Day 2 :
Keynote Forum
Ada Yonath
Weizmann Institute, Israel
Keynote: Novel antibiotics against species-specific resistance, microbiome conservation and environmental preservation
Time : 09:30-10:30
Biography:
Ada Yonath is focusing on protein biosynthesis and the antibiotics hampering it. In the seventies she established the first structural-biology laboratory in Israel. She is the Director of Kimmelman Center for Biomolecular Structure. During 1986-2004 she was the Head of Max-Planck Research Unit for Ribosome Structure in Hamburg. She is a Member of US National Academy of Sciences, Israel Academy, German Science Academy and Pontificia Accademia-delle-Scienze (Vatican). She holds honorary Doctorates from Oslo, NYU, Mount-Sinai, Oxford, Cambridge, Hamburg, Berlin-Technical, Patras, De-La-Salle, Xiamen, Lodz universities. Her awards include the Israel Prize, Louisa-Gross-Horwitz Prize, Linus-Pauling Gold Medal, Wolf-Prize, UNESCO/L’Oreal Award, Albert-Einstein World Award for Excellence and Nobel Prize for Chemistry.
Abstract:
The current global escalation in resistance to antibiotics is a serious threat, as it seems that the world is headed for a post antibiotic era, in which common infections and minor injuries that have been treatable for decades could become fatal again. Ribosomes, the universal cellular machines that translate the genetic code into proteins, are paralyzed by many clinically useful antibiotics. The structures of ribosomes from non-pathogenic bacteria, used as models for genuine pathogens, illuminated the antibiotics binding modes, inhibitory actions, synergism pathways, the differentiation between patients vs. pathogens and mechanisms leading to bacterial resistance. However, as species specific diversity was detected in susceptibility to infectious diseases and in developing specific resistance mechanisms, our structural studies have been extended to ribosomes from genuine pathogens. The high resolution structures of ribosomal particles from multi-resistant pathogens and from eukaryotic parasites with several antibiotics, highlighted subtle, albeit highly significant structural elements that can account partially or fully for species specificity and may be exploited for improving known antibiotics and for the design of novel compounds.
Keynote Forum
Reiko Kuroda
Tokyo University of Science, Japan
Keynote: How is the chirality information transferred during the mollusk development?
Time : 10:30-11:00
Biography:
Reiko Kuroda has obtained her PhD in Chemistry from The University of Tokyo and carried out Postdoctoral studies at King’s College London. Her research focuses on chirality both in the field of chemistry and biology: Chirality recognition, transfer and amplification in the solid state, development of chiroptical spectrophotometers to follow aggregation processes of e.g. β-amyloids and the molecular basis of snail coiling. She has 320 papers published to her credit.
Abstract:
Body handedness of Gastropod Lymnaea stagnalis is determined by a single gene locus that functions maternally. We have previously shown that the gene dictates the cytoskeletal dynamics at the third cleavage and have created fertile snails of mirror-image body plan by mechanically manipulating the blastomeres at this stage. In this talk, we shall show that nodal expression started at the 33-49 cell stage and was confined only to the left or the right side of embryos, in a mirror-image manner for the sinistral and the dextral snails, throughout the development until the shell was getting formed. The mechano-manipulation reversed the entire expression. Thus, the blastomere arrangement at the 8-cell stage regulates the asymmetric expression of nodal/Pitx genes. At the 24-cell stage, the organizer macromere 3D moved to the central location of vegetal side, filled almost entire cleavage cavity where it contacted with micromeres. MAPK (mitogen-activated protein kinase) was activated in 3D macromere 30 - 60 min. after embryo reached the 24 cell stage. Treatment with U0126, a specific inhibitor of MEK (MAPK/ERK kinase), at the 4-8 cell stage completely inhibited the expression of nodal and its downstream Pitx, whereas, similar treatment at the 24-cell stage after the activation of MAPK did not inhibit the gene expressions. These results clearly indicate that chirality determined at the 8-cell stage is transferred to the 24-cell stage via spiral cleavage and MAPK at the 24-cell stage controls the left-right asymmetric expression of nodal/Pitx genes.
- Molecular Biology | Structural Biology
Location: Experience II
Chair
Michael Kolbe
Max Planck Institute for Infection Biology, Germany
Co-Chair
Alexei V Finkelstein
Russian Academy of Sciences, Russia
Session Introduction
Alexei V Finkelstein
Russian Academy of Sciences, Russia
Title: There and back again: Two views on the protein folding puzzle
Time : 11:15-11:35
Biography:
Alexei V Finkelstein was graduated in 1970 and received his PhD degree in 1976 from the Moscow Institute of Physics and Technology. He is the Head of the Protein Physics Lab at the Institute of Protein Research, Russian Academy of Sciences and is a Professor of the Lomonosov Moscow University awarded by the State Prize of Russia; elected to the Russian Academy of Sciences, Alumnus of LMB MRC (Cambridge) and HHMI Scholar (1995-2010). He has published 200+ papers in reputed journals. His two books on protein physics has been published in English, Russian and Chinese. He is serving as an Editorial Board Member of several reputed journals.
Abstract:
The intricate protein 3D structure results from spontaneous folding (both in vitro and in vivo), though more than the life-span of the Universe would have been required to find it by sampling of amino acid conformations, this is called "the Levinthal's paradox". To resolve it, various models of folding were proposed during decades. However, they fail to overcome this paradox when the 3D structure’s stability is close to that of the unfolded chain, as it is in reality. The folding rate problem was first solved using unfolding (not folding!). The trick is that, firstly, the rates of the forward and reverse reactions coincide when the structure stability equals to that of the unfolded chain (according to the “principle of detailed balance” well-known in physics) and it is much easier to imagine and investigate how the thread unfolds than how it obtains a certain fold. Recently, the folding problem has been also solved “from the viewpoint of the folding chain”: it has been proved that the necessary, search for the most stable fold, sampling is by many orders of magnitude smaller when considered at the level of formation and packing of secondary structures than at the Levintal-considered level of amino acid conformations. The estimated rate of such a sampling turned out to be physically and biologically reasonable for proteins of various sizes.
Michael Kolbe
Max Planck Institute for Infection Biology, Germany
Title: The bacterial type 3 secretion system: Structure function analysis of a molecular syringe
Time : 11:35- 11:55
Biography:
Michael Kolbe has studied Chemistry at the Universities of Paderborn and Hamburg. Thereafter he has completed his Doctorate on the structure and function of the chloride pump Halorhodopsin at Max-Planck-Institute for Biochemistry and the Ludwig-Maximilians University in Munich. After his Postdoc at the Max-Delbrück Centre in Berlin he joined the Max-Planck-Institute for Infection Biology as Leader of a Junior Research Group. Since the beginning of the year 2015, he is a Professor at the University Hamburg and Head of the Department for Structural Infection Biology at Helmholtz Center for Infection Research.
Abstract:
Many different bacterial pathogens use protein secretion systems to transport effector molecule into host cells. Effector transport through these systems is essential to establish and maintain host cell infection. To understand host-pathogen interaction and pathogenesis during infection, we analyzed the structure and function of the type 3 secretion system (T3SS) of Gram-negative bacteria including Shigella flexneri and Salmonella typhimurium. The T3SS is a highly conserved virulence machinery of Gram-negative bacteria that initiates infection by subverting host cell defense mechanisms. Although great efforts were made to elucidate the structure of the T3SS in the last decades, the mechanistic details of the protein transport remains elusive. By using structural biology methods in combination with biochemical and cellular assays we analyzed the assembly and the architecture of the T3SS. Our studies explain the assembly of the T3SS needle and they provide a model of substrate molecule transport. We also developed tools to block substrate molecule transport through the T3SS channel which might help to characterize important steps in the secretion process.
Biography:
Cordula Enenkel has completed her PhD at the University of Stuttgart, Germany and Postdoctoral studies from Rockefeller University, New York, USA. She has worked as Privatdozent at the Humboldt University, Berlin, Germany, before she joined the University of Toronto as an Associate Professor in 2011. She has published 24 papers in reputed journals and has been serving as Reviewer for journals and funding agencies.
Abstract:
Proteasomes are key proteases in regulating protein homeostasis. During cell cycle progression, proteasomes exist as holo-enzymes in the nucleus and are engaged in the degradation of poly-ubiquitylated proteins. Proteasomes can be imported into the nucleus as mature or immature sub-complexes dependent on the growth conditions. Most of our body’s cells do not proliferate and remain in quiescence during aging. The transition from proliferation to quiescence is accompanied by significant metabolic changes and the rearrangement of sub-cellular structures. Proteasomes are exported from the nucleus into reversible and motile granules in the cytoplasm. Studies in quiescent yeast revealed that proteasome granules are not surrounded by membranes and rapidly resolve upon exit from quiescence. Proteasome granules either represent proteolytic centers for unwanted and toxic proteins or serve as storage compartments, thus names proteasome storage granules (PSGs). Their presence strengthens the resilience of quiescent yeast towards proteo and genotoxic stress and confers fitness during aging. The organizing principle of PSGs remains a mystery. We searched for proteins orchestrating PSG formation and performed high throughput microscopy screens in the yeast null mutant collection and GFP library. The hit genes involved in PSG organization are required for the regulation of nuclear transport, ubiquitin concentration, genotoxic stress and metabolic energy. We purified cross-linked PSGs and subjected them to mass spectrometry analysis. Our data suggest that PSGs mainly consist of proteins of the ubiquitin-proteasome-system and provide an interesting link between PSG formation and the availability of free ubiquitin.
Hermona Soreq
The Hebrew University of Jerusalem, Israel
Title: Non-coding RNAs: An integrative biology hope for anxiety-related syndromes
Time : 12:15- 12:35
Biography:
Hermona Soreq was trained at The Weizmann Institute of Science and the Rockefeller University. In 1980, she has joined the Weizmann Institute as a Senior Lecturer and joined the Faculty of The Hebrew University in 1986. She pioneered the application of molecular biology and neurogenomics to the study of cholinergic signaling with a recent focus on the evolutionary and functional aspects of brain microRNA regulation over cholinergic coding genes and pseudogenes under stress and in mental diseases. She has authored over 300 publications and won Advanced ERC Award and Israeli I-Core Center of Excellence Award on Mass Trauma.
Abstract:
The discovery of non-coding RNAs revolutionized our view of how the brain works and holds great promise for rapid progress in the run for therapeutics of anxiety-related diseases. We now know, unlike previous beliefs that our DNA includes several families of non-coding genes with regulatory functions; that these functions are differentially impaired under diverse anxiety reactions and most importantly, that the new genes can serve as scaffolds for developing new diagnostic tools and synthesizing novel therapeutics. I will discuss the family of microRNAs, tiny blockers of brain signaling pathways that govern cognition and anxiety reactions and will focus on those microRNAs that malfunction in mental diseases and their target genes with a special emphasis on the pathway of cholinergic signaling.
Byung-Dong Kim
Seoul National University, South Korea
Title: The foldback intercoil DNA
Time : 12:35- 12:55
Biography:
Byung-Dong Kim is a Professor Emeritus of the Seoul National University and a Member of the Korean Academy of Science and Technology. He is the chair of the Advisory Board of the Plant Genomics and Breeding Institute of Seoul National University. He has authored a book entitled “Foldback Intercoil (FBI) DNA” in 2008. Currently he is pioneering the biological meaning of the FBI DNA in the age of genomics and epigenetics.
Abstract:
Foldback intercoil (FBI) DNA is a unique and transient configuration of duplex DNA that consists of four functional parts: Foldback head, intercoil stem, intercoil blunt end tail and heteroduplexes. FBI DNA was first found in rare TEM configurations of pearl millet mtDNA and was tested out for its feasibility by space-filling model of DNA. FBI DNA is formed by foldback bending at one location of a duplex DNA, to lead the flanking anti-parallel double helixes to intertwine in each other’s major groove to form an intercoil. Repeat sequences in the intercoil make four-stranded base pairing. Base flipping of the repeats leads to heteroduplexes formation and homologous recombination. This subtle transformation of the double helix into FBI DNA structure is instrumental in mediating four major DNA-DNA transactions, namely, α deletion by direct repeats, Ω site-specific inversion by inverted repeats, FBI tip insertion in site-specific insertion and non-homologous end joining and gap filling (EHEJ-GF) in transposition. Most significantly, FBI DNA transfiguration into heteroduplexes offers an effective mechanism of paranemic separation of daughter strands after DNA replication which otherwise would get stuck after unwinding a plectonemic double helix DNA at the replication fork. Biochemical and bioinformatics evidences in the literature that support the FBI DNA for the inevitable presence and working in the real biology will be presented. Put together, realization of the dynamic and diverse transformation of the FBI DNA structure would allow a new understanding of diverse DNA function, their interaction at genome level and at cellular differentiation. Suggestions for future research strategies would be discussed.
Larry Mark Fisher
St George’s, University of London
Title: Structural basis of DNA gating by gyrase and topoisomerase IV and its inhibition by antibacterial therapeutics
Time : 12:55- 13:15
Biography:
Larry Mark Fisher has obtained his PhD in Chemistry from Harvard University and was a Damon Runyon Walter Winchell Cancer Fellow at NIH working with Dr Martin Gellert. He is the Dean of Research at St George’s, University of London. He has published more than 100 papers on DNA topoisomerases and DNA supercoiling in bacteria, yeast and human cell systems.
Abstract:
Gyrase and topoisomerase IV (topo IV) are bacterial type II topoisomerases that regulate cellular DNA topology and are key targets of antimicrobial therapeutics. The enzymes share close mechanistic and architectural similarity notably a DNA gating mechanism that involves the formation of a covalent enzyme-DNA complex known as the ‘cleavage complex’. Antibacterial fluoroquinolones and quinazolinediones stabilize the cleavage complex triggering cell death. To gain insight into the reaction cycle and drug action and resistance, we solved the first crystal structures of drug-DNA cleavage complexes with several different quinolones. We present structures for topo IV and gyrase revealing details of the cleaved DNA gate and drug binding pockets. Recently, we have also solved structures for the three-gate ‘open clamp’ state of topo IV, a key intermediate that suggests a mechanism for how DNA is captured and transported through the enzyme complex. Our studies provide new insights on how DNA is manipulated by these complex molecular machines to mediate DNA supercoiling and chromosome segregation.
Tali E Haran
Technion-Israel Institute of Technology, Israel
Title: The relationship between the structural properties of p53 binding sites and p53-dependent gene expression
Time : 14:05- 14:25
Biography:
Tali E Haran is an Associate Professor at the Department of Biology, Technion-Israel Institute of Technology. She holds PhD and Master Degree from the Weizmann Institute of Science, studying the crystal structure of DNA oligomers. She has completed her Postdoctoral studies at Yale University, studied DNA bending and indirect recognition in protein-DNA interactions. She has been a Visiting Scientist at the Department of Biochemistry, Cambridge University, UK at the Department of Biochemistry and Molecular Biology, Columbia University, USA and at the Centre for Integrative Biology, University of Trento, Italy. Her main research interest is the interactions between sequence-specific transcription factors and their DNA binding sites, focusing on the mechanism by which DNA structure contributes to the recognition of particular sites by regulatory proteins.
Abstract:
The tumor suppressor protein is a central hub protein in human cells. In response to stress, it functions mainly as a transcription factor (TF), binding to more than 200 response elements (REs), activating and repressing adjacent genes. The REs are made of two sequence-specific half-sites, separated by a variable number of base pairs. It is currently unclear how p53 chooses its target sites, however, p53 multi-functional activities are directly linked to its ability to function as a sequence-specific TF. It is now well appreciated that the intrinsic physical properties of the DNA double helix can affect binding of TFs and transactivation of adjacent genes. We have previously shown that DNA structure and flexibility vary among p53 REs, affecting their binding characteristics. We carried out experimental measurements of global structure and flexibility properties of p53 REs using cyclization kinetics of DNA minicircles, together with binding studies and transactivation assays in yeast and human cells. We propose that functional selectivity is conferred at least in part through p53/DNA binding and that differential structural characteristic of p53 REs play a role in this selectivity. I will present recent experimental results showing how p53 uses the structural properties of its binding sites to differentiate between functional classes of p53 REs, that p53 REs are allosteric effectors of p53 transactivation and the novel role of the spacer sequences in these interactions. These recent findings reveal how non-canonical sites, such as half-sites can be functional and hence expand the “universe” of p53 binding sites.
Gali Prag
Tel Aviv University, Israel
Title: Bacterial genetics and structural biology for decoding the ubiquitin code
Time : 14:25- 14:45
Biography:
Gali Prag has earned his PhD from the Hebrew University, Israel. With his supervisor Prof. Oppenheim, he has studied bacterial genetics and obtained two EMBO Fellowships to determine the structures of several chitin-degrading enzymes in complex with their native substrates. He has completed his Postdoctoral studies at the NIH with Professor James Hurley, where he determined the first structures of ubiquitin-receptor complexes with ubiquitin and some of the ESCRT complexes. In 2008, he returned to Israel and established a structural-biology and bacterial genetic laboratory in TAU.
Abstract:
Ubiquitin (Ub)-signals virtually regulate all cellular pathways. However, two major challenges impede our ability to identify and characterize associations within ubiquitylation cascades: Ubiquitylation cascades are multiplex, i.e., few E1s, dozens E2s and hundreds of E3s ubiquitylate thousands of substrates. Moreover, many substrates possess more than one cognate E3-ligase. About a hundred deubiquitylases rapidly and efficiently reverse the ubiquitylation. To circumvent these limitations we took an integrating biology approach including structural based in silico search, bacterial genetics, biochemical, biophysicals and X-ray crystallography to establish a productive interdisciplinary research. A novel bacterial genetic selection system for ubiquitylation and its utilization in identifying and characterizing new E3s, Ub-receptors and ubiquitylation substrates will be presented. Using bacterial expression of a functional ubiquitylation apparatus we purified and crystallized and determined the structure of an ubiquitylated-Ub-receptor for the first time. We took a multidisciplinary approach and uncovered a novel UBD within this receptor. A surprising function of the Ub-receptor ubiquitylation will be presented. As the findings derived from the genetic selection system we developed and the crystal structure of ubiquitylated-ubiquitin-receptor are still under review so I will share the full result and discussion at presentation time.
Sapun H Parekh
Max Planck Institute for Polymer Research, Germany
Title: Mechanically-induced protein structural changes in fibrin hydrogels using hyperspectral CARS microscopy
Time : 14:45-15:05
Biography:
Sapun H Parekh has joined the MPIP in February 2012. Before coming to the MPIP, he has worked as a Science Policy Fellow in the National Science Foundation in Washington, DC and as a Visiting Scientist at the National Institutes of Health. Prior to this, he was a Postdoctoral Researcher in the Biomaterials Group at the National Institute for Standards and Technology where he has worked on mechanobiology of stem cell differentiation and development of label-free imaging techniques. He has received his BS in Electrical Engineering from the University of Texas at Austin in 2002 and his PhD in Bioengineering from the University of California at Berkeley, San Francisco in 2008. His PhD thesis focused on force generation and mechanics of semi-flexible actin networks that are ubiquitous in biology. During his PhD, he has developed and applied a new atomic force microscopy system with improved long-term stability to measure these biophysical systems. At the MPIP his group focuses on molecular imaging and his research interests include applications of non-linear microscopy in biology and molecular biophysics.
Abstract:
Fibrin is a protein hydrogel material responsible for stabilizing the platelet-rich blood clot over a wound in blood coagulation. Its utilitarian mechanical properties: Being stiff or deformable as needed result from the unique hierarchical organization of the polymer network. Experimental and theoretical studies have shown that single filament stretching, network rearrangements and protein unfolding are responsible for this unique mechanical behavior. In this work, we investigate the biophysics of fibrin unfolding in response to uniaxial stretch in situ using coherent Raman imaging. Fibrin hydrogels are stretched to regime where unfolding transitions of α-helical structures to β-sheet occur and spatially resolved broadband coherent anti-Stokes Raman scattering (B-CARS) spectra of fibrin gels are acquired and analyzed. The secondary structure is calculated from peak analysis of the amide I and amide III regions, which are sensitive to subtle changes in protein structure. Experiments on gels of different polymer concentration and cross-linker density show unfolding transitions that correlate with non-linear effects seen in shear rheology. Finally, imaging results show an inhomogeneous unfolding distribution near inert beads in the gel, suggesting that local stresses on platelets within biological clots is different from the bulk mechanical response. This demonstrates the use of hyperspectral B-CARS microscopy to measure local, mechanically-induced conformational changes in proteins.
Henrik Devitt Møller
University of Copenhagen, Denmark
Title: Extrachromosomal circular DNA: A key player in creation of copy number variation
Time : 15:05-15:25
Biography:
Henrik Devitt Moller has completed his PhD at the University of Copenhagen, Denmark in 2015 and continued his work on extrachromosomal circular DNA as Post doctorate in the Regenberg Laboratory. He has published 8 papers in peer-reviewed journals.
Abstract:
Numerous human cancers are caused by copy-number variations (CNVs) of proto-oncogenes. Yet, detecting chromosomal CNVs before they reach establishment in large cell populations is a major challenge. By screening for a potential deletion-byproduct of CNVs, the so-called extrachromosomal circular DNA (eccDNA), we reasoned that we might elucidate some of the early ongoing processes in genomic rearrangements. We developed a highly sensitive eccDNA purification method, Circle-Seq that relies on removal of all linear DNA and next-generation sequencing of circular DNA. More than a thousand eccDNAs larger than 1 kb were recorded in the eukaryotic model Saccharomyces cerevisiae (yeast) increasing the number of known eccDNAs in eukaryotes more than a hundred fold. Now we present hundreds of eccDNA profiles from a distant related yeast subspecies. A number of eccDNAs are found to be identical between the two yeast strains, advocating for conserved hotspots for DNA circularization and potential genomic reintegration. We reveal that CNVs in the form of eccDNAs are common in S. cerevisiae and we hypothesize that eccDNAs could be important players in genetic variation and evolution of eukaryotic genomes.
Nadine Provencal
Max Planck Institute of Psychiatry, Germany
Title: Exposure to early life stress and glucocorticoids: Mechanisms of system-wide epigenetic effects
Time : 15:25-15:45
Biography:
Nadine Provencal has completed her PhD in Epigenetics at McGill University in collaboration with the Research Group on Psychosocial Maladjustment in Children (GRIP) in 2013. Thereafter, she continued her training through Post-doctoral research at the University of Montreal and Max-Planck Institute of Psychiatry. She has published more than 15 papers in high impact international journals and received a prestigious Postdoctoral Fellowship from the Canadian Institute of Health Research (CIHR) and has been awarded the 2014 Richard Todd Award from the International Society of Psychiatric Genetics for her outstanding contribution to the genetics of child psychiatry.
Abstract:
Exposure to early life stress (ELS) is a well-known major risk factor for developing psychiatric and behavioral disorders later in life. Both prenatal and postnatal stressors have long-lasting impact on adult pathological states. Epigenetic mechanisms have been shown to be involved in the embedding of these long-term changes. In a model of early life adversity in rhesus macaques, we have shown that differential rearing leads to long-lasting epigenetic alterations in two different tissues, the prefrontal cortex (PFC) and T cells. One of the mechanisms that might lead to these epigenetic alterations in multiple tissues is a long-lasting disruption of the stress hormone system by excessive glucocorticoids (GCs) release after ELS exposure. Using human hippocampal progenitor cells (HPCs), we have recently identified long-lasting DNA methylation alterations induced by GCs exposure, where a significant portion of these marks were initiated early during cellular proliferation and differentiation stages and persisted in mature neurons. Moreover, a significant overlap was observed between our GR-induced epigenetic changes in HPCs and sites previously associated with child abuse in postmortem human hippocampus and blood cells, suggesting similar long-lasting GR-induced epigenetic alterations in the brain and in peripheral tissues reflecting GC actions during ELS. A mechanistic understanding of the long-term epigenetic consequences of stress using a translational approach may allow novel, targeted intervention and prevention strategies for behavioral, psychiatric and other stress-associated disorders.
Jinyan Du
Merrimack Pharmaceuticals Inc., USA
Title: Identification of non-intuitive biomarkers predicting dependency on AKT vs. MAPK signaling of ERBB2+ cancer cells using an integrated experimental and computational framework
Time : 16:20-16:40
Biography:
Jinyan Du has completed her PhD at Harvard University and Postdoctoral training at the Broad Institute. She is currently a Principal Scientist at Merrimack Pharmaceuticals, a fully integrated biopharmaceutical company that is building one of the most robust oncology pipelines in the industry. She has published over 30 papers in reputed journals.
Abstract:
Understanding the molecular pathways by which oncogenes drive cancerous cell growth and how dependence on such pathways varies between tumors, could be highly valuable for the design of anti-cancer treatment strategies. In this work, we study how dependence upon the canonical PI3K and MAPK cascades varies across HER2+ cancers and define biomarkers predictive of pathway dependencies. A panel of 18 HER2+ (ERBB2-amplified) cell lines representing a variety of tumor types was used to characterize the functional and molecular diversity within this oncogene-defined cancer. PI3K and MAPK-pathway dependencies were quantified by measuring in vitro cell growth responses to combinations of AKT (MK2206) and MEK (GSK1120212; trametinib) inhibitors, in the presence and absence of the ERBB3 ligand heregulin (NRG1). A combination of three protein measurements comprising the receptors EGFR, ERBB3 (HER3) and the cyclin-dependent kinase inhibitor p27 (CDKN1B) was found to accurately predict dependence on PI3K/AKT vs. MAPK/ERK signaling axes. Notably, this multivariate classifier outperformed the more intuitive and clinically employed metrics, such as expression of phospho-AKT and phospho-ERK and PI3K pathway mutations (PIK3CA, PTEN and PIK3R1). The predictability of the three protein biomarkers for differentiating PI3K/AKT vs. MAPK dependence in HER2+ cancers was confirmed using external datasets (Project Achilles and GDSC), again out-performing clinically used genetic markers. Measurement of this minimal set of three protein biomarkers could thus inform treatment and predict mechanisms of drug resistance in HER2+ cancers. More generally, our study provides an integrated framework for identifying non-intuitive biomarkers in cancer patients.
Chaohui Dai
Yangzhou University, China
Title: Effect of porcine CD14 gene RNAi on the expression of toll-like receptor 4 pathway-related genes, the level of pro-inflammatory cytokines and adhesion ability to Escherichia coli
Time : 17:00-17:20
Biography:
Chaohui Dai is currently a Postgraduate student of Yangzhou University, China. She is mainly engaged in pig disease-resistant breeding and reproduction.
Abstract:
Three full-sib individuals of the resistance and susceptibility to E. coli F18 in Meishan piglets were obtained by challenging with the pathogens through feeding F18ab and F18ac strains and then the transcriptome sequencing of intestinal tissue in sensitive and resistant pigs to E. coli F18 was analyzed. The pig small intestinal epithelial cell line (IPEC-J2) with CD14 gene silencing were established by Lentivirus-mediated RNAi and the transcription and protein expression level of TLR4 pathway-related genes (MyD88, IFN-α, IL-1β, TLR4 and TNF-α) were detected; the levels of proinflammatory cytokines IL-6, IL-8, IL-12, MIP-1α and MIP-1β in cell culture supernatants were measured; the E. coli F18ab and F18ac’s adhesion ability to IPEC-J2 were detected. The transcriptome sequencing results revealed that toll-like receptor 4 (TLR4) signaling pathway especially CD14 gene played important roles in immune process of E. coli F18-invasion. After CD14 gene silencing, the transcription and protein expression levels of IFN-α, IL-1β, TLR4 and TNF-α were significantly down-regulation (P<0.05); the levels of IL-6 and IL-12 in cell supernatants were significantly reduced (P<0.05); the E. coli F18ab’s adhesion to IPEC-J2 enhanced highly significantly (P<0.01). In summary, the establishment of IPEC-J2 cell line with CD14 gene silencing stably mediated by Lentivirus offered important material for mechanism research of CD14 gene and TLR4 signal pathway. These results suggested that CD14 gene played an important role in not only TLR4 signaling pathway but also regulating the immune process of E. coli F18-invasion.
Ying Liu
Yangzhou University, China Panel
Title: MicroRNA transcriptome of duodenum tissues in Chinese Meishan weaned piglets challenged with E. coli F18
Time : 17:20-17:40
Biography:
Ying Liu is a Doctor of Yangzhou University, China. She is mainly engaged in pig disease-resistant breeding and reproduction.
Abstract:
Escherichia coli F18 is mainly responsible for post-weaning diarrhea (PWD) in piglets. The molecular regulation of E. coli F18 resistance in Chinese domestic weaned piglets is still obscure. We used Meishan piglets as model animals to test their susceptibility to E. coli F18. Small RNA duodenal libraries were constructed for E. coli F18-sensitive and resistant weaned piglets challenged with E. coli F18 and sequenced using Illumina Solexa high-throughput sequencing technology. Sequencing results showed that 3,475,231 and 37,198,259 clean reads were obtained with 305 known and 681 novel microRNAs (miRNAs) differently expressed in resistant and sensitive groups, respectively. Twenty-four miRNAs including 15 up-regulated and 9 down-regulated demonstrated more than a 2-fold differential expression between the F18-resistant and sensitive pigs. Stem-loop RT-qPCR validation indicated that most of the differently expressed miRNAs were consistent with the high-throughput sequencing data, including significantly expressed miRNAs, such as miR-136, miR-196b, miR-499-5p and miR-218-3p (p<0.05). KEGG pathway analysis for target genes revealed that differently expressed miRNAs were involved in infectious diseases, signal transduction and immune system pathways. According to the function of the target genes and previous sequencing results, miR-196b, miR-499-5p and miR-218-3p appear to be very promising candidates for miRNAs involved in response to E. coli F18 infection. The present study provides improved database information on pig miRNAs, better understanding of the genetic basis of E. coli F18 resistance in local Chinese pig breeds and lays a new foundation for identifying novel markers of E. coli F18 resistance.
- Poster Presentation
Session Introduction
Nehad Mahmoud Gumgumjee
King Abdulaziz University, KSA
Title: Synergistic antimicrobial effects and GC-MS analysis of phytocomponents of Commiphora quadricincta
Biography:
Nehad Mahmoud Gumgumjee has completed her PhD and Postdoctoral studies from King Abdulaziz University, Saudi Arabia. She has published more than 10 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
The Commiphora quadricincta, a small tree, belong to Burseraceae family is traditionally known for its medicinal properties. The present study was therefore carried out to investigate the synergistic antimicrobial activities and the phytochemicals of the bioactive components in the extract of this plant species. The antimicrobial activities of stem bark and leaves extract was investigated against 7 medically important bacterial strains, namely Bacillus subtilis, MRSA, Micrococcus, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aurues and Klebsella pneumoniae and five fungi (Aspergillus niger, A. fumigatus, A. flavus, Candida albicans and Saccharomyces spp.). The antibacterial activity was determined using agar well diffusion method. The most susceptible bacteria to this extract were Escherichia coli, while the most susceptible fungi were A. flavus. GC-MS analysis revealed that the ethanol extract of Commiphora quadricincta contained mainly; 2-Methyl-3-pentanol (2.84%); Butyl hydroxy toluene (22.32%); 9,12,15-Octadecatrienoic acid, 2-phenyl-1, 3-dioxan-5-yl ester (5.90); Ethyl isoallocholate (11.21%); à-Amyrin (2.22%) and Flavone 4-OH,5-OH,7-dioglucoside (11.21%). Most identified compounds are known to have antimicrobial activity.
Biography:
Abdul Rahman Hajar has completed his PhD and Postdoctoral studies from King Abdulaziz University, Saudi Arabia. She has published more than 45 papers in reputed journals and has been serving as an Editorial Board Member of repute.
Abstract:
Qutran (Wood tar) oils and vapor of Olea europaea subsp. cuspidate extracted medicinal plants were screened for their activity against seven strains of bacteria. Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Micrococcus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia using agar well diffusion method were investigated. Results showed that the effect of oils extract was higher than that of vapor. The tar oils have great antibacterial activity against all the investigated strains. The growth inhibition rate ranged from 16.33 to 46.00 mm .The wood tar oil showed higher activity against tested organisms than Streptomycin. In this study the most pronouns effect was shown by that of Olea europaea subsp. cuspidata wood tar oil. The most susceptible bacteria was Pseudomonas aeruginosa, flowed by Micrococcus luteus, while the most resistant bacteria was Staphylococcus aureus with 16.33 mm of inhibition zone. The results showed that wood tar oil of Olea europaea subsp. cuspidate was more effective than Streptomycin. GC-MS analysis revealed that the Qutran (Wood tar) oils contained mainly Octadecenamide (5.77%); Lucenin 2 (5.46%); Docosane and Nonacosane (3.75%); Cyclopropene (3.50%); Hematoporphyrin (2.68%); Tetratetracontane (2.36%); Dotriacontane (1.57); Acetic acid (1.53); N-methylglycine (1.49%); Propyne antimicrobial (1.41%). All identified compounds are known to have antimicrobial activity.
In-Su Park
Dankook University, South Korea
Title: Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice
Biography:
In-Su Park is currently working in Stem Cell Biology/Tissue Engineering Department at Korea Institute of Science and Technology (KIST) in Seoul, South Korea. He has completed his PhD in 2012 at Korea University. He has developed growth factor-immobilized substrate (fibroblast growth factor, heparin-binding peptide substrate) as a bioartificial material for stem cells differentiation. He has published more than 10 papers in reputed journals.
Abstract:
Human adipose-derived mesenchymal stem cells (hASCs), which are found in adipose tissue, are an attractive cell therapy source for the regeneration of damaged tissues because they are the
ability of self-renewal and the ability to differentiate into various cell lineages. Transplanting hASCs induces neovascularization and improves blood flow to ischemic tissue in animal models. In spite of the angiogenic potential of hASCs for treatment of ischemic wounds, these cell sources have limitations for therapeutic angiogenesis. Although ASCs are favorable with regard to obtaining the number of cells required for transplantation, few transplanted stem cells have been found to differentiate into endothelial cells (ECs) and incorporate into vascular structures in ischemic sites. Thus, the foremost mechanism by which stem cells participate in tissue repair seems to be related to their trophic factors. Indeed, stem cells provide the microenvironment with a wide range of growth factors, cytokines and chemokines, which can broadly defined as the stem cells secretome. In in vitro condition, these molecules can be traced from the conditioned medium or spent media harvested from cultured cells. Conditioned medium now serves as a new treatment modality in regenerative medicine and has shown a successful outcome in some diseases. Low-level light therapy (LLLT) has been used for a long time for various purposes, such as reduce inflammation and improvement in the local circulation. Moreover, many studies have demonstrated positive bio-stimulatory effects of LLLT on stem cells. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on stem cells secretome. With the emergence of this approach, we described the possibility of using stem cells conditioned medium as a novel and promising alternative to skin wound healing treatment.
Daniel Neiman
The Hebrew University-Hadassah Medical School, Israel
Title: Identification of tissue specific cell death using methylation patterns of circulating DNA
Biography:
A Phd student under the guidance of Prof Aharon Razin & Prof Yuval Dor lab & Ruth Shemer at The Hebrew University-Hadassah Medical School, Israel.
Abstract:
Minimally-invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA), released from dying cells, is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on DNA sequence differences in source tissues, so that cell death in tissues with a normal genome cannot be identified. We developed a method of detecting tissue-specific cell death in humans, based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures, and developed a method to detect these in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated with bisulfite, PCR-amplified and sequenced to quantify cfDNA carrying the methylation markers of the cell-type of interest. Pancreatic beta-cell DNA was identified in the circulation of recently diagnosed type-1 diabetes patients and islet graft recipients, oligodendrocyte DNA in patients with relapsing multiple sclerosis, neuronal/glial DNA in patients after traumatic brain injury or cardiac arrest, and exocrine pancreas DNA in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be measured in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally-invasive window for monitoring and diagnosis of a broad spectrum of human pathologies, as well as better understanding of normal tissue dynamics.
Joice de Faria Poloni
Federal University of Rio Grande do Sul, Brazil
Title: Evaluation of somatic point mutations associated to splicing mechanisms in colorectal cancer
Biography:
Joice de Faria Poloni is currently a PhD candidate in the Cellular and Molecular Biology Post-graduation program of the Federal University of Rio Grande do Sul, Brazil. Her area of expertise is in the bioinformatics field with emphasis on systems biology, systems chemo-biology and transcriptomic analysis to study tumoral processes and developmental mechanisms. She also works with somatic point mutations and alternative splicing isoforms in different tissues. In this sense, her scientific production involves book chapters, research and review articles in these fields.
Abstract:
Coloretal cancer (CRC) is a multifactorial disease and the third more common cancer worldwide. Cancer development involves numerous abnormal regulations, such as gene expression, mRNA processing and somatic point mutations. To improve the comprehension of these factors, we evaluated the correlation of differential splicing with differentially expressed genes (DEG), differentially exon usage (DEU) and somatic point mutations through interatomic network analysis. For this purpose, the RNA-seq dataset (GSE50760) of 12 paired tumor and adjacent normal tissue samples was employed. After reads alignment on the reference genome, the data was submitted to DEG analysis supported with gene and transcripts quantification and DEU analysis assisted with exonic quantification. Additionally, samples were analyzed for somatic point mutations detection. The results were integrated in an interatomic network for the proceeding analysis. Furthermore, the variants effects prediction was estimated to understand their impact in the splicing mechanisms of CRC. According to the obtained results, it was possible to elaborate an interatomic network composed by DEG, which was assigned to gene ontology, hub-bottlenecks and clustering analysis. These analyzes allowed the detection of DEG that are highly representative for CRC development and maintenance. The DEG was mainly involved in processes such as cell cycle regulation, extracellular matrix organization and immune response. The data also permitted the identification of DEG with specific somatic point mutations in essential regions for splicing mechanism. Hence, the methods employed in this work promoted a better and innovative understand of cancer development.
Biography:
Nermeen M. Arafa has completed her PhD at the age of 34 years from faculty of Agriculture, Cairo university and postdoctoral studies from National Research Center. She has published three papers in reputed journals.
Abstract:
Cassava (Manihot esculenta) is a major industrial crop which are rich in lot of therapeutic applications such as antitumor activity. Leaf, stem and petiole of Cassava in vitro plantlets were cultured on solid MS-medium containing different combinations and concentrations of 2.4-D, picloram or NAA as auxins and BA as cytokinins. Stem was chosen as the best explant for callus induction, stem derived calli (25.892 ± 0.117g) on the best selected medium for callus production (5 mg/l 2.4-D and 0.2 mg/l BA) with subculture intervals each 40 days of culture. The phytochemical components such as fatty acids and their ester derivatives constituents were extracted from calli of cassava using different solvents, ethylacetate as aprotic polar solvent, chloroform and hexane as non-polar solvent to examine the efficiency of these extracts for inhibition the growth of the breast cancer cell. All extracts recorded positive results for inhibition cancer cell proliferation. Ethylacetate extracts have maximum efficiency for inhibition breast cancer (2.63 ug IC50), followed by hexane extracts (3.44 ug IC50), then chloroform extracts (6 ug IC50) recorded the least value. The objective of this study was analysed the composition of lipid profile components for ethylacetate extract, chloroform extract and hexane extract by GC-Mass and determine the efficiency of these extracts as anti-breast cancer cell growth. Ethylacetate extracts showing higher concentration of the total fatty acids (76.53%), also, included caprylic acid (14.52%) and caproic ester (3.10%), while that hexane and chloroform extracts recorded less values of total fatty acids (69.26% and 39.77%, respectively) and recorded no value of both of caprylic acid and caproic ester. Therefore it could be recommended to use ethylacetate as aprotic solvent for the extraction of calli, as including most amounts of fatty acids and their derivatives compounds due to the most effect as anti-breast cancer.