Day 2 :
Universitat de les Illes Balears, Spain
Keynote: First insights into the discovery of novel toxin-antitoxin systems from comparative genomics of non-tuberculous mycobacteria
Time : 10:05-10:30
Antoni Bennasar-Figueras has completed his PhD at the Universitat de les Illes Balears (UIB, Spain) and Post-doctoral research at the Gesellschaft für Biotechnologische Forschung (GBF, Germany). He is a Professor of Microbiology and member of the Microbiology Research Group at the UIB. He has specialized in the application of Next-Generation Sequencing (NGS) technologies to the genome and comparative analysis of several microorganisms (Pseudomonas, Mycobacterium and Streptococcus). He has published more than 30 articles in well-known journals and has been serving as an Editorial Board Member of recognized journals like Environmental Microbiology, Systematic and Applied Microbiology.
The non-tuberculous mycobacteria (NTM), also known as rapid growing mycobacteria (RGM), include several species that are environmentally widely distributed. Some of them are even considered as emerging opportunistic pathogens. Many bacteria genomes, including those belonging to the Mycobacterium genus, contain a considerable number of toxin-antitoxin (TA) systems, specially the important slow growing pathogen M. tuberculosis. The TA systems are small genetic modules based on two genes, one coding for the toxin and another for its cognate antitoxin. These elements are well-known systems that can be related to the virulence of bacteria and are considered interesting targets for alternative treatments of infections. A detailed study from a genomic, functional and structural point of view of the TA systems could be useful for finding alternative targets in the treatment of hard-to-treat infections, such the ones caused by emergent opportunistic NTM pathogens. The comparative analysis of information based on the application of next-generation sequencing (NGS) technologies, has been useful to explore the presence of potential TA systems in clinical strains of NTM. Effectively, the genome sequencing of NTM isolates, the identification of gene families, functional characterization, and comparison, has revealed the presence of TA systems in several of the strains considered. Interestedly, most of the TA systems discovered in NTM are also present in M. tuberculosis. The potential toxic effect of the proposed toxins and its neutralization with the hypothetical antitoxins has been tested through their expression in Escherichia coli cells. A brand new functional type II epsilon/zeta toxin-antitoxin system has been discovered in a strain closely related to Mycobacterium chelonae. Additionally, in the vicinity of the new TA system are genes coding for proteins containing β-lactamases and integrases/transposases domains, representing a potential mobile genetic element stabilizing antibiotic resistance genes. Conclusively, this is a good example of how the new NGS based information may lead to important breakthroughs, both in biotechnology and clinical microbiology.
- Drug Discovery and Development | Pharmaceutical Biotechnology | Antibiotics
B Koray Balcioglu is a senior Molecular Biologist with experiences in Antibody Engineering, Phage Display Technology, Molecular Biology and Recombinant Protein and Antibody Purification. His research projects experience includes the development of antio-angiogenic peptides and recombinant antibodies for cancer therapy, the development of gold binding bi-functional antibodies for biosensor applications. He is interested in new biopharmaceutical drug development, biosimiliars and theranostic agents. He has completed his PhD in Molecular Biology and Genetics Department from Technical University of Istanbul. He has one national patent and one international patent regarding the development of peptides and recombinant antibodies against angiogenesis.
Signal transduction through vascular endothelial growth factor (VEGF) and its receptor play a crucial role in Angiogenesis. Therefore, the development of a biotechnological product with anti-angiogenic properties is widely investigated for fighting cancer. For that purpose, antibodies, recombinant antibodies and peptides are generated or selected from various libraries. Previously, we have discovered anti-angiogenic peptides against VEGF by using a 7-mer phage displayed peptide library. The sequence of the peptides was determined after DNA sequence analysis. The anti-proliferative activity of the selected peptides was monitored in vitro on Human Umbilical Vascular Endothelial Cells (HUVEC). Here, we have synthesized and purified several peptides derived from the original anti-VEGF peptide by changing one by one all the amino acids by Alanine. This process so called “alanine scanning” was done for the determination of critical amino acids responsible in the binding of the peptide to VEGF and the ones necessary for blocking the activity of VEGF. Each of the synthesized peptides was tested in vitro on human umbilical vein endothelial cells for their anti-proliferative effect. With this work, new peptides with higher affinity and anti-angiogenic properties are intended to be developed.
TUBITAK Marmara Research Center, Turkey
Time : 11:15-11:40
Berrin Erdag has expertise on Antibody Engineering, Phage Display Technology, Molecular Biology and has successfully completed projects in different field of applications such as Cancer Therapy, Biosensor, in-vitro monitoring. She has a national patent entitled, “Peptide Structures that Bind and Block the Activity of Vascular Endothelial Growth Factor (VEGF)”, where an anti-angiogenic peptide is protected. She has also an international patent entitled, “Recombinant antibody structures binding to and blocking the activity of vascular endothelial growth factor-2 (VEGFR-2/KDR)” US patent No: 9193792, 24/11/2015, China patent No: ZL 2010800690064, 29/06/2016, where two anti-angiogenic recombinant antibodies with a potential anti-cancer properties are expected to be protected. She is the co-leader of a Biosimilar development Landmark project which is the first nationally funded biosimilar project in Turkey. The project consists on the development and production of an anti-cancer biosimilar antibody. Right after the support of the biosimilar project, she has been granted by the Ministry of Development for the development of a Centre of Excellence for Medical Biotechnology. She is still working hard for the development of a biotechnological drug development ecosystem in Turkey.
Statement of the Problem: Even though cancer cells are abnormal, they still require oxygen and nutrients. Angiogenesis, the development of blood vessels, is an essential step in the growth of a tumor. Without vessels, tumors cannot grow to be larger than a small fraction of an inch. Vascular endothelial growth factor (VEGF) and its receptor (VEGFR2/KDR) are major mediators of angiogenesis associated with tumors and other pathological conditons, including age-related macular degeneration and proliferative diabetic retinopathy. It is well known that inhibition of VEGF induced angiogenesis is a valid strategy for the treatment of solid tumors and other disorders in humans. In this context, two peptides and two recombinant antibodies able to inhibit angiogenesis have been developed in MRC.
Methodology: In this study, we identified two single chain variable fragments (scFvs) that directly bind VEGFR-2 and inhibit VEGF-dependent cell proliferation and quantified their receptor-binding affinities. Phage display method was used to construct recombinant single-chain antibodies, which are smaller in molecular size, but still retain the VEGF-blocking property of larger antibodies. Two specific single-chain antibodies (KDR1.3 and KDR2.6 scFvs) recognizing the extracellular immunoglobulin-like domains 1–7 of VEGFR-2 were selected from a V-gene phage display library constructed from mice immunized with the commercially available soluble extracellular domains 1–7 of VEGFR-2.
Findings: KDR1.3 and KDR2.6 scFvs were characterized at the DNA and protein levels by ELISA, DNA sequencing, and surface plasmon resonance (SPR) spectroscopy. Both anti-KDR scFvs bind to sKDR D1–7, block VEGF binding to sKDR D1–7, and show potent inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells (HUVECs) by a rat cornea angiogenesis assay (CAA).
Conclusion: Our results demonstrated that KDR1.3 and KDR2.6 antibodies could inhibit angiogenesis via. interaction with the VEGFR-2 extracellular domain. Thus the identified recombinant antibodies may have potential to be used as angiogenesis inhibitors.
National Polytechnic Institute, Mexico
Title: Dioxo/isoindoline derivatives as novel drugs for neurodegenerative disease: Parkinson’s and Alzheimer's disease (in silico, in vitro and in vivo study)
Time : 11:40-12:05
Erik Andrade Jorge is a Doctorate student in the Department of Biochemistry at Instituto Politecnico Nacional. He is a Chemist-Pharmaceutical-Biologist and has a Master’s degree in Pharmacology and is currently pursuing Doctorate research in medicine. He has two different research lines, one is cancer cell proliferation and another one is Parkinson’s disease. His work focuses on the rational drug design based on the molecular mechanisms of different pathologies and in the physicochemical properties of the ligands.
Neurodegenerative diseases are a heterogeneous group of disorders that are characterized by the progressive loss of the structure or function of neurons, including the death of neuron cells. Common neurodegenerative diseases include Alzheimer's disease and Parkinson's disease. Isoindoline represents an important family of compounds presents in a wide array bioactive molecules and also it has been seen this kind of compounds has effect in the central nervous system. This is the main reason they have attracted the attention of many researchers, even our work group. Therefore, the aim of the present study was to design and evaluate a series of isoindoline to test their selectivity for the dopamine D2 receptor, and a series of dioxoisoindoline as possible inhibitors of acetylcholinesterase. In the study of the molecular and toxicological properties, dioxoisoindoline and isoindolines showed that possess favorable characteristics as potential drugs. Taking into account the in silico results, the synthesis of the molecule Ia1 (isoindoline) and Da1 (dioxoisoindoline), and their structures were confirmed by IR, 1H and 13C NMR and mass spectroscopy was carried out. The in vivo evaluation for the isoindoline (Ia1) showed that this compound has an effect on the motor activity of male C57BL/6 mice in the MPTP model. While molecule Da1 (dioxoisoindoline) was tested in an intro experiment, results showed that this molecule has the ability to inhibit acetylcholinesterase. These results allow us to try other candidates who might possess the same properties and also perform others studies that can give us more evidence about selectivity.
Bribiesca-Carlos José is a Medical student at Escuela Superior de Medicina at Instituto Politécnico Nacional. He is working at the Biochemistry Research Laboratory in order to improve his knowledge about the chemistry of life and the synthesis of drugs.
Cancer is a disease that has a multifactorial origin, actually, the scientist in the entire world are working for one effective treatment or a cure, but the idea seems so far because in one-day thousands of new cases are detected and in the moment we only have a small idea of confronting it. The new discoveries to stop cancer have a central element, this is the genetic regulation, and one that has great interest is blocking the function of histone deacetylase which has as their central role withdrawing an acetyl group, which helps temporarily silencing gene expression, as it returns to a state of super curl DNA. Our work comprises selective inhibitors of HDAC8, family member number one of the HDACs, which depends on a zinc molecule to perform its function; this specific protein has been implicated in the development of metastasis in cancer breast, besides participating in the differentiation and proliferation of cells by yet unknown mechanisms. We had been designed two molecules that have structural relation, the results obtained in our tests are positive for inhibition of HDAC8 with a ∆G=-5.290 Kcal/mol, we obtained favorable results in terms of reducing the load of transformed cells at a concentration of 1x10-4 M, in 24 and 48 hours, for one of our two molecules, data were shown by spectroscopy at 540 nm. Our second molecule proved to be nothing effective and opens the gap to a discussion on how to change the structure a little can affect the biological response.
Medical University of Lublin, Poland
Time : 12:30-12:55
Urszula Kosikowska (PhD) is a Pharmacist, specialist in Medical Microbiology. She is a Lecturer in the Department of Pharmaceutical Microbiology with Laboratory for Microbiological Diagnostics, Medical University of Lublin, Poland. She has published scientific publications in reputed journals, popular articles and conference reports. Her research interests include issues of respiratory microbiota, diagnostics and drug resistance of bacteria and antimicrobial activity of newly synthesized compounds on planktonic and biofilm-forming cells mainly of the genus Haemophilus and other selected members of the family Pasteurellaceae.
Antibiotic-resistant Enterococcus faecalis present in poultry can pose hazard health serving as the risk of transmitting these strains to humans. The aim of the study was to provide data on antimicrobial resistance in E. faecalis isolated from the hearts of broiler chickens, aged from 3 to 6 weeks. 57 isolates of E. faecalis were investigated. First, the isolated bacteria were identified using MALDI-TOF mass spectrometry (Bruker Daltonics, Germany). Then, the identification was confirmed by genus- and species-specific multiplex PCR. Susceptibility testing was carried out to determine sensitivity to vancomycin and ampicillin (0.125-64 µg/ml), gentamicin (2-1024 µg/ml), kanamycin and streptomycin (4-2048 µg/ml), erythromycin and tetracycline (0.25-128 µg/ml), ciprofloxacin, lincomycin and chloramphenicol (0.5-256 µg/ml) using the minimal inhibitory concentrations (MICs) assessment. Resistance to two or more antibiotic agents was demonstrated. High resistance (>50%) was shown in the isolates including resistance to lincomycin (100%), tetracycline (63.2%), erythromycin (54.4%) and gentamicin (52.6%). Furthermore, high-level aminoglycoside (gentamicin - 1.8%, kanamycin - 1.8%, streptomycin - 5.3%) resistance was noted. Resistance to ciprofloxacin (21.1%) and chloramphenicol (8.8%) was classified as low (<25%). Moreover, a certain percentage of isolates exhibited intermediate sensitivity, particularly to gentamicin (36.8%), erythromycin (29.8%), and ciprofloxacin (19.3%). Vancomycin and ampicillin resistant isolates were not detected.
University of Pretoria, South Africa
Time : 13:55-14:20
Oleg Reva has obtained his PhD in 1995 from Institute of Microbiology and Virology, National Academy of Science of Ukraine. He has his Post-doctoral fellowship in 2002-2004 in High School of Medicine, Hannover, Germany. He is an Associated Professor in the Centre for Bioinformatics and Computational Biology at the University of Pretoria. He has published more than 90 papers cited in 1700 in other publications. Fields of interest are bioinformatics for biotechnology and medicine, genome sequencing, comparative genomics and molecular docking.
Development of new antibiotics is recognized as a global problem. We have to admit that we have been rather prodigal with the natural resources of antibiotics. The idea that one bioactive compound may exhibit many useful activities by targeting many unrelated molecular targets is getting popularity. This work was focused on two antibiotics, mupirocin and batumin, – synthesized by Pseudomonas, which are active against drug resistant Staphylococcus aureus. Our study was driven by a curiosity: what could be the biological sense for the rhizobacterial Pseudomonas to produce these rather complex and energy expensive compounds, which specifically inhibit growth of bacteria not common for the rhizosphere? Pharmacophore based search, molecular docking and experimental trials demonstrated that these antibiotics may have multiple molecular targets. Pathogenic staphylococci were inhibited by targeting tRNA synthetases and fatty acid biosynthesis. Another possible target was the bacterial MotB flagellar motor protein. It was shown that batumin can immobilize phytopathogenic bacteria leaving them alive but not active. Moreover, possible molecular targets were found among eukaryotic cancer related proteins. Mupirocin and batumin showed an extremely high and specific cytotoxicity against several cancer cell lines including melanoma MEL-1 and Lewis lung carcinoma. By preliminary results it was supposed that these compounds caused a cell cycle arrest at G1/S checkpoint via activation of retinoblastoma protein and down-regulation of the cyclin D1. Our results showed that the same antibiotics, or their target specific derivatives, may find much more proper and broader use in medicine, veterinary and agriculture.
Rajaratnam College for Women, India
Time : 14:20-14:45
U Umadevi is an Assistant Professor of Botany. She has published 35 research articles in national and international journals. She is an Editorial Board Member of Virology Research Journal. She has Co-authored a scientific book, “The Multi-science 22nd Century Inventions” and also published book on divinity. She has received awards, fellowships, prizes from government and private organizations. She has chaired international conference organized by World Academy of Science, Engineering and Technology, London on 17th and 18th October, 2016 and got best paper award. She has received Alexander Fleming Award from USA.
Efficacies of newly evolved antibiotics are basically assessed under in vitro by antibiotic sensitivity test (ABST). In this study, recently invented universal potentiator was incorporated with various antibiotic discs with control. These antibiotics were challenged with various virulent bacteria (hospital isolates). By ABST, the zone of inhibition measured. The universal potentiator was found to have potentiated the antibiotics up to 3-166%. This discovery has enormous application to the living beings.
Vienna University of Technology, Austria
Alexandra Hofer is recently doing her PhD thesis at the Technical University of Vienna, Institute of Chemical Engineering. She has done her Diploma thesis in Pharmacy, focusing on the development of novel methods in order to describe the glycol-recognitive drug delivery systems of targeted therapy of urothelial diseases. Thus, she has a strong background in biochemical analytics. At TU Vienna, her research focuses on the characterization of different raw material and their impact on process performance, concentrating on filamentous processes.
Statement of the Problem: Processes using Penicillium chrysogenum for the production of antibiotics are established since the 1940’s and are already well described in literature. Generally, these processes are performed in two phases, a batch phase where complex nutrients – mainly corn steep liquor (CSL) are used to enhance spore germination and a fed-batch phase using defined media for product formation. Especially in the batch phase, the mechanism of the positive impact of CSL on growth is still poorly understood and accordingly also the effect of complex media supplements that are transferred in the fed-batch phase. This study focuses on the investigation of CSL and its impact on growth and process performance in both process phases.
Methodology & Theoretical Orientation: Varied methods were developed in order to quantify specific nutrients in CSL with the special focus on amino acids. Batch and Fed-batch cultivations with P. chrysogenum, producing Penicillin V, were performed and analyzed concerning variations in its metabolite pattern. Three main topics were investigated in detail: (1) utilization of complex nutrients by the mold, (2) influence of CSL on physiology and (3) morphology of the fungus.
Findings: Correlations in the beforehand mentioned topics of nutrient utilization; physiology and morphology could be found and lead to higher process robustness. Especially information about limiting amino acids enabled an enhanced process control and led to an improved productivity in the main culture.
Conclusion & Significance: Robustness of fungi fermentations is still a challenge in bioprocess development. Therefore, this contribution focuses on a better process understanding in respect to the influence of complex media ingredients on process performance. Especially the results concerning bioavailability of the CSL and its influence on physiology and morphology of the fungus enable an overall deeper understanding of the fermentation.