Day 2 :
National Institute of Standards and Technology, USA
Time : 09:00-09:40
Miral Dizdaroglu has obtained his PhD in physical chemistry at the Karlsruhe Technical University, Germany, and subsequently worked for seven years at the Max-Planck-Institute for Radiation Chemistry, Germany, before moving to USA. He has been at the National Institute of Standards and Technology (NIST) for more 30 years. In 2006, Dr. Dizdaroglu was conferred upon the rank of NIST Fellow. To date, he published highly cited 247 papers and gave numerous presentations on his work around the world. Dr. Dizdaroglu received numerous scientific awards including the Hillebrand Prize of the American Chemical Society, and the Silver and Gold Medal Awards of the US Department of Commerce. He was also awarded two Honorary Doctorates.
Most chemotherapeutic agents kill cancer cells by damaging DNA. Cancer cells overexpress DNA repair proteins and thus increase DNA repair capacity that can cause resistance to therapy by removing DNA lesions before they become toxic. DNA repair proteins constitute targets for inhibitors to overcome the therapy resistance. Inhibition of DNA repair proteins is a promising approach to enhance the efficacy of cancer therapy. Despite the successes with other proteins, the development of inhibitors has been lagging for DNA glycosylases involved in the base excision repair mechanism. The purpose of this study was to discover small molecule inhibitors of the major human DNA glycosylases NEIL1, NTH1 and OGG1. First, we developed a fluorescence-based assay using double-stranded oligodeoxynucleotides containing one substrate lesion to detect both glycosylase and apyrimidinic/apurinic lyase activities of DNA glycosylases. From a screen of ~400,000 compounds, many inhibitors were identified. Subsequently, we applied gas chromatography/isotope-dilution tandem mass-spectrometry to measure the glycosylase activities of NEIL1, NTH1 and OGG1 using damaged DNA containing multiple lesions. Four purine analogs were found to be potent inhibitors of excision of the main substrates of NEIL1. Three of NEIL1 inhibitors also inhibited the excision of NTH1 substrates, but did not affect OGG1 activity. From a screen of ~50,000 molecules, five hydrazydes were identified as potent inhibitors of OGG1 (Figure 1). These compounds exhibited no inhibition of NEIL1 and NTH1 activities for all their substrates analyzed by two different methodologies used in this work. Overall, this work forms the foundation for future drug discovery for the entire family of DNA glycosylases. The inhibitors identified serve as a proof-of-concept for the initial phase of the drug discovery process. Future work will involve the screening of additional compound libraries for different types of inhibitors of DNA glycosylases.
- Workshop on
National Institute on Aging, NIH, USA
Title: Biochemical and biological assays for the discovery and characterization of DNA helicase inhibitors
Time : 09:40-10:25
Robert M Brosh has his expertise in DNA Repair and Genome Stability Maintenance. He leads a Research group at the National Institute on Aging, NIH that is focused on characterizing the roles of clinically relevant human DNA helicases in cellular nucleic acid metabolism. This work has yielded insights into how DNA repair helicases promote phenotypes consistent with healthy aging and cancer resistance.
The growing number of helicases implicated in hereditary disorders and cancer is indicative of their fundamentally essential roles in DNA transactions, genomic stability, and cellular homeostasis. Indeed, molecular and cellular evidence demonstrates that helicases catalytically unwind or remodel a variety of nucleic acid substrates and interact with numerous proteins to perform their functions in replication, DNA repair, recombination, and transcription. Understanding how helicases operate in unique and overlapping pathways is a great challenge to researchers. Identification and characterization of biologically active small molecules that modulate the catalytic activity of a target helicase represents a unique approach to studying helicase function in human cells. In this review, we describe a series of experimental approaches and methodologies to identify and characterize DNA helicase inhibitors which collectively provide an alternative and useful strategy to explore their biological significance in cell-based systems. These procedures were used in the discovery of biologically active compounds that inhibited the DNA unwinding function catalyzed by the human WRN helicase-nuclease defective in the premature aging disorder Werner syndrome. Our studies with newly discovered WRN-specific helicase inhibitors have provided proof-of principle evidence for how these compounds can be used in synthetic lethal approaches with other pharmacological agents or in defined genetic mutant backgrounds. In this Nucleic Acids 2017 Workshop, I will describe in vitro and in vivo experimental approaches to characterize helicase inhibitors with WRN as the model, anticipating that these approaches may be extrapolated to other DNA helicases, particularly those implicated in DNA repair and/or the replication stress response. Helicase inhibitors provide an alternative strategy for investigating the molecular and cellular functions of their targets, and in a broader scope, the sophisticated orchestration of overlapping and intersecting DNA metabolic pathways. In addition, my lab and others visualize helicases as suitable small molecule targets that might enhance existing anti-cancer strategies or emerge as novel therapeutic treatments.
- Track 7: Chemical Biology
Track 8: DNA Damage and Repair
Track 9: RNA and DNA Nanotechnology
Track 10: Sequencing
Track 11: Molecular Therapeutics
Track 12: Molecular Biologics
Track 13: Computational Molecular Biology
Saint Petersburg State University, Russia
Time : 10:25-10:50
Oleg Tikhodeyev is the author of the original approach for resolving multiple ambiguities and contradictions in current genetic concepts. He has shown that the key source of such ambiguities and contradictions is the erroneous belief that the same genetic term (for example, mutation) is able to comprise both specific phenomenology and the underlying mechanisms (Tikhodeyev, 2015). This belief became widely accepted after 1952, when the hereditary role of DNA had been demonstrated. In modern genetic concepts, the terms describing molecular mechanisms should be clearly distinguished from those describing phenomenology because there is no strict correlation between phenomenology and molecular mechanisms
Statement of the Problem: It has long been accepted that any hereditary factor in any organism is represented by DNA sequences. This idea became fundamental in molecular genetics, and was implicitly transformed into the DNA theory of inheritance. All basic genetic terms (genotype, gene, allele, mutation, recombination, etc.) were considered as specific DNA sequences or their alterations. However, multiple examples of stable epigenetic inheritance lacking any distinctions in DNA sequences were recently discovered, and the most exciting among them is protein inheritance. Amyloid hereditary prions in fungi were considered as “protein-only” hereditary factors, which features were determined entirely by protein conformation. As a result, the principal question arises whether the DNA theory of inheritance is wrong or not.
Methodology & Theoretical Orientation: Considering different variants of the same hereditary prion as prion alleles, we examined the molecular nature of such variety.
Findings: To perpetuate stably in cell generations a certain prion allele requires two entities: specific state of the prion protein, and the corresponding DNA sequence to provide reproduction of the prion particles. We name these entities as the DNA determinant and the epigenetic determinant, respectively. Thus, a certain prion allele is a bimodular hereditary system depending on both the DNA determinant and the epigenetic determinant. Alteration of any of these two determinants may result in establishment of a novel prion allele. Moreover, similar regularities are characteristic to all other cases of epigenetic inheritance, irrespective to the underlying mechanisms.
Conclusion & Significance: The hereditary role of DNA is fundamental for any known mechanisms of inheritance, including epigenetic. However, it becomes an element of a more complicated concept: in addition to “DNA-only” hereditary factors, various bimodular hereditary factors also exist.
De Montfort University in Leicester, United Kingdom
Time : 11:10-11:35
Carika Weldon is a Lecturer in Biomedical Science at De Montfort University in Leicester. She has obtained her BSc in Medical Biochemistry and her PhD in Biochemistry from the University of Leicester. Her Doctoral work focused on alternative splicing of the apoptotic gene Bcl-X.
Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.
NMS Labs, USA
Title: Sexual assault kit backlog in the United States: A history of and efforts to reduce the backlog
Time : 11:35-12:00
Megan Foley is working as a Forensic Biologist at NMS Labs. She possesses a BA in Biology from the College of St. Benedict in St. Joseph, Minnesota, and a MSFS from Arcadia University in Glenside, Pennsylvania. Her current works include examination and interpretation of casework samples, as well as leading validations and projects for the Forensic Biology Department at NMS. She also is a Fellow for the Center for Forensic Science Research and Education where she assists in Teaching Forensic Biology methods and assists with research. Her areas of research have focused on optimizing the DNA recovery from pipe bombs after being rendered safe but also have included optimizing and evaluating a variety of current DNA and serological methods. She has also received her certification through the American Board of Criminalistics in Molecular Biology.
Due to years of mishandling and inadequate funds to process sexual assault kits (SAK), the backlog of these cases has launched to the forefront of the current news. An unknown number of kits, estimated to be around 400,000, across the US have been sitting untested. The true number is unknown due to the poor tracking of kits in different storage facilities. Due to this fact, a heavy push has occurred in legislation over the past years to evaluate and address the backlog. This demand to remedy the problem has created funding opportunities at different levels and strategies for how the different states and laboratories can handle them. Some labs have pioneered the way (NYC OCME and Detroit) and others have followed their recommendations. Many laboratories have chosen to outsource the backlog, which is where labs like NMS have come in to be involved with this reduction. Currently, NMS Labs assists two separate jurisdictions in the testing of their sexual assault kits. Testing of these samples is designed to utilize a streamlined approach aimed at obtaining the best possible result from a high volume of samples with a relatively short processing time. To achieve this, a Y-Screen protocol has been utilized and optimized. Some laboratories, like NMS, have internally developed such Y-Screen protocols, modeled after those recommended by other laboratories as well as manufacturers of forensic DNA instruments and reagents. These protocols all work around the same concept to initially screen the samples for male DNA and then choose the best samples to send forward for STR analysis. Utilizing these techniques, NMS Labs has been processing SAK backlog samples for over a year and had success in developing DNA profiles foreign to the victim from these kits which have been sitting untested for over a decade.
National University of Singapore , Singapore
Time : 12:00-12:25
Yvonne Tay began her research career in Bing Lim’s lab at the Genome Institute of Singapore and her PhD work led to key insights into the scope and mechanisms of microRNA activity. Subsequently, she has pursued her Post-doctoral training in Pier Paolo Pandolfi’s lab at Harvard Medical School and Beth Israel Deaconess Medical Center under a Special Fellow award from the Leukemia and Lymphoma Society and this research led to the discovery that protein-coding transcripts can co-regulate the tumor suppressor PTEN by competing for shared microRNAs. She has recently returned to Singapore to begin her independent research career. She has been awarded the Singapore National Research Foundation Fellowship and NUS President’s Assistant Professorship to continue her research into this previously uncharacterized layer of gene regulation.
Pseudogenes, non-coding homologs of protein-coding genes, were once considered non-functional evolutional relics. Recent studies have shown that pseudogene transcripts can regulate their parental transcripts by sequestering shared microRNAs, thus acting as competing endogenous RNAs (ceRNAs). In this study, we utilize an unbiased screen to identify the ferritin heavy chain 1 (FTH1) transcript and multiple FTH1 pseudogenes as targets of several oncogenic miRNAs in prostate cancer. We characterize the critical role of this FTH1 gene: pseudogene: microRNA network in regulating tumorigenesis in prostate cancer, and show that impairing microRNA binding and subsequent ceRNA crosstalk results in complete phenotype rescue. Our results also demonstrate that pseudogenes can regulate intracellular iron levels, which are crucial for multiple physiological and pathophysiological processes. In summary, we describe a novel and extensive gene: pseudogene ceRNA network comprising multiple microRNAs and multiple pseudogenes derived from a single parental gene, which regulates iron storage and tumorigenesis in prostate cancer.
Jagiellonian University, Poland
Time : 12:25-12:50
Jolanta Jura (maiden name: Kwiatkowska by 2000) is professor at Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University in Krakow. After completing her Ph.D. in 2004 in Human Genetic Institute in Poznan, Jolanta Jura joined the group at Brigham and Women's Hospital of Harvard Medical School in Boston for two years, focusing on identification and characteristic of gene involved in tuberous sclerosis. Currently, the laboratory of Jolanta Jura studies the molecular mechanism of regulation of inflammation and inflammation related disorders focusing on gene expression and transcript stability, role of RNases in regulation of RNA profiles, signaling pathways.
The response of cell to external and internal stimuli results in the global changes in gene expression profile and accumulation of molecules that are essential for cell to adjust to a new conditions.One of recently discovered endonuclease capable of mRNA cleavage is Monocyte Chemoattractant Protein-induced protein 1 (MCPIP1), known also as Regnase-1 (Reg1). The protein is essential for degradation of short-living transcripts coding for inflammation-related proteins, including IL-1β, IL-6, IL-2, IL-8, IL-12b, IER-3, c-Rel and many other transcripts coding for proteins engaged in many other cellular processes such as: cell differentiation, metabolism and apoptosis. Besides mRNA, MCPIP1 regulates also miRNA processing. Ribonucleolytic activity of MCPIP11 has been attributed to a PIN (PilT N terminus) like domain, where four Asp residues (D141, D225, D226, D244) in the catalytic center determine RNase activity. It has been proven that MCPIP1 binding of an mRNA depends on a conserved stem-loop structures present in the 3’UTR of mRNA templates. Level of MCPIP1 changes during inflammatory processes generated by pathogen infections and also during sterile inflammation induced for example by cancer development. We found that the MCPIP1 levels are significantly lower in ccRCC samples than in surrounding tissues. Furthermore, lower level of MCPIP1 influences very important signaling pathways what corresponds to higher level of proangiogenic factors and enhanced metabolism and proliferation rate. Overall, these results suggest that MCPIP1 is an important player in ccRCC development.
Columbia University, USA
Time : 13:40-14:05
Pankaj Kumar Giri is a Research Scientist at Columbia University, New York. His work is on exploration of live cell super resolution imaging, design and development of effective tools for genome editing based on CRISPER-Cas9 and mouse model based studies in the field of “non-coding RNA metabolism” for antibodies diversification. Within his exploration of identity, he is devolving new methods to study chromosome and protein dynamics in 3D nuclear space at sub-nanometer resolution with the help of super resolution imaging techniques. He holds an MTech in Biomedical Engineering from the Indian Institute of Technology, Bombay and a PhD in Molecular Biophysics from the National University of Singapore, Singapore.
Strand specific DNA mutations determine whether programmed DNA rearrangements diversify antigen receptor loci genes. However, patients with various malignancies demonstrate DNA mutagenesis skewed toward the sense strand genome wide. Using single-molecule super-resolution microscopy, we have identified sub nuclear compartments in B cells where biologically programmed strand-specific DNA mutagenesis are engineered at focal DNA/RNA hybrid structures. The strand specific distribution of DNA mutations is determined by the coupled activities of two RNA helicases, Mtr4 and Senataxin, along with the noncoding RNA processing function of RNA exosome. Our study envisions that the regulatory mechanism of strand specific DNA mutagenesis in sub nuclear compartments during programmed and aberrant DNA mutagenesis events will play a major role in other undiscovered aspects of organismic development.
The Wistar Institute, USA
Time : 14:30-14:50
Prashanth Shastrula has his expertise in protein nucleic acid assemblies that maintain the integrity of the ends of our chromosomes referred to as telomeres. His long-term goal is to understand how naturally occurring mutations on the human telomeric shelterin complex contribute to cancer and identify methods to treat this disease.
The telomeric shelterin, POT1-TPP1 sub-complex is involved in telomere length regulation and maintenance. POT1 binds the telomeric overhang with high affinity and specificity and represses ATR-dependent DNA damage response (DDR). TPP1 is involved in telomerase recruitment to telomeres. Several naturally occurring POT1 mutations are implicated in chronic lymphoid leukemia and familial glioma and melanoma. However, the mechanism of POT1-TPP1 binding and how naturally occurring POT1 mutations contribute to cancer remains unknown. Here, we report the crystal structure of the interacting portions of POT1-TPP1 which includes the POT1 C-terminus (POT1C) and the POT1 binding domain of TPP1 (TPP1 (PBD)). POT1C consisting of an OB-fold and a holiday junction resolvase domain. TPP1 (PBD) consists of several loops and helices involved in extensive interactions with POT1C. The structure reveals that several of the POT1C cancer associated mutations partially disrupt the POT1-TPP1 complex. Biochemical and cell-based assays show that disruption of POT1-TPP1 leads to decreased POT1 telomere binding efficiency resulting in persistent telomerase activity at telomeres. This leads to longer and fragile telomeres resulting in undesired DDR, genomic instability and cancer.
University of Southern Denmark, Europe
Title: Amplification-free detection of miRNA associated with rheumatoid arthritis by simple hybridization assay
Time : 14:50-15:10
Jesper Uhd is a Master’s student of Science & Engineering in Advanced and Applied Chemistry at Technical University of Denmark. He is a current Member of Dr. Kira Astakhova’s group at Technical University of Denmark. He has completed his BS degree in Chemistry at University of Southern Denmark in 2017 and working in the group of Dr. Kira Astakhova since 2016.
In our on-going project, we aim at rational design of novel oligonucleotide probes that target miRNA and developing a diagnostic method for clinically relevant microRNA (miRNA, Fig. 1). In my talk, I will discuss our recent studies on the development of highly specific LNA/DNA probes and present the results in testing miRNA levels by the new assay in clinical samples. Nucleic acids and their interactions are the basis of life. To build a reliable theoretical model on the structure and function of nucleic acids, and to apply this model to emerging tasks of life sciences, the detection and studies of nucleic acids must be carried out under biologically relevant conditions. Currently nucleic acids are most often detected by enzymatic amplification. Amplification has multiple disadvantages. First, it affects stoichiometry of the initial sample and cannot be carried out in cells. Secondly, not every sequence can be amplified. Thirdly, enzymes often make mistakes which results in biased detection. At last, amplification is time consuming and costly. As an alternative, efficient probe including modifications, i.e. locked nucleic acids (LNA), can be applied in non-enzymatic assays. The experiments proposed in this project open an exciting opportunity to learn about nucleic acid structure and interactions at biologically relevant low abundance levels and directly in biofluids. Detection and absolute quantification of disease related miRNA presented herein could benefit early diagnosis and treatment of the diseases such as cancer and rheumatoid arthritis.
Jinnah University for Women Karachi, Pakistan
Title: Development of a PCR-based rapid method for the detection of L. monocytogenes in food samples after enrichment steps
Time : 15:10-15:30
Shumaila Naz Malik has completed her MS successfully in 2015. After that she started working as Research Scholar at Jinnah University for Women, Karachi, Pakistan. She has strong academic and research background in the areas of Food Microbiology. Particularly, she has worked on Listeria monocytogenes. Her MS thesis was on “Rapid detection of Listeria monocytogenese in food sample after enrichment step”. She worked as a Lecturer in the Department of Microbiology from 2011 till 2013. She has attended and organized several workshops, research posters exhibitions, conferences and seminars for the students and faculty, while in Pakistan.
Listeria monocytogenes is a Gram positive, facultative, and opportunistic pathogen causing food borne infections in humans worldwide. It causes major problems in immunocompromised individuals such as pregnant women, neonate and elderly. The old conventional methods for the identification of L. monocytogenes in foods are laborious, and require almost 3-5 days giving ready results. To overcome this problem we have demonstrated a fast, non-conventional, simple, sensitive and rapid Polymerase Chain Reaction (PCR)-based method by using the primers for prfA gene sequence for the detection of Listeria in food samples. Experiments were to observe the sensitivity of this primer in number of combinations. Optimization studies were conducted using milk samples spiked with different inoculum size and for different time intervals. It was observed that this method efficiently detects minimum contamination of Listeria as tested with spiked samples in around 14 hours. Comparable results were observed when this method was applied to detect Listeria in naturally contaminated samples along with conventional methods. The proposed method can be employed to detect Optimization studies in parallel to standard conventional methods.