Day 1 :
Keynote: Mobilome and resistome analysis of canine multidrug resistant methicillin-Resistant Staphylococcus sciuri strain C2865 and comparative genomics of S. sciuri species group
Time : 09:15-09:45
Elena Gómez-Sanz is an expert on staphylococcal antimicrobial resistance and currently focusses her research on animal and environmental antimicrobial resistance from a One Health perspective following next-generation approaches. She has participated in six national and international projects, and is currently Principal Investigator for the Swiss National Science Foundation NFP72 (“Antimicrobial Resistance”). She has developed her research career in Spain, UK, Germany, Australia and Switzerland, and has been awarded a number of grants and fellowships in competitive, merit-based calls from national and international organizations. Dr. Gómez-Sanz has participated in more than 40 scientific publications and contributed with 75 presentations in international and national conferences. She participates as reviewer for several scientific editorials and is member of several scientific societies. Since December 2016 Dr. Gómez-Sanz is a postdoctoral fellow at the “Swiss Federal Institute of Technology Zurich (ETHZ)” thanks to a Marie Skłodowska-Curie Individual Fellowship Global Fellowship within the Horizon 2020 program.
Statement of the Problem: Antibiotic resistance is one of the biggest threats to global health, food security, and development today. Staphylococci are common member of our skin and mucosa but they are also a common cause of severe infections and resistance to first line antimicrobial to treat their infections is widespread. Here, a multidrug- and methicillin-resistant Staphylococcus sciuri strain C2865 (canine nasal sample, Nigeria) showed thrimethoprim resistance for which all staphylococcal dfr genes were negative. This strain was subjected to whole-genome-sequencing (WGS) for resistome and mobilome profiling and to comparative genomics with all NCBI-deposited S. sciuri species group genomes for diversity analysis.
Methodology & Theoretical Orientation: Illumina Miseq was used for C2865 WGS and in-house pipelines (SPAdes, Prodigal, tRNAscan-SE, RNAmmer, NCBI NR, COG, TIGRfam, RAST, ISsaga2) were followed for data processing and analysis. Plasmid contigs identification and plasmids reconstruction were achieved by contig coverage, sequence similarity and composition. PCR plus sequencing was done for scaffolding regions of interest. Average Nucleotide Identity (ANI) between all 22 available S. sciuri group strains and comparative and pangenome analysis of S. sciuri group strains was calculated using JSpecies. progressiveMauve, ClonalFrame and Roary, respectively.
Findings: S. sciuri C2865 revealed 2,937,715 bp in size, a GC content of 32.7%, and 3316 CDSs, with 1887 genes categorized into COG functional groups. Two small resistance plasmids and two novel mobilizable plasmids were reconstructed. p2865-3, a multidrug resistance plasmid, revealed a trimethoprim resistance gene described for the first time in marine Exiguobacterium (order Bacillales). p2865-4 carried the intercellular adhesion gene cluster involved in biofilm formation. A novel staphylococcal cassette chromosome (SCCmec) was identified. Additional chromosomal resistance genes (antibiotic, metal, biocide) and mobile genetic elements (MGEs) were detected.
C2865 shared highest ID with S. sciuri Z8 and SNUDS-18 (99-98%), comprising a different branch within the species. WG alignment among all S. sciuri genomes revealed a core genome of 1.7 Mb (60.7%). Synteny was preserved among these genomes while there were several genomic islands with distinct gene content.
Conclusion & Significance: 1) Several novel MGEs are detected revealing, among others, a novel dfr gene conferring thrimethoprim resistance not only in S. sciuri but also in an environmental species. 2) WGS reveals a trustful tool to identify and characterize novel and already known resistance & virulence determinants and MGEs. 3) Comparative genomics shows high S. sciuri intraspecies diversity and high genome plasticity. 4) Commensal staphylococci represent a reservoir for mobilizable AMR & virulence determinants.
- Biopharmaceuticals | Computer Aided Drug Design (CADD) | Medicinal Chemistry in Modern Drug Discovery | Drug Metabolism and Drug Designing
Vladimir A Baulin
Universitat Rovira I Virgili, Spain
Wolfgang B Fischer is Professor at the Institute of Biophotonics, School of Biomedical Science and Engineering, National Yang-Ming University, Taipei, Taiwan. He has obtained his PhD in Chemistry at Heidelberg University, Germany, working in the field of vibrational spectroscopy in 1991. After years in the US, he has completed his Postdoctoral, working on bacteriorhodopsin using vibrational spectroscopy in Boston University, Germany. Then he has worked in Analytical Chemistry, working on ion channels as potential biosensors, UK (Oxford University as EU Marie Curie Research Fellow and later as Lecturer, working on viral ion channels using bilayer recordings and molecular dynamics simulations. He has moved to Taiwan. The field of research is on biophysical aspects describing dynamics and energetic of protein-protein interactions (PPIs) of membrane proteins. The focus is on the development of computational platform technologies to support drug discovery and design as well as materials sciences.
Many viral membrane proteins interact with membrane proteins of the host to steer the cell for a successful mass production of novel virions. The viral proteins rely on selective interactions of their transmembrane domains (TMDs) with those of the host protein. An understanding of the modalities of recognizing the proper host target, on the revers, can be turned against the virus. Getting insights into the specificity of binding, the interaction of oncoprotein E5 of Human papillomavirus-16 (HPV-16), an 83-amino acid membrane protein containing 3 TMDS, with a peptide corresponding to the fourth TMD (TMD4) of the 16 kDa subunit of the ATP6V0C is investigated as an example. HPV-16 is the major cause of cervical cancer diagnosed today. E5 is a membrane protein which is expressed at an early (hence the letter E) stage of the infectivity cycle when the virus turns the cell malignant. The protein interacts with a series of host factors, but has also been identified experimentally to allow channel activity when most likely in a hexameric assembled form. Computational modeling suggests a weak selectivity of the channel. Docking approaches as well as coarse grained molecular dynamics (CGMD) simulations of the peptides within a hydrated lipid membrane specify the mode of binding of TMD4 with either E5 protein or its individual TMDs. From potential of mean force calculations (PMF) and statistical analysis enthalpy and entropy contributions are attributed of TMD4 binding to TMD3 and TMD2 of E5, respectively.
Pasteur Institute of Iran, Iran
Sepehr Soleymani has graduated from Tehran University of Medical Science in Clinical Laboratory Science. Currently, he is a Master student of Medical Biotechnology in Blood Transfusion Organization and also as a Research Assistant in Pasteur Institute of Iran for 3 years. His activity focuses on natural and synthetic antiviral agents, drug delivery system and vaccine research.
Human immunodeficiency virus type 1 (HIV-1) belonging to the retrovirus family is the major agent of acquired immunodeficiency syndrome as a public health problem in the world. There are more than 253 types of approved anti-HIV drugs, but further development of novel anti-HIV agents would be needed especially in low-income countries without anti-retroviral treatment. Some limitations of the recent viral therapies include high risk of resistant viruses, and adverse side effects in long-term therapy. Therefore, it is necessary for improvement of novel potent and safe anti-HIV drugs with decreased side effects especially tolerability and toxicity. Furthermore, other problem in treatment of HIV-infected patients is their susceptibility to Herpes simplex virus (HSV) infection; thus, both anti-HSV and anti-HIV drugs with novel modes of action are required. Recently, saffron components have been proposed to treat various pathological conditions. In this study, crocin, a major carotenoid of saffron, was extracted from the ethanolic saffron extract by adsorption chromatography using neutral aluminum oxide 90 active. Then, the anti-HSV-1 and anti-HIV-1 activities of crocin were assessed as well as its cytotoxicity in vitro. The data indicated that crocin was active against HIV-1 and HSV-1 virions at certain doses. Crocin inhibited the HSV replication at before and after entry of virions into Vero cells. Indeed, crocin carotenoid suppressed HSV penetration in the target cells as well as disturbed virus replication after entry to the cells. This sugar-containing compound extracted from saffron showed to be an effective anti-herpetic drug candidate. In general, crocin would be a promising anti-HSV and anti-HIV agent for herbal therapy against viral infections.
Eastern Mediterranean University, Turkey
Şükrü Tüzmen is a Molecular Biologist and Geneticist. He has more than 28 years of multi-disciplinary research experience integrating studies of the molecular basis of human diseases, including cancer genetics. He has a passion for advancing the molecular genetics of diseases by studying the associations between drugs, genes, pathways, and diseases. His mission is to discover and validate links between gene states and disease phenotypes, and further use these links to identify druggable targets to be utilized as biomarkers in the early diagnostic stages of genetic diseases. He has focused his career on developing and applying cutting edge methods and technologies to ensure excellence in translation of his basic scientific research including cancer genetics, from bench to bedside. He has been invited to deliver talks in many national and international settings, and he has served on many expert panels including The Research Grant Council, Hong Kong, China.
A classical technique to determine the function of a gene is to experimentally inhibit its gene expression in order to examine the resulting phenotype or effect on molecular endpoints and signaling pathways. RNA interference (RNAi) is one of the recent discoveries of a naturally occurring mechanism of gene regulation facilitated by the induction of double stranded RNA into a cell. This event can be utilized to silence the expression of specific genes by transfecting mammalian cells with synthetic short interfering RNAs (siRNAs). siRNAs can be designed to silence the expression of specific genes bearing a particular target sequence and may potentially be presented as a therapeutic strategy for inhibiting transcriptional regulation of genes, which in such instances constitute a more attractive strategy than small molecule drugs. Low dose drug and siRNA combination studies are promising strategies for the purpose of identifying synergistic targets that facilitate reduction of undesired gene expression and/or cell growth depending on the research of interest. Commercially available RNAi libraries have made high-throughput genome-scale screening a feasible methodology for studying complex mammalian cell systems. However, it is crucial that any observed phenotypic change be confirmed at either the mRNA and/or protein level to determine the validity of the targeted genes. Currently, qPCR is widely utilized for accurate evaluation and validation of gene expression profiling. In this study, we describe a high-throughput screening of RNAi based gene knock-down approach and qPCR validation of specific transcript levels. Considering such advantageous applications, siRNA technology has become an ideal research tool for studying gene function in research fields including Pharmaceutical Biotechnology, and holds the promise that the utilization of siRNA-based therapeutic agents will accelerate drug discovery in clinical trials.
Prince Sattam Bin Abdulaziz University, Saudi Arabia
Sunitinib malate (SM) is reported as a weakly soluble drug in water due to its poor dissolution rate and oral bioavailability. Hence, in the current study, various “self-nanoemulsifying drug delivery systems (SNEDDS)” of SM were prepared, characterized and evaluated for the enhancement of its in vitro dissolution rate and anticancer efficacy. On the basis of solubilization potential of SM in various excipients, “Lauroglycol-90 (oil), Triton-X100 (surfactant) and Transcutol-P (cosurfactant)” were selected for the preparation of SM SNEDDS. SM-loaded SNEDDS were developed by spontaneous emulsification method, characterized and evaluated for “thermodynamic stability, self-nanoemulsification efficiency, droplet size, polydispersity index (PDI), zeta potential (ZP), surface morphology, refractive index (RI), the percent of transmittance (% T) and drug release profile.” In vitro dissolution rate of SM was significantly enhanced from an optimized SNEDDS in comparison with SM suspension. The optimized SNEDDS of SM with droplet size of 42.3 nm, PDI value of 0.174, ZP value of −36.4 mV, RI value of 1.339% T value of 97.3%, and drug release profile of 95.4% (after 24 h via dialysis membrane) was selected for in vitro anticancer efficacy in human colon cancer cells (HT-29) by MTT assay. MTT assay indicated significant anticancer efficacy of optimized SM SNEDDS against HT-29 cells in comparison with free SM. The results of this study showed the great potential of SNEDDS in the enhancement of in vitro dissolution rate and anticancer efficacy of poorly soluble drug such as SM.
University of Zabol, Iran
Nima Sanadgol is expert in field of Cell and Molecular Neurobiology. His recent research emphasis is in treatment of neurodegenerative disease with use of new natural compounds. He has particular interest in evaluation of mechanisms of neuron-glia interactions, in order to fascinating myelin repair and control of neuro-inflammatory and neuro-degenerative diseases (Multiple sclerosis, Alzheimer, Parkinson, etc.). He has already gained so much experience in neuro-immune and circuit-specific signaling in glial-neuron networks (T cell biology, NF-κB, Nrf2, MAP kinase, AMP kinase, apoptosis and autophagy).
Pharmacological approaches to inhibit brain acute inflammation may represent important strategies for the control of autoimmune diseases. Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating and autoimmune disease of the central nervous system (CNS). Cucurbitacin B (CuB), an oxygenated tetracyclic triterpenoid compound extracted from Cucurbitaceae plant species, is a bioactive agent by disruption of microtubule polymerization and inhibition of JAK/STAT signaling. However, there has been little information about impact of CuB on MS treatment. In this research, for the first time we examine effects of CuB (specific STAT3 blocker), in experimental autoimmune encephalomyelitis (EAE) mouse model of MS. EAE was induced by subcutaneous immunization of MOG35-55 in 8-week-old C57BL/6 mice. CuB was administered at different doses (0.25, 0.5 and 1 mg/kg body weight/day/i.p) from the first day of the experiment. Inflammatory responses were examined using qRT-PCR, western blot and immunohistochemistry (IHC) analysis of specific markers such as p-STAT3, IL-17A, IL-23A, CD11b and CD45. CuB reduced STAT3 activation, leukocyte trafficking, and also IL-17/IL-23 immune axis in this model. Treated mice with lower doses of CuB exhibited a considerable depletion in the EAE clinical score which correlated with decreased expression of IL-17, IL-23 and infiltration of CD11b+ and CD45+ cells into the CNS. Our in vivo results suggest that STAT3 inhibition by CuB will be an effective and new approach for the treatment of neuro-inflammatory disease such as MS.
Universitat Rovira I Virgili, Spain
Vladimir A Baulin has completed his graduation with honors from the Physics Department at Moscow State University in 2000. He spent three years in the Commissariat à l’Energie Atomique, Grenoble, France, pursuing his PhD in theory of polymer physics and received a PhD in Physics in 2003. In 2004-2006 he was a Postdoctoral Researcher at the Institut Charles Sadron, Strasbourg, France. Since 2008, he leads a group of Soft mater theory at the University Rovira i Virgili, Tarragona, Spain. He is a Coordinator of EU funded initial training network SNAL: smart nano-objects for alteration of lipid bilayers.
Design of nanomaterials able to cross lipid bilayers is a challenging task in nanotechnology. Large variety of shapes, sizes and surface coatings are used for the design of nanomaterials to overcome this barrier. However, the potential barrier is quite high for carbon nanotubes and nanoparticles to cross the lipid bilayer to translocate by thermal motion. It is generally accepted that small hydrophobic nanoparticles are blocked by lipid bilayers and accumulate in the bilayer core, while nanoparticles with sizes larger than 5 nm can only penetrate cells through a slow energy-dependent processes such as endocytosis, lasting minutes. In one example, we show how variation of hydrophobicity of the nanoparticles can lead to passive translocation of nanoparticles through lipid bilayer. This adsorption transition through reversible destabilization of the structure of the bilayer induces enhanced permeability for water and small solutes. In another example, we demonstrate that lipid-covered hydrophobic nanoparticles may translocate through lipid membranes by direct penetration within milliseconds. We identified the threshold size for translocation: nanoparticles with diameters smaller than 5 nm stay trapped in the bilayer, while nanoparticles larger than 5 nm insert into bilayer, open transient pore in the bilayer. Using the Single Chain Mean Field (SCMF) theory a mechanism of passive translocation through lipid bilayers is proposed. Observing individual translocation events of gold nanoparticles with 1-dodecanethiol chains through DMPC bilayers, we confirm the particle translocation and characterize the kinetic pathway in agreement with our numerical predictions. Mechanism relies on spontaneous pore formation in the lipid bilayer. The observed universal interaction behavior of neutral and chemically inert nanoparticles with bilayer can be classified according to size and surface properties.