal expression patterns at the

cellular level. We introduced a transcriptome-based single-cell atlas, and a web-

based pipeline that accurately defines cell types based on single-cell digital

expression. Additionally, we mined key factors of lncRNA regulating renal tumor

metastasis based on single-cell RNA-seq data.

46

Drug taxonomy based on large pharmacogenomic data

Benjamin Haibe-Kains1,2,3,4 1Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario,

Canada 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 3Department of Computer Science, University of Toronto, Toronto, Ontario, Canada 4Ontario Institute for Cancer Research, Toronto, Ontario, Canada

5Vector Institute for Artificial Intelligence, Toronto, Ontario, Canada Identification of drug targets and mechanism of action (MoA) for new and

uncharacterized anticancer drugs is important for optimization of treatment efficacy.

Current MoA prediction largely relies on prior information including side effects,

therapeutic indication, and chemoinformatics. Such information is not transferable

or applicable for newly identified, previously uncharacterized small molecules.

Therefore, a shift in the paradigm of MoA predictions is necessary toward

development of unbiased approaches that can elucidate drug relationships and

efficiently classify new compounds with basic input data. I will present a new

integrative computational pharmacogenomic approach, referred to as Drug Network

Fusion (DNF), to infer scalable drug taxonomies that rely only on basic drug

characteristics toward elucidating drug-drug relationships. DNF is the first framework

to integrate drug structural information, high-throughput drug perturbation, and

drug sensitivity profiles, enabling drug classification of new experimental compounds

with minimal prior information. Application of DNF succeeded in identifying pertinent

and novel drug-drug relationships, making it suitable for investigating experimental

drugs with potential new targets or MoA. The scalability of DNF facilitated

identification of key drug relationships across different drug categories, providing a

flexible tool for potential clinical applications in precision medicine. Our results

support DNF as a valuable resource to the cancer research community by providing

new hypotheses on compound MoA and potential insights for drug repurposing.

47

Advances and Challenges in Single Cell RNA-Seq Analysis

Datta S., Sekula M. and Gaskins, J. College of Public Health & Health Professions College of Medicine, University of Florida, Gainesville, FL, USA; 2) Department of Bioinformatics and Biostatistics,

School of Public Health, University of Louisville, KY, USA susmita.datta@ufl.edu

While the bulk RNA-sequencing (RNA-Seq) data with average (over millions of cells)

transcriptomic abundance measurements have been valuable in countless studies,

they often conceal cell-specific heterogeneity in expression signals that may be

paramount to new biological findings. Fortunately, with single cell RNA-sequencing

(scRNA-Seq) data from individual cells are now accessible, providing opportunities to

investigate functional states of cells and identify rare cell populations. However,

there are challenges in analyzing such data with multimodality, sparsity and

heterogeneity. We will describe ways of modeling such data, finding differentially

expressed genes and possible ways of constructing gene-gene interaction network.

We will compare the performance of our modeling and differential analysis with

respect to some other existing methods.

48

Resolution of the concept of interaction

Fernandez-de-Cossio, J1, Fernandez-de-Cossio-Diaz, J2, Perera, Y3 1) Bioinformatics Department, Center for Genetic Engineering and Biotechnology (CIGB), PO Box 6162, CP10600, Havana (Cuba). 2) Systems Biology Department,

Center of Molecular Immunology, PO Box 6162, CP10600, Havana (Cuba). 3)Molecular Oncology Group, Pharmaceutical Division, Center for Genetic

Engineering and Biotechnology (CIGB), PO Box 6162, CP10600, Havana (Cuba). jorge.cossio@cigb.edu.cu

Prevailing interaction measures in large scale experiments diverge conceptually and

mathematically. The input of such experiments setup solely consists of the incidence

of (causal) factors at one end of the causation process, with the corresponding

outcome at another far end of an inquired effect. These data alone cannot produce

evidence of direct physical contact of the factors, neither when, where or how the

interaction arise. These large-scale studies tacitly convey that interaction arise when

the events triggered by the factors interconnect somewhere in their pathways to the

effect. We attempt here a resolution of the concept as a primary basic need, and

identify a very elementary probabilistic requirement, consistent with our notion of

interaction. The rules of probability theory determine a model multiplicative in the

complement of the effect, which is general, simple and uniquely determined.

Important theoretical properties are revealed along these derivations that has not

been noticed before: 1) intermediating mechanistic details are not required to assess

for interaction, 2) invariance to a priori distribution of factors not related to the

effect, 3) spurious “interaction” due to hidden correlation between the factors are

prevented. Further, the log-linearity of the neutral (no-interaction) model is

demonstrated.

49

Functional Analysis and Network approaches elucidate the

mechanism of action of Heberferon in the U87MG Glioma cell line.

Miranda, J1, Vázquez-Blomquist, D2, Bringas, R1, Fernández de Cossío, J1, Palenzuela, D2, Novoa, LI2, Bello-Rivero, I3.

1Bioinformatic Department, 2Pharmacogenomic Department, 3Clinical Assay Division. Center for Genetic Engineering and Biotechnology, Havana, Cuba.

jamilet.miranda@cigb.edu.cu The anti-tumor effects of interferons have been intensively studied. . Previously it

was reported the higher in vitro antiproliferative response of a combination of alpha

and gamma IFNs (CIGB128-A/Heberferon) compared to the effect of individual IFNs.

A microarray experiment was designed aiming to elucidate the molecular

mechanisms underlying this behavior in the U87MG cell line derived of glioblastomas.

We present the bioinformatics analysis of microarray data on the effect of individual

interferons and the combination of both. Greater emphasis we did on what

distinguishes the combination, so we selected a set of genes with high positive and

negative expression fold change for the combination only. Using an integrative

bioinformatics framework containing differential expression, functional-pathways

enrichment analysis and networks visualization led us to propose model of

mechanism activated by the combination only. A set of genes primarily involved in

cell cycle progression was identified, some with key functions in cycle checkpoints, as

CCNB1 strongly inhibited only by CIGB128-A in both microarray and qPCR

experiments. Signaling downstream of the cell cycle progression were identified,

more precisely in the Prometaphase stage, justifying an antimitotic behavior of

treatment. Furthermore, the relevance of the master regulator of mitosis: FOXM1,

was predicted. Network enrichment analysis revealed several of FOXM1 target genes

downregulated by the combined treatment. Our results suggest new insight into the

mechanism of action of the interferon combination and eventually new treatment

strategies for the treatment of initiation, progression and drug resistance in GBM.

50

CE-MS in clinical proteomics: application in oncology, kidney and

cardiovascular diseases Harald Mischak

Mosaiques Diagnostics, Germany, and University of Glasgow, United Kingdom In depth investigation has demonstrated that naturally occurring peptides demonstrate significant association with several pathologies. These peptides represent excellent biomarkers for specific diseases and have the potential of mirroring biology/pathophysiology. Among the technologies employed to analyze peptides in depth, capillary electrophoresis coupled mass spectrometry (CE-MS) has established itself as a leading technology, due to its robustness and reproducibility. In direct comparison, complementarity of CE- and LC-MS becomes evident, due to the different separation principles. Urine has emerged as a prime source for proteomic biomarkers, also due to its ease of collection. Urinary peptides are produced either by the kidney, or a result of plasma filtration. To date, over 100000 peptides have been tentatively identified in urine, over 5000 are sequenced. Large multicenter studies involving over thousand individuals indicate a significant added benefit of urinary proteomic biomarkers. More than 70.000 samples have been analyzed to date in a comparative way using CE-MS, enabling the generation of the worldwide by far largest peptidome database that serves as basis for the identification of biomarkers based on large numbers of independent samples, a prerequisite when aiming to identify valid biomarkers. The results from these studies also demonstrated that single biomarkers are of limited value as a result of moderate specificity and high variability. However, combining multiple biomarkers into specific classifiers has presented itself as an the ideal approach to assess disease in a non-invasive way with high precision. Since more that two thirds of the urine peptides appear to originate in the kidney, an obvious target for urinary clinical proteomics is the application in chronic kidney disease (CKD). CKD is a major health burden with associated costs exceeding 100 bio € per year worldwide. Urinary peptide biomarkers enable early and accurate detection of CKD (2-5 years prior to clinical diagnosis) and prognosis of future development. Specific urine collagen fragments show the highest predictive value at very early stage CKD. This opens a therapeutic window at an early point in time, possibly even enabling curative treatment. The results have led to the initiation of a first large, multicentric, randomised controlled clinical trial, PRIORITY, aiming at preventing the development of diabetes-associated CKD by early intervention guided by urinary proteomics. Additional fields of clinical application are in the (early) detection of cardiovascular diseases (heart failure, and coronary artery disease) and in the detection and, in some cases, monitoring of malignancies like prostate or bladder carcinoma.

51

Heberprovac, a GnRH based vaccine directed to prostate Cancer.

Clinical trials results and new challenges. Jesús A. Junco1, Ranfis Rodríguez4, Franklin Fuentes1, Idania Baladrón2, Lesvia

Calzada1, Carmen Valenzuela5, Eddy Bover1, Eulogio Pimentel2, Roberto Basulto1, Maria D. Castro1, Francisco Sariol3, Ayni Rodríguez6, Hilda Garay2, Osvaldo Reyes2,

Matilde López2, Ana Campal1, Verena Muzio2, Gerardo Guillén2. 1. Centro de Ingeniería Genética y Biotecnología de Camaguey. Ave Finlay y

Circunvalación Norte, CP 70100, Camaguey, Cuba. 2. Centro de Ingeniería Genética y Biotecnología. Ave 31 e/ 158 y 190, Playa, P.O. Box 6162. Habana 10600, Cuba. 3. Hospital Oncológico Provincial Marie Curie. Carretera Central Oeste esquina

Madame Curie. CP. 70700, Camaguey, Cuba. 4. Instituto Nacional de Oncología y Radiobiología (INOR) Calle 29 esq. a F. Vedado, CP. 1040. La Habana, Cuba.

5. Centro de Inmunología Molecular, Calle 15 esq. 216 S/N, Siboney, Playa, A.P 16040, La Habana, Cuba. 6. Universidad Médica de Camaguey. Carretera Central

Oeste entre Madame Curie y Calle 9. Camagüey. CP 70100. Camaguey, Cuba jesus.junco@cigb.edu.cu

GnRH-based vaccines represent a promising anti-hormonal treatment alternative in prostate cancer, because they can reduce serum testosterone to castrating levels, avoid the "hot flushes” produced by GnRH analogues and can be administered in acute and complicated forms of prostate cancer. The present study assesses the application of Heberprovac, a GnRH based vaccine candidate for patients suffering from advanced prostate cancer and their following up to ten years in 2 clinical trials. The main objective of the first clinical trial was to evaluate the safety and possible efficacy indicators and the second, Phase II clinical trial, to evaluate the safety and efficacy of this vaccine candidate in 4 levels of dosage. To this aim, 6 patients affected by advanced prostate cancer diagnosed by biopsy, were included in the first clinical trial and 56 were treated in the second one. As result of the first clinical trial it was generated a 100% of effective anti GnRH immune response that corresponded with a significant testosterone reduction until castration levels, normalization of PSA and full clinical response in the whole patients subset. On the other hand, the Phase II clinical trial revealed that after the first immunizations, the patients exhibited anti GnRH antibodies and in turn, testosterone levels reduction. In concordance with the hormonal and immunological response, the patients exhibit a decrease of both; the number of obstructive symptoms as well as the severity of them. There was also a normalization of the prostatic specific antigen (PSA) in the 80% of the patients after they finished the last immunization and the clinical evaluation demonstrated the significant reduction of the primary tumor from grades III/IV to I/II in all the patients that respond biochemically. Currently a Phase II/III controlled clinical trial is planned to be carried out in more than 300 advanced prostate cancer patients in Cuba.

52

Overpowering multiple inhibitory immune checkpoints with a

single peptide inhibitor. Dr. Michael Olin, PhD

Assistant Professor in the Division of Pediatric Hematology and Oncology University of Minnesota.

olin0012@umn.edu Given the modest effects current immune checkpoint-inhibitors have on patients

with CNS tumors, we focused our studies on the novel CD200 immune checkpoint.

The CD200 checkpoint suppresses the immune system through binding of the

inhibitory CD200 protein to the inhibitory receptor (CD200R1) expressed on immune

cells. In addition to CD200R1, the CD200 checkpoint includes activation receptors

(CD200AR), which we are targeting with a peptide ligand (CD200AR-L) to reverse the

inhibitory effects of the CD200 protein. We showed that spontaneous high-grade

glioma dogs receiving a canine specific CD200AR-L in combination with autologous

tumor lysate vaccine had an increased median overall survival of 9 months, compared

to 6.3 months with lysate alone, resulting in an overall 36-month survival of 21%.

However, we have a 45% event-free (progression-free) group of dogs that died of

non-tumor related deaths with an extended median survival of 18 months. Six dogs

remain on trial, 87% of the dogs receiving tumor lysate only died of tumor recurrence

within 6 months. Serum chemistry profiles and physical examinations showed that

the peptide did not induce any systemic toxicity. To determine how we obtained

these unprecedented results, we focused on the mechanism of CD200AR-L/CD200AR

binding. We discovered DAP10/12 signaling pathways and CD200ARs are

upregulated, while the inhibitory CD200R1 is downregulated. We discovered the

molecular connection between CD200, PD-1 and CTLA4. We demonstrated that our

murine and human CD200AR-L downregulates the inhibitory PD-1 and PD-L1 and

inhibits the upregulation of CTLA4. Therefore, we attribute the success of CD200AR-

L to the ability to concurrently modulate multiple immune checkpoints. We have now

developed human GMP grade CD200AR-L peptides to develop an immunotherapy

approach for patients with CNS tumors; we will conduct a Phase I trial utilizing the

peptide ligand concomitantly with an allogeneic GBM6-AD cell line as a tumor vaccine

and the adjuvant imiquimod.

53

CRISPR/Cas9-mediated gene knockout of COMMD1 decreases

sensitivity of H460 and HCT-116 cell lines to the antitumoral

peptide CIGB-552. Garcia G, Fernández JR; Palenzuela D, Guerra M.

Centro de Ingeniería Genética y Biotecnología (CIGB), La Habana, Cuba julio.fernandez@cigb.edu.cu

Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a critical role across

many cellular processes including embryonic and neuronal development, cell

proliferation, apoptosis, immune responses to infection, and inflammation.

Dysregulation of NF-κB signaling is associated with inflammatory diseases and certain

cancers. Constitutive activation of NF-κB signaling has been found in some types of

tumors including breast, colon, prostate, skin and lymphoid, hence therapeutic

blockade of NF-κB signaling in cancer cells provides an attractive strategy for the

development of anticancer drugs. CIGB-552 is a novel synthetic peptide derived by

modification of its primary structure from the antimicrobial peptide LALF32-51

(Limulus sp) which has been shown to be a potential candidate for the anticancer

therapy and one of its useful property is the cell-penetrating capacity. CIGB-552 binds

and stabilizes COMMD1 protein, which inhibits the transcription factor NF-kB, a

factor linked to the proliferation, survival and metastatic capacity of the cancer cell.

The accumulation of COMMD1 promotes the ubiquitination of the RelA subunit of

NF-kB and its proteosomal degradation, causing apoptosis of the cancer cells. CIGB-

552 modulates the expression of genes related to the cell cycle, apoptosis and

oxidative stress among others. CIGB-552 peptide blocks cell proliferation followed by

death by apoptosis and causes oxidative damage to lipids and proteins. Here, we

provided the expression profile data of COMMD1 knockout in a H460 cell line treated

with CIGB-552 and shows that the Knockout of COMMD1 decreases sensitivity of

H460 and HCT-116 cell lines to the antitumoral peptide CIGB-552.

54

Integrated ‘Omics Strategies to Advance Alzheimer’s Disease

Disparities Research Khan, MJ; Stepler, KS; Robinson, RAS, *

Department of Chemistry, Vanderbilt University Nashville TN 37235, USA Rena.as.robinson@vanderbilt.edu

Racial disparities in Alzheimer’s disease exist for certain subpopulations whereby for

example, African Americans have two to three times incidence of disease compared

to NonHispanic Whites. Genetic, socioeconomic, and comorbidities can contribute to

this incidence however if there are biological differences that can explain this

disparity do not exist. Our laboratory has been working to explain these differences

using an integrated proteomics and lipidomics strategy. The approach takes

advantage of tandem mass tags and comprehensive shotgun proteomics as well as

both untargeted and targeted lipidomics mass spectrometry analysis. This

presentation will discuss these approaches and their application to post-mortem

brain tissue and plasma from a cohort of African American and White Alzheimer’s

disease and cognitively normal patients.

55

Modulation of genes for the combination phycocyanobilin and ifn

beta promises greater expectations for a new therapy for multiple

sclerosis G Pentón-Rol (1), N Pavón-Fuentes (2), J Marín-Prida (3), M Cervantes-Llanos (1), H

Camacho- Rodriguez (1), B Piniella-Matamoros (1) JR Fernandez-Massó (1), MM Teixeira (4)

(1) Center for Genetic Engineering and Biotechnology (CIGB), Ave. 31 e/ 158 y 190, Cubanacán, Playa, Havana, PO Box 6162, Cuba (2) International Center for

Neurological Restoration (CIREN), Ave. 25 e/ 158 y 160, Playa, Havana, PO Box 11300, Cuba (3) Center for Research and Biological Evaluations (CEIEB), Institute of

Pharmacy and Food, University of Havana, Ave. 23 e/ 214 y 222, La Lisa, Havana, PO Box 430, Cuba (4) Laboratory of Immunopharmacology, Department of

Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Pampulha, Belo Horizonte, MG, Brazil

giselle.penton@cigb.edu.cu Introduction: Multiple sclerosis (MS) is leading causes of neurological disability among adults. We have evaluated the effect of combination Phycocyanobilin (PCB) and IFN beta (PCB/IFNb) on genes modulation related to the events of MS pathogenic mechanism such as: demyelination, immunomodulation and oxidative stress in animal models of MS (EAE). Material and Methods: EAE was induced in C57BL6 mice with MOG peptide. PCB was administered by oral route daily and IFN Beta subcutaneously every second day. Total RNA from brain was extracted and the expression of the genes LINGO1, CXCL12 (demyelinating/remyelinating processes), Tbet, GATA3, RORg, Foxp3 (effector/regulator balance) and Hmox1 and Nfr2 (oxidative stress) were evaluated by Real-time-quantitative-PCR (qPCR). Furthermore, we screened spleens for regulatory T cell subset modulation/induction using flow cytometry. Results: There was an effect of combination PCB/IFNb demonstrated by a downregulation of the genes linked to demyelination and an upregulation of genes involved in remyelination. The ratio CXCL12/LINGO1 was upregulated. The genes for the transcriptional factors GATA3 and RORg were downregulated only by o after the treatment of PCB/IFNb combination; a similar regulation was observed for the Hmox1 gene. Moreover, an induction of regulatory T cells in the spleen of combination treated animals of EAE model was verified by flow cytometry. Conclusion: The genes evaluated are currently the most important within the most novel pathways and targets for MS research and can support the link between Treg and remyelination in MS

56

Effects of phycocyanobilin on genes expression reveal

neuroprotective mechanisms in a rat model of ischemic penumbra Marín-Prida J1*, Pavón-Fuentes N2*, Llópiz-Arzuaga A3, Fernández-Massó JR3,

Polentarutti N 4, Riva F 5, Pentón-Arias E3, Pentón-Rol G3. 1Center for Research and Biological Evaluations (CEIEB), Institute of Pharmacy and

Food, University of Havana, Cuba; 2International Center for Neurological Restoration (CIREN), Havana, Cuba; 3Center for Genetic Engineering and

Biotechnology (CIGB), Havana, Cuba; 4Istituto Clinico Humanitas (IRCCS), Rozzano, Italy; 5 Department of Veterinary Science and Public Health, University of Milano,

Italy (DIVET). *equal contribution javier.marin@ifal.uh.cu

Introduction: Stroke remains as one of the main causes of mortality and disability worldwide without effective treatments. The main objective of acute neuroprotective interventions is to preserve the penumbra brain tissue from ischemic injury. Here, we aimed to assess the effects of the open-chain tetrapyrrole Phycocyanobilin (PCB), on genes expression in acute hypoperfused rat brains. The drop in cerebral blood flow during the acute phase of this model is comparable to ischemic penumbra, thus offering a reliable tool for studying molecular mechanisms. Material and Methods: The animals were subjected to permanent bilateral common carotid artery occlusion (BCCAo), and then treated with saline or PCB intraperitoneally in equal subdoses for 30 min, 1, 3 and 6h post-surgery. Samples were taken 24h after the surgery, and processed for RNA extraction. The analysis of the entire genome expression was carried out with GeneChip Rat Gene ST 1.1 from Affymetrix, and qPCR served to assess the expression of particular genes by using the Fast SYBR Green PCR Master Mix method. Results: The PCB treatment at 213 µg/Kg significantly modulated 190 genes (93 up- and 97 down-regulated) in the anterior cerebral cortex associated to several immunological processes in both the regulatory and effector subsets. The qPCR analysis showed a dose-dependent PCB positive modulation of 13 genes in three brain areas, the olfactory bulb, the anterior cerebral cortex and the hippocampus, revealing important neuroprotective mediators associated to remyelination, energetic metabolism, anti-apoptotic pathways, synaptic plasticity and angiogenesis. Conclusion: Our results support the view of major inflammatory mediators in acute cerebral hypoperfusion, effectively counteracted by PCB. This compound was also able to effectively modulate the gene expression related to brain protective mechanisms. These results point into the potential application of PCB as a new acute disease modifying therapy against ischemic stroke.

57

POSTER SESSION BioInfOMICS-01 Association of rs4278932 polymorphism of MERKT gene with

susceptibility to multiple sclerosis in Cuban population. Cintado A, Fernández-de-Cossío ME1, Ale A1, Díaz T1, Villarreal A1, Grass D2, Cervantes M1, Pavon N2, Cabrera JA2, Pentón G1.

BioInfOMICS-02 Glycosylation pattern of the Human Epidermal Growth Factor Receptor obtained in Human Embryonic Kidney tissue derived cell line (HEK293). Cabrera, G. and Musacchio, A.

BioInfOMICS-03 Analysis of a library of compounds with possible inhibitory properties in the MyD88-mediated signaling pathway. Colarte, A.B; Fernandez, JR; Mejias, C; Guirola, O; Penton, G.

BioInfOMICS-04 Comparative genomics with Neisseria Meningitidis serogroup B: insights into cross reactivity of VA-MENGOC-BC. Gregorio, ADJO; Gómez, RID; Peña, EAE

BioInfOMICS-05 PROINFLAMMATORY CYTOKINES IN ALZHEIMER’S DISEASE PATIENTS Piniella Matamoros B1, Rodriguez Tanty Ch2, Pentón Rol G1

BioInfOMICS-06 Structural insights into High Affinity PD-1 (HACPD1) and PD-L2 interaction. An in silico approach Mejías, C.1; Guirola, O.1

BioInfOMICS-07 Rational basis for MyD88 inhibitors development: an in silico approach Mejías C.1; Guirola O.1

BioInfOMICS-08 Challenges of Pharmacoeconomics towards Precision Medicine García-Díaz, D

BioInfOMICS-09 The impact of flow regime over dengue virus shadow effect of propeller shapes proteins E. Navas-Conyedo1, ∗, V. Huerta-Galindo2, M. Pupo-Meriño1, J. Gulín-González1, O. Guirola2 and N

BioInfOMICS-10 Molecular Dynamics Simulations of the cyclic and antiparallel dimer analogues of the antifungal peptide Cmp5 in a fungal membrane model Díaz, E1; Mejías, C2; Morales, F. E3; Garay, H.E1

58

BioInfOMICS-11 Strategy based on pan-genomics for rational design of vaccines against Neisseria Meningitidis serogroup B. Sánchez, FF; Ramírez, RP; Bermúdez, PER

BioInfOMICS-12 Mass spectrometric characterization of Bm86-Cys1pP0: A bivalent vaccine candidate to control the cattle-tick population. Gleysin Cabrera1, Alina Rodríguez Mallón1, Satomi Pouza1, Fabiola Almeida1, Osmany Guirola1, Samanta Jiménez2, Alexei Licea-Navarro2, Madelón Portela3, Rosario Durán3, Ania Cabrales1, Hilda Garay1, Mario Pablo Estrada1 and Luis Javier González1

BioInfOMICS-13 Expression profile of TLR pathway genes in a colitis biomodel. Camacho H1*; Roca J1*; Aguilera A2*; Guillen IA1; Bermudez Y2; Bacardí D3; Suarez JA3; Delgado Y1 and Palenzuela DO1

BioInfOMICS-14 CGWork: a tool for the search of the common genome with applications in the rational design of vaccines. Garí, JAJ; Mena, VV

BioInfOMICS-15 Mass spectrometric characterization of low‐abundance species in the active pharmaceutical ingredient of synthetic peptides Espinosa LA1; Garay H2; Perera Y3; Sánchez A1; Diago D2; Perea S3; Domínguez MC4; Junco JA5; Besada V1; González LJ1

BioInfOMICS-16 PHYCOCYANOBILIN MOLECULAR MECHANISMS ON A DAMAGE MODEL INDUCED WITH GLUTAMATE IN SHSY5Y CELLS Cervantes-Llanos M; Camacho H; Piniella-Matamoros B; Pentón Rol G.

BioInfOMICS-17 An integrative analysis of evolutionary pressures over 3C and VP1 segments in Coxsackievirus A24v Pupo-Meriño M1, Fonseca M2 and García LA3

BioInfOMICS-18 Characterization by mass spectrometry of the KLH1-Cys1pP0 and KLH2-Cys1pP0 conjugates: a vaccine candidate against ticks. Satomy Pousa1, Vladimir Besada1, Jacek Wisniewski2, Osmany Guirola1, Ania Cabrales1, Hilda Garay1, Alina Rodríguez-Mallón1, Mario Pablo Estrada1 and Luis Javier Gonzalez1.

BioInfOMICS-19 INTEGRATIVE MODEL OF RHODANINE DERIVATIVES AS TAU AGGREGATION INHIBITORS IN ALZHEIMER’S DISEASE Álvarez-Ginarte Y. M, Lecrerc Fabrice and Montero-Cabrera L.A.

59

BioInfOMICS-20 BitClust: Fast conformational clustering of long Molecular Dynamics simulations. Roy González-Alemán†*, David Hernández-Castillo†, Alejandro Rodríguez-Serradet†, Julio Caballero‡, Erix W. Hernández-Rodríguez ɸ, Luis Montero-Cabrera†

BioInfOMICS-21 PREDICTION OF MOLECULAR INTERACTIONS AND PHYSICOCHEMICAL PROPERTIES RELEVANT FOR VASOPRESSIN V2 RECEPTOR ANTAGONISM de la Nuez A, Álvarez-Ginarte Y. M, Rodriguez R and Montero-Cabrera L.A

BioInfOMICS-22 Metabolome Analysis of Breast Tumors for Discovery of Oncometabolites Ambs, S

BioInfOMICS-23 Toward the uses of antimicrobial peptides against multidrug resistant bacteria Montero-Alejo V*, Perdomo-Morales R*, Vázquez A*, Rodríguez-Viera, L**, Roque-Díaz Y*, Bello-Madruga R*, Garay-Pérez H§, Álvarez-Valcárcel C+.

BioInfOMICS-24 Proteotranscriptomics Reveals Associations of Protein-mRNA Concordance with Breast Cancer Subtypes and Survival Tang, W

BioInfOMICS-25 LC-MS/MS characterization of two conjugated vaccine candidates against ticks obtained by different crosslinking strategies between the carrier protein p64K from N. meningitidis and a peptide derived from the acidic ribosomal P0 protein from R. sanguineus. Wendy Machado1, Luis Ariel Espinosa1, Gleysin Cabrera1, Alejandro Leyva2, Nobuaki Okumura3, Rosario Durán2, Toshifumi Takao3, Alina Rodríguez-Mallón1, Mario Pablo Estrada1,Hilda Garay Perez1,Ania Cabrales Rico1, and Luis Javier González1.

BioInfOMICS-26 Integrated Genomics to Uncover Clinically-relevant Liver Cancer Drivers Wang, XW

60

BioInfOMICS-27 Epidermal Growth Factor regulates LPS-induced inflammatory scenario in fibroblasts derived from diabetic foot ulcers Mendoza-Marí, Y, García-Ojalvo, A, Fernández-Mayola, M, Berlanga-Acosta, J

BioInfOMICS-28 NETWORK INTERACTIONS AND SIGNALING PATHWAYS ANALYSIS FOR A PHOSPHOPROTEOMICS EXPERIMENT WITH HEBERFERON IN GLIOBLASTOMA CELLS Hardy A1*, Palomares S, Baez S, Besada V, Guirola O, Bello I, Vazquez-Blomquist D, Ramos Y, Wisniewski J and Gonzalez, LJ

BioInfOMICS-29 FUNCTIONAL ANALYSIS OF A PHOSPHOPROTEOMICS EXPERIMENT WITH HEBERFERON IN GLIOBLASTOMA CELLS Baez S*, Palomares S, Hardy A1, Besada V, Guirola O,Bello I, Vázquez-Blomquist D, Ramos Y, Wisniewski J and González LJ.

BioInfOMICS-30 Oxidative stress parameters as biomarkers for new multiple sclerosis therapies Herrera, T1,*; Lagumersindez, N2; Delgado, L2; Marín, J2; Rodríguez, H1; Cepero, J1; Valenzuela, C3; Cintado, A4; Pentón, G3.

BioInfOMICS 31 Phylogenetic and phylodinamic of Coxsackievirus A24 variant in Cuba over a 23- year period. 1986-2009. Fonseca MC1, Pupo M2, Norder H3, García LA2, Resik S1, Hung LH1, Muné M1, Rodríguez H1, Morier L4, Sarmiento L5

61

POSTER SESSION ABSTRACTS Association of rs4278932 polymorphism of MERKT gene with

susceptibility to multiple sclerosis in Cuban population.

Cintado A, Fernández-de-Cossío ME1, Ale A1, Díaz T1, Villarreal A1, Grass D2, Cervantes M1, Pavon N2, Cabrera JA2, Pentón G1.

1Pharmacogenomics Department. Biomedical Research Division. Center for Genetic Engineering and Biotechnology, 31 entre 158 & 190, Cubanacán, Playa, Cuba.

2International Center of Neurological Restoration. Ave 25 no 15805 entre 158 & 160 Cubanacan Playa.

alberto.cintado@cigb.edu.cu

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system. The risk of developing MS is influenced by environment and genetic factors with the HLA-DRB1*1501-DQBI*602 haplotypes being the predominant genetic risk factor. Other genetic associations have been identified using genome-wide association studies (GWAS). For example, nucleotide variation in IL7R, IL2RA, CLEC16A genes has been consistently associated with MS in some populations. .In the present study, we analyzed allelic associations between polymorphisms of MERKT and NR1H3 genes selected by GWAS and susceptibility to MS on Cuban patients. Blood samples were collected from 100 MS patients and 200 unrelated controls. Genomic DNA was isolated from blood using wizard Genomic DNA purification Kit. SNPs genotypes were determined by Taqman assay method and allele frequencies were estimated by means of expectation maximization algorithm. Statistic associations were calculated using the Chi-square test. Odd ratios (OR) with 95% confidence intervals (95%) were calculated using Fisher's method. The result showed only association between rs4278932 of MERKT gene and MS (p<0,05). No significant differences were observed between MS patients and controls (p>0,05) regarding to rs61731956 (p.Arg415Gln) and other SNP of NR1H3 gene. The results suggested that rs427893 could be considered as a risk factor in the susceptibility of MS in Cuban population.

62

Glycosylation pattern of the Human Epidermal Growth Factor

Receptor obtained in Human Embryonic Kidney tissue derived cell

line (HEK293). Cabrera, G. and Musacchio, A.

System Biology Department, Center for Genetic Engineering and Biotechnology. La Habana, Cuba.

alexis.musacchio@cigb.edu.cu

The human epidermal growth factor receptor (EGFR) belongs to the erbB family of

four closely related cell membrane receptors, also known as the type I tyrosine kinase

receptor family. It consists of an extracellular ligand binding -, a transmembrane - and

an intracellular domain with tyrosine kinase activity for signal transduction. EGFR

plays an essential role in the regulation of both development and neoplastic

processes. By interacting with their specific ligands, there is an induction of receptor

activation, modulation of cell proliferation and differentiation in normal tissues and

tumors. Glycosylation is one of the most common post-translational modifications,

and almost half of all known proteins in eukaryotic cells are glycosylated. Changes in

oligosaccharide structures are associated with several physiological factors and

pathological events. Similarly, glycosylation patterns for the same protein vary

between cells of different origin. Results: The N- and O- glycosylation in the EGFR

extracellular domain obtained in the HEK293 cell line, were determined. The

identified N- glycan structures are from oligomanosidic and complex types, while the

O- glycan structures are of the mucin type. O-mannosylation can also be found. The

HEK293 cell line contains all the glycosyltransferases necessary to form

multiantennated, hyperfucosylated and bisected complex N- glycans. The conditions

to obtain the EGFR extracellular domain were established, containing only N-

acetylglucosamines linked to asparagine residues, corresponding to the N-

glycosylation sites, after successive treatments with exoglycosidases, Endo H and O-

glycosidases, as well as the complete deglycosylated receptor variant from successive

treatments with PNGase F and O-glycosidase.

63

Analysis of a library of compounds with possible inhibitory

properties in the MyD88-mediated signaling pathway.

Colarte, A.B; Fernandez, JR; Mejias, C; Guirola, O; Penton, G. Pharmacogenomics Department. Biomedical Research Division. Center for Genetic

Engineering and Biotechnology, 31 entre 158 & 190, Cubanacán, Playa, Cuba. amanda@cigb.edu.cu

Current anti-inflammatory therapies cause adverse events, which has stimulated the search for more selective and potent drugs. MyD88 is an important therapeutic target to control undesirable immune responses. The use of cell lines that express a reporter gene under the control of the transcription factor NF-kB plays a key role in the identification of inhibitory compounds of MyD88-mediated signaling. The activation of NF-κB has direct applications in the discovery of drugs for various therapeutic indications. In our work we generated and characterized a HEK293-SEAP reporter cell line and also analyzed a library of compounds with possible inhibitory properties in the MyD88-mediated signaling pathway. Organic molecules from VS database were used in a virtual screening against the cavity E in the TIR domain of MyD88. We applied the scoring function implemented in AutoDock Vina3 program to order the compounds. This is one of the most used tools for molecular coupling studies, due to its efficiency and correlation with experimental energy values of protein-ligand affinity. The NF-kB activation assay in the stable cell line HEK293-SEAP show that MyD88-mediated activation is selective for IL-1β. We use the SEAP detection assay to evaluate 76 products at 50 µM. As a result, none of the compounds was able to selective inhibit Myd88 mediated signaling. Cytotoxicity was observed in 22 compounds. The possible inhibitory capacity of these molecules should be evaluated at lower concentrations.

64

Comparative genomics with Neisseria Meningitidis serogroup B:

insights into cross reactivity of VA-MENGOC-BC.

Gregorio, ADJO; Gómez, RID; Peña, EAE Departamento de Bioinformática, Facultad de Ciencias y Tecnologías

Computacionales, Universidad de las Ciencias Informáticas (UCI), Carretera a San Antonio de los Baños, Km 2 ½, Torrens, Boyeros, La Habana, Cuba.

antoniodjog@estudiantes.uci.cu

Meningococcal meningitis is a serious bacterial infection of the membranes that surround the brain and spinal cord. It can cause significant brain damage and is fatal in 50% of untreated cases. Although there are effective vaccines against most serogroups of this pathogen, in the case of serogroup B, vaccines based on outer membrane vesicles (OMV) are generally strain-specific, they continue to be the ones with the best results. Cuba has a vaccine of this type patented in 1991 that served to contain the epidemic caused by the strain CU385 in the early 90s of the last century and is part of the national vaccination program. Since cross-reactivity responses were observed against other strains of serogroup B induced by this vaccine preparation, interest in this also makes it possible to continue its commercialization internationally. Recently as part of a European study the genome of this strain was sequenced and published in the NCBI database by segments. The availability of this genome together with several of this serogroup offers the possibility of carrying out a comparative genomic study that allows the identification of membrane proteins present in the outer membrane vesicle (OMV) of VA-MENGOC-BC that justify its cross-reactivity. This paper presents a preliminary result made with a subset of genomes available from said serogroup, in which light is shed on the possible causes of cross-reactivity.

65

Proinflammatory cytokines in alzheimer’s disease patients

Piniella Matamoros B1, Rodriguez Tanty Ch2, Pentón Rol G1 1 Center for Genetic Engineering and Biotechnology, Pharmacists Department,

Havana, Cuba. 2 Cuba Neurosciences Center, Havana, Cuba. beatriz.piniella@cigb.edu.cu

Introduction: Alzheimer's disease (AD) is accompanied by a chronic inflammatory process, including activation of microglia and astrocytes that express pro-inflammatory cytokines. Altered cytokine levels might reflect the neuropathological changes in patients. It is unclear to what extent this inflammation is a reaction to the pathology of AD. For this reason an exploration of the relationship between cytokines and AD stage may facilitate our understanding of AD pathogenesis and contribute to improved treatment strategies. Methods: Levels of cytokines, IL-6, IL-10, IL-17, TNF-α and neuronal damage marker Enolase(2-phospho-Dglycerate hydrolase), were assessed by High Sensitivity ELISA in serum of AD patients (mean age 70 ± 10 years) with mild and moderate dementia and compared to the control group (mean age 65 ± 9 years) without cognitive deficit. The correlation between cytokine levels and the cognitive impairment severity was studied. Results: Mean levels of IL-6, IL-10, IL-17 and enolase were higher in patients with AD compared to control group. However TNF-alpha showed lower levels in patients than healthy controls. Conclusion: Our results suggest the dual role of cytokines in AD progression. Pro-inflammatory cytokines as IL-6 could promote the disease-associated pathways, meanwhile regulatory IL-10 might be involved in dementia prevention, IL-17 is increased at disease onset and development and TNFα induce neurodegeneration as it is evidenced in our work.

66

Structural insights into High Affinity PD-1 (HACPD1) and PD-

L2 interaction. An in silico approach

Mejías, C.1; Guirola, O.1 1Bioinformatic Group, System Biology Department, Center for Genetic Engineering

and Biotechnology, Havana, Cuba. claudia.mejias@cigb.edu.cu

The immune-checkpoint receptor PD1 and its ligands PD-L1 and PD-L2 have gained a lot of attention in the field of cancer immunotherapy. Blocking these interactions with antibodies led to impressive and unprecedented clinical success. Up to date the number of patents, articles and preclinical studies related with drug design and binding mechanism for PD-1/PD-L1 (2) has dramatically increased. Alternative therapeutic variants such as macrocycles, peptides and small molecules are also being studied. The affinity of PD-1 for PD-L1 is 8.2uM , and for PD-L2 is 2.3 uM. However, a mutated variant of PD-1 was developed by Pascolutti et al. with binding affinity of 210 pm. Interestingly, even when mPD-L2 has a similar binding surface interface with PD-1,HAPD-1 doesn’t significantly interacts with hPD-L2. There is no current structural explanation for this experimental findings, hence we applied molecular modeling techniques to predict the most probable binding interface between HAPD1 and PDL2 and to explain its low affinity interaction. Our methodology combines molecular dynamics simulations, protein-protein docking and MMPBSA binding energy calculations. We performed three independent MD simulations of HAPD1, PD-L1 and hPD-L2 for 200ns. We clustered the trajectories and extract centroids of more populated clusters. We employed those conformations to perform flexible local docking and predict HAPD1-PDL2 interaction specificity and differences to HAPD1-PDL1 crystal structure. This research offers important leads for the design of selective inhibitors targeting exclusively PD-L2 or PD-L1.

67

Rational basis for MyD88 inhibitors development: an in silico

approach

Mejías C.1; Guirola O.1 1 Bioinformatic Group, System Biology Department, Center for Genetic Engineering

and Biotechnology, Havana, Cuba claudia.mejias@cigb.edu.cu

Chronic inflammation, the seventh hallmark of cancer, may affect all phases of tumor development and provoke genetic mutations and epigenetic mechanism that promote malignant cell transformation. Toll like receptors (TLR) activation by DAMPS, PAMPS, LPS and others pathogens, activate inflammatory responses and its overactivation is related to chronic diseases such as Crohn’s disease and ulcerative colitis. Adaptor protein MyD88 is recruited by all TLR (excepting TLR3) to activate signaling, so this protein has been recognized as a molecular target for autoimmune and inflammatory diseases. In this work, we performed druggability analysis of MyD88 adaptor protein with the aim to search druggable hot spots that could be suitable for antinflammatory drug design. To validate the site proposed by us, we docked the RDVLPGT peptide inhibitor of MyD88 into detected druggable sites. By combining co-solvents molecular dynamics simulations, molecular docking and free energy calculations, we identified the cavity near helix E of MyD88 as a druggable site. Residues in these pocket (W284, K282, R288) are reported as key for MyD88 biological functions, so we propose this site as putative binding site for a reported inhibitor: RDVLPGT peptide. We analyzed stability, interactions and estimated binding energy of the heptapeptide to conclude that Helix E is a suitable cavity to design new inhibitors. This research provides the first dynamic search for hotspots on MyD88 adaptor protein and the first model of interactions for heptapeptide RDVLPGT. This could offer valuable insights for the rational development of selective and potent inhibitors of chronic inflammation diseases.

68

Challenges of Pharmacoeconomics towards Precision

Medicine

García-Díaz, D Head of the Health Technology Assessment Department, Center for Engineering and

Biotechnology, Havana, Cuba darien.garcia@cigb.edu.cu

Precision medicine, as a field in vertiginous development, has enabled the switch from traditional concepts of general treatments to a patient tailor-made concept. The health benefits are markedly significant when treating affected persons with personalized therapeutic options, thus reducing the risks and uncertainty of treatments. Nevertheless, the approval of such new technologies must still overcome the barriers that health decision makers presuppose when examining their efficiency through the relationship among the new added health value, costs and the impact on health budgets. This work aims to examine the main challenges facing precision medicine in pharmacoeconomic evaluations. By thoroughly reviewing documents of international pharmacoeconomic agencies, we have identified key areas that could be developed so as to improve value assessment, reimbursements, and patient access decisions for precision medicine. Next steps for evolving health economics and outcomes research practices in precision medicine are also discussed.

69

The impact of flow regime over dengue virus shadow effect

of propeller shapes proteins

E. Navas-Conyedo1, ∗, V. Huerta-Galindo2, M. Pupo-Meriño1, J. Gulín-González1, O. Guirola2 and N

1 Centro de Estudio de Matemática Computacional (CEMC). Grupo de Matemática y Física Computacionales, Facultad de Ciencias y Tecnologías Computacionales,

Universidad de las Ciencias Informáticas, Carretera a San Antonio de los Baños, 2 21 Km, Torrens, La Lisa, La Habana, Cuba. 2 Center for Genetic Engineering and

Biotechnology, Cuba enavas@uci.cu

Some viruses like dengue have the ability to interact with the proteins presented in the blood plasma, which are sited over and avoid the direct recognition of antibodies from immunologic system, we call this effect as virus shadow effect. How the proteins are coupled to the virus surface depend of the flow regime and of the proteins structures itself. A collection of blood plasma proteins which interact with dengue 2 virus structures were recognized in previous work, all of them have a propeller structures. For propeller shapes the flow obstacles gaits additional hydrodynamics interaction patterns. We propose a general coarse grained model interaction for proteins-virus interaction and for flow hydrodynamics based on multiparticle collision dynamics mesoscopic technique. Proteins are modeled as a collection coarse grained bounded colloids particles with head point which interact with virus vesicle membrane particles via a Morse like potential. The interaction of proteins and virus vesicles with the flow particles are consider over the collision step for a Poiseuille flow channel. The radial and angular distribution of the collection of propeller shapes proteins according to the max flow rate is evaluated. The simulations reveals a relation between the propeller protein shape, flow pattern and virus-protein interaction strength, which can be used to explain the virus shadow effect.

70

Molecular Dynamics Simulations of the cyclic and antiparallel

dimer analogues of the antifungal peptide Cmp5 in a fungal

membrane model

Díaz, E1; Mejías, C2; Morales, F. E3; Garay, H.E1 1Synthetic Peptides Group-Center for Genetic Engineering and Biotechnology,

Havana, Cuba 2Department of Bioinformatics-Center for Genetic Engineering and Biotechnology, Havana, Cuba 3Faculty of Chemistry-University of Havana, Havana,

Cuba erbio.diaz@cigb.edu.cu

Cm-p5, a derived peptide from a coastal tropical snail Cenchritis Muricatus, has shown antifungal activity against some Candida species and other fungus. This peptide exhibited nontoxic action against a mammalian cell line in vitro but it may barely be therapeutically applied as drug because of their easy proteolytic degradation. A Cm-p5 derivative by Cys substitution of Glu and His from its basic sequence was synthesized and cyclized with the unexpected generation of two dimers. Despite antiparallel dimer showed a helical secondary structure in water whereas the cyclic peptide only showed the same behavior in SDS, the cyclic peptide was more active against Candida species than the Cm-p5 peptide as well as the antiparallel dimer. In spite of many experimental studies, the molecular mechanism and interaction of these two Cmp5 derivatives with the fungal membrane is not yet fully understood. In this work we performed Molecular Dynamics simulations (600ns) of two Cm-p5 derivatives, the cyclic peptide and the anti-parallel dimer, in the presence of fungal membrane model. We found that the cyclic peptide maintain an alpha helix structure for 10 ns but the dimer maintain its structural definition over half of the simulation. The interactions established with the fungal membrane model revealed a higher number of salt bridges and hydrogen bonds for the cyclic peptide, not being so for the antiparallel dimer. This in turn, displayed more intramolecular interactions in order to stabilize its secondary structure. Finally, energetic calculation with the MM-PBSA yielded the complex membrane-cyclic peptide as the more stable complex.

71

Strategy based on pan-genomics for rational design of vaccines

against Neisseria Meningitidis serogroup B.

Sánchez, FF; Ramírez, RP; Bermúdez, PER Departamento de Bioinformática, Facultad de Ciencias y Tecnologías

Computacionales, Universidad de las Ciencias Informáticas (UCI), Carretera a San Antonio de los Baños, Km 2 ½, Torrens, Boyeros, La Habana, Cuba.

fernandofs@estudiantes.uci.cu

In the world, meningitis is a disease that causes a high level of mortality, mainly caused by serogroup B of the Neisseria Meningitidis. While effective polysaccharides-based vaccines exist against serogroups A, C, W135, and Y, major vaccine efforts against serogroup B are based on outer membrane vesicles (OMVs). No vaccine developed so far against serogroup B is universally protective due to the high variability of outer membrane proteins. The rational design of a possible vaccine (serogroup B) of universal character goes through the search for external membrane proteins common to all subtypes and with low variability. Due to the high development of the technologies, the available information of Neisseria genomes has increased; which creates an adequate framework for the search for such proteins. This framework consists of a new approach to comparative genomics called pangenomics, which several genomes of organisms of the same species or genus are compared in order to find the common and distinctive part between them. This paper presents a search strategy for potential vaccine candidates based on a pangenomic approach. A preliminary study using this workflow allowed to identify, in addition to the main surface proteins already known as immunogenic determinants, other proteins with potential for use as a vaccine target.

72

Mass spectrometric characterization of Bm86-Cys1pP0: A bivalent

vaccine candidate to control the cattle-tick population. Gleysin Cabrera1, Alina Rodríguez Mallón1, Satomi Pouza1, Fabiola Almeida1,

Osmany Guirola1, Samanta Jiménez2, Alexei Licea-Navarro2, Madelón Portela3, Rosario Durán3, Ania Cabrales1, Hilda Garay1, Mario Pablo Estrada1 and Luis Javier

González1 1 Center for Genetic Engineering and Biotechnology. Havana, Cuba. 2 Departamento

de Innovación Biomédica, CICESE, Ensenada, México. 3 Instituto Pasteur de Montevideo. Montevideo, Uruguay.

gleysin.cabrera@cigb.edu.cu Chemicals are widely used to control cattle tick population despite they are not friendly for the environment, contaminate the leather, meat and milk and induce the emergence of resistant tick species. Vaccination has become an alternative strategy. There is only one commercial vaccine probed in field conditions which is based on a Bm86 hidden antigen located in the intestine of ticks and it is expressed as a recombinant protein in P. pastoris. However, this vaccine is only effective for the R. microplus tick species. Recently, a peptide from the ribosomal acidic P0 ribosomal protein has been proposed as a new anti-ticks vaccine candidate. In this manuscript we obtained a bivalent vaccine by chemical conjugation of two antigens Bm86 and Cys1P0 peptide using the MPS chemistry. SDS-PAGE analysis demonstrated that Bm86-Cys1pP0 showed a broad band (93-127 kDa) and contains an average of 9 units of Cys1pP0. The glycosylated and deglycosylated Bm86-Cys1pP0 conjugate were reduced; S-alkylated and digested separately and in tandem with trypsin, and V8 and analyzed by LC-MS/MS. This procedure allowed the verification of 90 % of amino acid sequence. Four software (pLink, Protein Prospector, StavroX and Peaks) were used to identify the conjugated lysine residues with a great reliability (FDR < 5%). Forty-six out of the 54 lysine residues of Bm86 and the N-terminal end of the carrier protein were found linked to Cys1pP0. Three-hundred and four different MS/MS spectra were assigned to 209 intermolecular cross-linked peptides. Sixty percent of these MS/MS spectra were coincidently assigned by the combination two different software and eighty-nine percent of the conjugated lysine were identified, at least, by the combination of three different software. Several PTMs, including N- and O-glycosylation, acetylation, pyroglutamic, carbamylation of the N-terminal end, phosphorylation, deamidation, methionine sulfoxide, and formylation of Lys residues were also found in the carrier protein.

73

Expression profile of TLR pathway genes in a colitis biomodel.

Camacho H1*; Roca J1*; Aguilera A2*; Guillen IA1; Bermudez Y2; Bacardí D3; Suarez JA3; Delgado Y1 and Palenzuela DO1

1. Pharmacogenomics group. Center for Genetic Engineering and Biotechnology CIGB. La Havana. Cuba. 2. Formulations group. Center for Genetic Engineering and

Biotechnology CIGB. La Havana. Cuba. 3. Preclinical Research and Animal Experimentation group. Center for Genetic Engineering and Biotechnology CIGB. La

Havana. Cuba. hamlet.camacho@cigb.edu.cu

Introduction: Animal models of ulcerative colitis are valuable because they provide information on the pathogenesis of the disease.. 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. Recent evidences highlight immune dysfunction as a central player in the pathogenesis of IBD. 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 15 genes related to TLR pathway. Results: During treatment with DSS, animals developed clinical and histological signs indicating colitis. 46% of the analyzed genes varied their expression, six were upregulated and none were downregulated. The increase in the level of expression of TLR2, 6, 8 and 9 has been correlated with the severity of inflammation in patients with ulcerative colitis. Three of these receptors showed a statistically significant increase in their expression. On the other hand, it has been suggested that the TLR3 signal has a protective effect on intestinal inflammation; interestingly this was the only receptor that did not increase its expression. Conclusions: These results point to a deregulation of the TLR pathway in this colitis model.

74

CGWork: a tool for the search of the common genome with

applications in the rational design of vaccines.

Garí, JAJ; Mena, VV Departamento de Bioinformática, Facultad de Ciencias y Tecnologías

Computacionales, Universidad de las Ciencias Informáticas (UCI), Carretera a San Antonio de los Baños Km 2 ½, Torrens, Boyeros, La Habana, Cuba.

jorgeajg@estudiantes.uci.cu

Due to the development of sequencing techniques and the accumulation of data from several genomes of microorganisms, the possibility of comparative genomics studies for the identification of vaccine candidates is opened. In these studies, universally present and little variable surface proteins are sought among organisms of the same species. This paper presents a tool capable of performing a process of clustering and analysis of intersections between genomes. This process is divided into two, rapid clustering for a preliminary analysis of possible results and exhaustive clustering to provide more specific results. It also allows the search for a specific gene and its location both genomically and in the clusters in which it is found, for the search of its possible orthologs. This tool was used in a preliminary study of the cross-reactivity of VA-MENGOC-BC.

75

Mass spectrometric characterization of low‐abundance species in

the active pharmaceutical ingredient of synthetic peptides Espinosa LA1; Garay H2; Perera Y3; Sánchez A1; Diago D2; Perea S3; Domínguez MC4;

Junco JA5; Besada V1; González LJ1 1Mass Spectrometry Laboratory, Proteomics Group, System Biology Department;

2Peptide Synthesis Laboratory, Physics and Chemistry Department; 3Molecular Oncology Laboratory, Pharmaceutical Department; 4Autoimmunity Group, System

Biology Department. Biomedical Research, Center for Genetic Engineering and Biotechnology, PO Box 6162, Havana, Cuba. 5Department of Cancer, Center for

Genetic Engineering and Biotechnology of Camaguey, CP 70100, Apdo 387, Camaguey, Cuba

luis.espinosa@cigb.edu.cu

Several projects at CIGB are based on the use of synthetic peptides for the treatment of various pathologies. Autoimmune diseases and cancer therapy are two of the most favored in this regard, with three candidates (CIGB-814, Pyr-GnRHm1-TT and CIGB-300) who have passed phase I clinical trials with promising results. ICHQ3A(R2) guidelines state the daily doses intake, abundance and identity of the impurities in synthetic peptides as pivotal nodes that define actions to be taken (reporting, identification and qualification). Purity was first assessed by RP-HPLC (98.49% CIGB-814, 97.41% Pyr-GnRHm1-TT and 99.16% CIGB-300) and individually low‐abundance impurities (≤0.72% CIGB-814, ≤1.2% Pyr-GnRHm1-TT and ≤0.27% CIGB-300) were collected and identified by ESI-MS. Peptide-related species of CIGB-814 and Pyr-GnRHm1-TT were generated during peptide synthesis with the N-terminal end truncated and/or modified. The most abundant fractions contain up to three impurities (0.72, 0.46 of CIGB-814 and 1.2, 0.66% of Pyr-GnRHm1-TT) due to amino acid deletions, insertions and/or modifications. The rest of fractions (0.07, 0.11, 0.16% of CIGB-814 and 0.35, 0.66% of Pyr-GnRHm1-TT) contained up to three peptide-related species. Most of the impurities of CIGB-300 were generated during peptide synthesis, air oxidation of free Cys and the lyophilization step. Parallel and antiparallel dimers of CIGB‐300 linked by two intermolecular disulfide bonds exhibited a higher SRB anti-proliferative activity (IC50=9.2±0.2 and 11.0±0.8 M) than the CIGB‐300 monomer (IC50=53±4 M) in the NCI-H125 tumor cell line. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB‐300 derived entities merits further investigation.

76

Phycocyanobilin molecular mechanisms on a damage model

induced with glutamate in shsy5y cells

Cervantes-Llanos M; Camacho H; Piniella-Matamoros B; Pentón Rol G. Biomedical Research Division of the Center for Genetic Engineering and

Biotechnology. La Habana, Cuba majel.cervantes@cigb.edu.cu

Introduction: Excitotoxicity is a form of neuronal death induced by increased Glutamate signaling which has been implicated mainly in Central Nervous System diseases. These include hypoxic-ischemic states, hypoglycemia and neurodegenerative disease among others. Neurodegeneration is the cause of many diseases of unknown aetiology and common pathogenic features characterized by disability and fatal endpoints with both economically and socially deleterious impact. Dissecting and modelling the sequence of pathogenic mechanisms contributing to neuronal damage, is a genuine approach in order to develop effective therapies for neurodegenerative diseases. Methods: In order to study the neuroprotective effect of Phycocyanobilin in excitotoxicity involved events, SHSY5Y cells were maintained in culture RPMI 1640 medium supplemented with foetal bovine serum, penicillin, streptomycin and L-glutamine. After 24 h of pre-stimulation PCB, media was replaced with freshly prepared PCB and Glutamate for another day. The medium was removed; cells were the washed using PBS and used to conduct gene expression analysis. Total RNA extraction was performed using the QIAcube platform. The RNA was employed in quantitative real time polymerase chain reaction (qPCR) to measure the differential expression of genes. Results: Phycocyanobilin was able to protect cells from the excitotoxic insult and it was confirmed morphologically and using MTT viability assay. Interestingly, PCB performed contrary to Glutamate, modulating mRNA expression of genes involved in apoptosis, redox imbalance and cellular stress. Conclusions: from our results it can be seen that the effect of the PCB is protective by generally inhibiting apoptosis and damage signals and activating detoxification mechanisms of glutamate-induced toxicity.

77

An integrative analysis of evolutionary pressures over 3C and VP1

segments in Coxsackievirus A24v

Pupo-Meriño M1, Fonseca M2 and García LA3 1 Departamento de Bioinformática, Universidad de las Ciencias Informáticas (UCI),

La Habana, Cuba) 2 Instituto Pedro Kouri, La Habana, Cuba. 3 Centro de Matemática Computacional, Universidad de las Ciencias Informáticas (UCI), La Habana, Cuba

mpupom@uci.cu

Coxsackievirus A24v (CV-A24v) has been implicated as a major causative agent of Acute Hemorrhagic Conjunctivitis outbreaks in many countries. Previous studies of selective pressures acting over CV-A24v have suggested the occurrence of sequential extinction of dominant lineages and replacement by newer immunity-driven selection escape lineages. So far, two factors have been stablished as limiting factor of limited CV-A24v gene pool: a narrow tissue tropism and outbreaks caused by single prevalent lineage. Here, an integrated analysis including Bayesian phylogenetic and phylodynamic inference, antigenic and functional sites prediction and structural modelling using VP1 and 3C coding regions is presented. Results show that phylogenetic behavior observed in some periods could be interpreted in terms of purifying selection instead of scape from immune system. There seems to be a balance between the need of scape from immune pressures in the short range and preservation of structural features associated with its narrow tissue tropism.

78

Characterization by mass spectrometry of the KLH1-Cys1pP0 and

KLH2-Cys1pP0 conjugates: a vaccine candidate against ticks.

Satomy Pousa1, Vladimir Besada1, Jacek Wisniewski2, Osmany Guirola1, Ania Cabrales1, Hilda Garay1, Alina Rodríguez-Mallón1, Mario Pablo Estrada1 and Luis

Javier Gonzalez1. 1 Center for Genetic Engineering and Biotechnology, Havana, Cuba. 2 Max Planck

Institute of Biochemistry, Munich, Germany satomy.pousa@cigb.edu.cu

The vaccine candidate composed by Cys1pP0 peptide from acidic ribosomal P0 protein of Ripicephalus spp linked to KLH carrier proteins (KLH1 and KLH2) from M. crenulata has shown high efficacy against two tick species challenged in two animal models (Rodriguez-Mallon et al.,2012; Rodriguez-Mallon et al.,2015). These conjugates were obtained by the chemical reaction between the Cys residue located at the N-terminal end of the P0 peptide and the maleyl group introduced at the Lys residues of the carrier proteins using MPS as a cross-linking reagent. Mass spectrometric characterization of vaccine candidates is a requirement requested by the regulatory entities. However, the characterization of the conjugates KLH1-Cys1pP0 and KLH2-Cys1pP0 by MS was an analytical challenge. On one side, the molecular mass of both monomers of the carrier proteins (390 kDa molecular mass) that can be assembled into compact structures of decamer, didecamers and multidecamers of megadalton molecular mass. Conjugates derived from these carrier proteins are even more complex due to the heterogeneity introduced by the chemical reactions. This work is aimed at the characterization of the KLH1-Cys1pP0 and KLH2-Cys1pP0 by LC-MS/MS to determine the conjugation sites. The conjugates were efficiently digested using MED-FASP procedure and analyzed by LC-MS/MS. The conjugation sites were identified by using the Kojak and pLink software previously used in functional proteomics for the identification of type II cross-linked peptides. The automatic assignments were further validated by inspecting manually each MS/MS and verifying the presence of diagnostic ions. The conjugation sites in KLH1-Cys1pP0 and KLH2-Cys1pP0 were found in 130 out of 169 lysine residues (77% Lys conjugated) and in 81 out of 150 lysine residues (51% Lys conjugated), respectively. In agreement with the literature charge distribution of type II cross-linked peptides were higher or equal than 3+ in 98 % of the cases.

79

Integrative model of rhodanine derivatives as tau aggregation

inhibitors in alzheimer’s disease

Álvarez-Ginarte Y. M, Lecrerc Fabrice and Montero-Cabrera L.A. Laboratory of Theoretical and Computational Chemistry, Faculty of Chemistry,

University of Havana, 10400, La Habana, Cuba. yoanna@fq.uh.cu

Tau aggregation is responsible for the formation of neurofibrillary tangles which is one hallmark of Alzheimer's disease. Short hexapeptides of Tau (PHF6) are known to be responsible for the formation of aggregates. Rhodanine-based compounds are known as Tau anti-aggregation inhibitors. However, their precise mode of action on Tau is still undefined, e.g. the binding site(s), binding mode(s), or stoichiometry remain undefined. To date, there is no structural data on Tau complex with any Rhodanine derivatives, and there is no rationale for their activity or toxicity. Three computational modelling approaches have been used to propose an integrative model for the mode of action of Rhodanine derivatives. First, QSAR studies were used to identify and quantify the molecular properties relevant to their activity and toxicity. The more active and less active compounds were then used in docking simulation to identify the possible binding modes and the contacts specific to the more active compounds. Finally, molecular dynamics simulations were performed to validate the 3D models of interaction between Tau (PHF6) and the more active Rhodanine compound and determine the more probable stoichiometry. Possible pathways for their insertion into the aggregated PHF6 peptides are also identified suggesting how they may act as Tau anti-aggregation inhibitors.

80

BitClust: Fast conformational clustering of long Molecular

Dynamics simulations.

Roy González-Alemán†*, David Hernández-Castillo†, Alejandro Rodríguez-Serradet†, Julio Caballero‡, Erix W. Hernández-Rodríguez ɸ, Luis Montero-Cabrera†

† Laboratory of Theoretical and Computational Chemistry, Faculty of Chemistry, University of Havana, 10400, La Habana, Cuba.

roy_gonzalez@fq.uh.cu

Grouping similar conformations together (clustering) is a key analysis when exploring the potential energy surface of molecules. Molecular Dynamics often return conformational sparse trajectories where every cluster can be important to detect and analyze. Democratization in the use of high performance computing, and the exploitation of graphical processing units have permitted to recurrently escape the nanoseconds scale, where conventional Molecular Dynamics were trapped, to irrupt into micro- and mili-second scales of time. Naturally, longer the simulation, bigger the size of produced trajectories. In that scenario, parallelized algorithms optimized to detect many clusters in a rational time are appreciated. In this work we propose BitClust, a fast implementation of Daura´s algorithm for clustering long molecular trajectories. It is able to calculate all the information needed to run a clustering job using parallelization and then store it in memory for further processing. The amount of sacrificed memory has been deeply optimized by encoding similarity distances as bits. This encoding results in a huge reduction of storage when comparing to algorithms that save similarity distances as single precision float values. Significant computation performance is also achieved by computing clustering-related steps as bitwise operations. A speed benchmark demonstrates that our implementation is able to outperform one of the fastest alternatives in at least two orders of magnitude, which sets BitClust as an attractive option among the state-of-the-art clustering software for long molecular trajectories.

81

Prediction of molecular interactions and physicochemical

properties relevant for vasopressin v2 receptor antagonism

de la Nuez A, Álvarez-Ginarte Y. M, Rodriguez R and Montero-Cabrera L.A Department of Biochemistry, Faculty of Biology, University of Havana, 10400, La

Habana, Cuba. ania.delanuez@fbio.uh.cu

Autosomic Dominant Polycystic Kidney Disease (ADPKD) is a genetic disease with an incidence of 1:400, to 1:1000 in the world population. Patients develop multiples fluid-filled cyst in both kidneys, increasing the total kidney volume and leading to Chronic Kidney Disease. The activation of vasopressin V2 receptor (V2R) is crucial for the development and growing of the cysts. There are several V2R antagonist described in the literature with tolvaptan as the only one approved so far, to treat advanced stages of ADPKD. We used a computational modeling approach to study the binding modes to V2R of antagonist and also the physicochemical properties relevant to the biological activity of this compounds. V2R was modeled by comparative modeling and further relaxed in a POPC membrane by molecular dynamic simulations. The protein-ligand docking was performed considering the ligand as flexible. The binding mode of the tested antagonists was similar. To study the stability of the protein-ligand interactions, selected complexes were inserted in to a POPC membrane, minimized, equilibrated and a molecular dynamic simulation was performed. QSAR studies were used to identify and quantify the molecular properties relevant to antagonist activity. These models will be used in further studies looking for potential new V2R antagonist molecules

82

Metabolome Analysis of Breast Tumors for Discovery of

Oncometabolites

Ambs, S Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda,

Maryland, U.S.A. ambss@mail.nih.gov

Metabolomics has emerged as a new discovery tool with the promise of identifying therapeutic targets in cancer. Breast tumors undergo metabolic reprogramming as part of malignant transformation. This reprogramming leads to increased availability of putative oncogenic metabolites that are used for the synthesis of biomolecules, energy metabolism, or as signaling molecules. Cancer metabolism has also been shown to modify the chromatin of cells, the tumor microenvironment, or anti-tumor immunity. We reported that metabolite patterns of breast tumors are greatly different from those in the adjacent non-cancerous tissue and vary between disease subtypes. A key discovery from our studies was the observation that 2-hydroxyglutarate (2HG) and bile acids, among other metabolites, can accumulate in breast tumors. We also observed that D-2HG is the predominant 2HG enantiomer in breast tumors and identified Alcohol Dehydrogenase Iron Containing Protein 1 (ADHFE1) as a Myc-linked and iron-inducible oncogene that promotes metabolic reprogramming with an increase in D-2HG, reactive oxygen formation and histone methylation, leading to cellular de-differentiation and mesenchymal transition. In my presentation, I will summarize some of the identified roles of 2HG and bile acids in breast cancer biology.

83

Toward the uses of antimicrobial peptides against multidrug

resistant bacteria Montero-Alejo V*, Perdomo-Morales R*, Vázquez A*, Rodríguez-Viera, L**, Roque-

Díaz Y*, Bello-Madruga R*, Garay-Pérez H§, Álvarez-Valcárcel C+. * Laboratory of Biochemistry and Molecular Biology. Center for Pharmaceutical

Research and Development, Havana, Cuba. ** Center for Marine Research, Havana University, Havana Cuba. § Laboratory of Synthetic Peptides. Center for Genetic

Engineering and Biotechnology, Havana, Cuba + Center for Protein Studies. Biology Faculty of Havana University, Havana, Cuba.

The emergence of multidrug resistance bacteria has become a global health problem with implication in the social and economic standard to the 21st century society. To face that phenomena, the scientific community has strongly encouraged to find out antibacterial agents with novel mechanism of actions. In this sense, the Center for Pharmaceutical Research and Development has leading in the field of development of novel peptides with potential use as antimicrobial agents in the treatment of complicated infection diseases. Our work is based on the finding of natural peptides from marine invertebrates that showing broad spectrum of antimicrobial activity against bacteria, fungus and eventually antiviral. The natural peptides have served as templates for the designs of novel chemical entities based on structure-activity relationships. The designed peptides improved the capacity of impair bacterial viability by diminish the minimal inhibitory concentration from micro to nanomolar concentration. They exert membrane disrupting mechanisms and can permeabilized membranes of Gram negative bacterial at lytic concentrations. The designed peptides retain the selective toxicity against bacterial membrane showed by the natural molecules. In addition, experiments have carried out with membrane models, mimicking the outer and inner membrane lipid composition of E. coli, using the intrinsic fluorescence of peptides. In all cases the peptides showed high affinity to interact with membrane models and they also carry out conformational changes in the membrane microenvironment, as was assessed by circular dichroism. Summarizing, there is demonstrated the effectiveness of structural determinants to disrupt the bacterial membrane for the treatment of multidrug resistant bacteria.

84

Proteotranscriptomics Reveals Associations of Protein-mRNA

Concordance with Breast Cancer Subtypes and Survival

Tang, W Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda,

Maryland, U.S.A. tangw3@mail.nih.gov

Analysis of the breast cancer transcriptome discovered distinct disease subtypes of clinical significance. However, it remains a challenge to define disease biology solely based on gene expression data because tumor biology is most often defined by protein function. Thus, we applied a quantitative liquid chromatography/mass spectrometry-based proteome analysis of 65 breast tumors and 53 adjacent non-cancerous tissues and performed an integrated analysis of the proteome and transcriptome to examine relationships between them and associations with disease characteristics and survival. We found that the proteome describes differences between cancerous and non-cancerous tissues that are not revealed by transcriptome. The proteome revealed an activation of infection-related signal pathways in triple-negative tumors. The integrated analysis of the two technologies further revealed a global increase in protein-mRNA concordance in tumors. Highly correlated protein-gene pairs were enriched in protein processing and disease metabolic pathways. The increased concordance between transcript and protein levels was additionally associated with aggressive disease, and decreased patient survival. Our study provides new insights into the relationship between protein and mRNA expression in breast cancer and shows that an integrated analysis of the proteome and transcriptome has the potential of uncovering novel disease characteristics.

85

LC-MS/MS characterization of two conjugated vaccine candidates

against ticks obtained by different crosslinking strategies between

the carrier protein p64K from N. meningitidis and a peptide

derived from the acidic ribosomal P0 protein from R. sanguineus. Wendy Machado1, Luis Ariel Espinosa1, Gleysin Cabrera1, Alejandro Leyva2, Nobuaki Okumura3, Rosario Durán2, Toshifumi Takao3, Alina Rodríguez-Mallón1, Mario Pablo

Estrada1,Hilda Garay Perez1,Ania Cabrales Rico1, and Luis Javier González1. Center for Genetic Engineering and Biotechnology, Havana, Cuba. Institut Pasteur de Montevideo, Uruguay. Institute for Protein Research, Osaka University, Japan.

wendy.machado@cigb.edu.cu

The pP0 peptide derived from the P0 acidic ribosomal protein from Rhipicephalus spp. conjugated to KLH protein was effective as a vaccine candidate to protect rabbits and cattle against the infection with R. sanguineus and R. micropolus ticks, respectively. This peptide was also conjugated to a recombinant carrier protein p64k from N. meningitidis by two different procedures. The conjugate p64k-Cys1P0 was obtained in two steps. Firstly, p64K was modified with MPS to introduce maleimide groups in the lysine residues. In a second step, the modified p64K reacted with the N-terminal free thiol group of the Cys1pP0 peptide. The other conjugate, p64K-bAla1pP0, was obtained in a single step by the direct reaction between the six free thiol groups of the carrier protein and the bAla1pP0 peptide functionalized with a maleimide group at the N-terminal end. SDS-PAGE analysis of p64kCys1-pP0 conjugate showed a broad band between 77-103 kDa suggesting the addition 1 and 14 units of Cys1pP0 peptide, respectively. In addition, a higher molecular band detected between 157 and 223 kDa suggested the cross-linking of two monomers during the conjugation reaction. SDS-PAGE analysis of p64K-bAla1pP0 showed a band between 79-96 kDa suggesting the addition 2-5 units of bAla1pP0 to p64K. The conjugates were digested with different proteases and the conjugation sites were determined by the combination of LC-MS/MS analysis and several software that have been used for the identification of cross-linked peptides (pLink, StavroX, Kojak, and Protein Prospector). The automatic assignment of cross-linked peptides were manually validated by considering the presence of some diagnostic ions in the MS/MS spectra. In p64k-Cys1pP0 the conjugation sites were found in 36 out of 39 lysine residues and the N-terminal end of the carrier protein. In the case of p64K-bAla1pP0 the conjugation site were exclusively found in four of the six free cysteine residues of p64k.

86

Integrated Genomics to Uncover Clinically-relevant Liver

Cancer Drivers

Wang, XW Laboratory of Human Carcinogenesis and Liver Cancer Program, Center for Cancer

Research, National Cancer Institute, Bethesda, Maryland 20892, USA xw3u@nih.gov

Genomic analyses of most solid tumors reveal a complex mutational landscape with vast inter-tumor (sample by sample) and intra-tumor (within each tumor) heterogeneity. This is especially relevant to hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), the two major histological types of liver cancer, where various etiological factors may elicit different molecular mechanisms to initiate carcinogenesis, which lead to different molecularly distinct subtypes and complex tumor cell communities. Such heterogeneity poses a major challenge to define drivers responsible for early stage hepatocarcinogenesis and develop diagnostic tools and effective treatment modalities for HCC and iCCA. The establishment of patient populations with associated well-annotated biobanks and well-characterized molecular features is essential to better define unique tumor subtypes, and integrated genomics, transcriptomics, proteomics and metabolomics may provide a superior resolution to distinguish molecular subtypes. Understanding molecular features of tumor cells at a single cell level may also provide a better understanding of tumor cell communities and help define key drivers and druggable targets responsible for tumor initiation and progression in HCC and iCCA. With the development of precision molecular technologies, we may be able to effectively identify actionable targets for early liver cancer intervention and ultimately find solutions to fundamentally improve outcomes of patients with liver cancer.

87

Epidermal Growth Factor regulates LPS-induced

inflammatory scenario in fibroblasts derived from

diabetic foot ulcers Mendoza-Marí, Y, García-Ojalvo, A, Fernández-Mayola, M, Berlanga-Acosta, J

Wound Healing and Cytoprotection Group, Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Havana, Cuba.

yssel.mendoza@cigb.edu.cu

Diabetes mellitus (DM) is considered one of the main non-communicable diseases that affects people worldwide. DM predisposes to lower extremities ulceration and impairs the healing process leading to wound chronification. Ulcerated patients show high levels of inflammatory mediators, resulting from sustained hyperglycemic insult, combined to microbial biofilm present in diabetic wounds. In this scenario, the intralesional administration of epidermal growth factor (EGF) constitutes an effective treatment that stimulates granulation and closure of diabetic foot ulcers (DFU), reducing the risk of amputation. Within the wound, fibroblasts play key role during healing process, as they secrete growth factors and extracellular matrix molecules, promoting granulation and contraction. Fibroblasts also express Toll Like Receptors, which mediate host defense by regulating both, innate and adaptive immune responses. The objective of this work was to study the effect of lipopolysaccharide (LPS) on the expression pattern of inflammation mediators in DFU-derived fibroblasts and to elucidate the possible anti-inflammatory effect of EGF. Levels of TLR4, TLR2, IL6, IL1B, NFKB, TNF, TGFB1, MYD88, CD14 and HMGB1 were measured by semi-quantitative qPCR, under normal and hyperglycemic conditions. We demonstrated that LPS significantly increased mRNA levels of pro-inflammatory genes. Besides, EGF alone or in presence of LPS, downregulated the levels of these genes, except for IL1B and TGFB1. For some genes, fold change showed more markedly in normal glucose condition. The results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblast, regardless glucose concentration. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena.

88

Network interactions and signaling pathways analysis for

a phosphoproteomics experiment with heberferon in

glioblastoma cells Hardy A1*, Palomares S1, Baez S1, Besada V1, Guirola O1, Bello I2, Vazquez-Blomquist

D3, Ramos Y1, Wisniewski J4 and Gonzalez, LJ1 1. Department of System Biology, Biomedical Research Division, 2. Clinical Assays Direction, 3. Pharmacogenomic Group, Department of System Biology, Biomedical

Research Division, CIGB, Havana, Cuba; 4. Department Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Germany.

anette.hardy@cigb.edu.cu

HeberFERON is a pharmaceutical formulation of interferons that combines IFN-a2b and -g based in their anti-proliferative synergism on several tumors cell lines. Clinical trials have demonstrated the efficacy of HeberFERON in Basal Cell Carcinoma (BCC) and in glioblastoma multiforme. There is a strong interest in understanding the key signaling pathways and cellular events that justify the anti-proliferative effect in glioblastoma cells. This work is aimed to identify and analyze relevant signaling pathways that support the anti-proliferative effect of HeberFERON in glioblastoma. To achieve this goal, we performed the functional analysis of the phosphoproteomics data that resulted from the studies of HeberFERON in glioblastoma cells. Appropriate bioinformatics tools (such as Cytoscape, Keeg Pathway, DisNor) were used to build protein-protein interaction networks and identify relevant signaling pathways. In particular, the results suggest that PI3K-AKT signaling pathway is a relevant phosphorylation pathway that could support the anti-proliferative effect of HeberFERON in glioblastoma cells.

89

Functional analysis of a phosphoproteomics experiment

with heberferon in glioblastoma cells Baez S1*, Palomares S1, Hardy A1, Besada V1, Guirola O1,Bello I2, Vázquez-Blomquist

D3, Ramos Y1, Wisniewski J4 and González LJ1. 1. Department of System Biology, Biomedical Research Division, 2. Clinical Assays Direction, 3. Pharmacogenomic Group, Department of System Biology, Biomedical

Research Division, CIGB, Havana, Cuba; 4. Department Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Germany.

saiyet.baez@cigb.edu.cu

Phosphorylation is one of the commonest post-translational modifications and serves as a basis for regulating a number of cellular processes. This aberrant regulation may result from overexpression of kinases or defects in negative regulatory mechanisms. Interferons are part of a family of proteins that have various biological effects such as the inflammatory process and the production of reactive oxygen species. HeberFERON is a pharmaceutical formulation produced at CIGB that contains IFN-a2b and IFN–g in a ratio that maximally inhibits tumor cell growth. There is evidence of the superiority of HeberFERON over the individual IFNs. Therefore, phosphoproteomics experiments have been made for the identification of HeberFERON mechanism of action. In this work, we used appropriated bioinformatics tools for the identification of relevant biological pathways and processes in the proteomic and phosphoproteomic data. We identified downstream effectors of kinases and deregulated signaling pathways. IFNs signaling start several downstream events involved in the anti-proliferative action and also stimulate immune response. There are activations of GTPasa, ATPsa and peptidases activities in an apparent absence of phosphatase activity. Some kinases changed their expression by phosphorylation processes [CAMK2D, PRKCA, GSK3B, MAPK8, RPS6KA3, CSNK1E] and Cyclin depend kinases could influence the regulation of cell proliferation. The axis of PI3K-AKT-mTOR-MAPK signaling could also be mediating Apoptosis as another antitumor effect of HeberFERON.

90

Oxidative stress parameters as biomarkers for new

multiple sclerosis therapies Herrera, T1,*; Lagumersindez, N2; Delgado, L2; Marín, J2; Rodríguez, H1; Cepero, J1;

Valenzuela, C3; Cintado, A4; Pentón, G3. 1Preclinical Studies Department, National Institute of Oncology and Radiobiology

(INOR), Havana, Cuba; 2Pharmacology Department, Center for Research and Biological Evaluations (CEIEB), Havana, Cuba; 3Biomedical Research Direction,

Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba; 4 Genomics Department, Center for Genetic Engineering and Biotechnology (CIGB), Havana,

Cuba. therrera@infomed.sld.cu

Multiple Sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system that leads to demyelination and axonal damage. Chronic inflammation increases production of reactive oxygen and nitrogen species so endogenous antioxidant defenses are overcome and occur oxidative stress, that plays an important role in pathogenesis of MS but also in its principal animal model, the experimental autoimmune encephalomyelitis (EAE). C-Phycocyanin (C-Pc) is a natural antioxidant, which has been shown to have therapeutic potential to contain the neuronal damage caused by reactive oxygen and nitrogen species, for this reason, it becomes a potential candidate to be combined with the IFN-alpha. In the present study, were evaluated biomarkers of protection and oxidative damage, as well as motor clinical signs in mice after induced with a chronic model of EAE, also treated with IFN-alpha, C-Pc and the combination of IFN-alpha and C-Pc (IFN-alpha/C-Pc). Results showed that the combination therapy improved the redox status more than the independent treatments in mice subjected to EAE. Combined therapy, directed towards the immune and neurodegenerative components, leads to an enhancement of the clinical motor signs caused in the animal model.

91

Phylogenetic and phylodinamic of Coxsackievirus A24

variant in Cuba over a 23- year period. 1986-2009 Fonseca MC1, Pupo M2, Norder H3, García LA2, Resik S1, Hung LH1, Muné M1,

Rodríguez H1, Morier L4, Sarmiento L5. 1. Virology Department, Pedro Kourí Institute of Tropical Medicine (IPK), La Havana,

Cuba. 2. Informatic Science University. La Havana, Cuba.

3. Department of Infectious Diseases, University of Gothenburg, Sweden. 4. Biology Faculty, Havana University. La Havana, Cuba.

5. Cellular Autoimmunity Unit, Department of Clinical Sciences, Skåne University Hospital, Lund University, Malmo, Sweden.

magile@ipk.sld.cu

Coxsackievirus A24 variant (CV-A24v) is the prevalent etiological enterovirus causing Acute Hemorrhagic Conjunctivitis. We present the phylogenetic and phylodinamic analysis of the partial genomic regions 3C and VP1 of CV-A24v strains isolated from five epidemic periods occurred in Cuba between 1986 and 2009. Phylogenetic trees and spatiotemporal dynamics of CV-A24v transmission were inferred by a Bayesian inference in the BEAST framework. A data set of 54 Cuban sequences and 83 worldwide sequences from 17 countries for 3C genomic region and 35 Cuban sequences and 95 worldwide sequences from 18 countries for VP1 genomic region were analyzed. The virus was repeatly introduce to the country and the genetic Cuban variants were homologous to those circulated in the Americas and Asia in similar periods within genotypes GIII and GIV. The virus showed an effective population size stable over time, peaking in the pandemic periods with a nucleotide substitution rate (substitutions/site/year) of 4.39×10-3 for the 3C genomic region and 5.80×10-3 for VP1 genomic region. Nineteen viral worldwide transmission routes based on 3C region and 25 routes based on VP1 were obtained. The introduction routes of the virus in Cuba in four epidemic periods were inferred by the ancestral relations of the Cuban sequences supported by networks of viral transmission. Both genomic region were informative and a new chronological origin for CV-A24v genotype IV was proposed. The results of this study contribute to the knowledge in the molecular epidemiology of the CV-A24v that circulated in Cuba and the world during the period studied.

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