2^nd International Conference on Molecular Biology , Nucleic Acids & Molecular Medicine August 30-31, 2017 Philadelphia, Pennsylvania, USA

Biography:

McClintock is a Professor and Director of Forensic Sciences in the Department of Biology and Chemistry at Liberty University in Lynchburg, VA where he teaches undergraduate and graduate courses in forensic sciences (forensic DNA analysis, trace evidence) and microbiology. The forensic DNA course focuses on current laboratory methods and applications in forensic DNA profiling and effective presentation of DNA evidence at trial. His latest book, entitled "Forensic Analysis of Biological Evidence: A Laboratory Guide for Serological and DNA Typing" focuses on the newest techniques available for the analysis of biological material. Dr. McClintock is also the founder of DNA Diagnostics, Inc., a forensic service company that provides DNA testing and the scientific review and analysis of DNA test results performed in forensic casework. In 2013, Dr. McClintock, was named among the top 15 DNA analysts in the country by ForensicsColleges.com, a leading website on forensics programs across the nation.

Abstract:

Bloodstain pattern analysis has been an integral part of criminal investigations for over a century. The use of chemiluminescent reagents such as luminol or Bluestar® to visualize presumed bloodstains in criminal investigations has provided useful investigative information. Newer technologies and recent advances in forensic DNA analysis have gained much notoriety over the past two decades as a tool in human identification and parentage verification. More recently, bloodstain analysis, coupled with methods to generate DNA profiles, have been applied to investigate samples of historical significance. This study investigated samples collected from America’s past conflicts to determine the biological origin and nature of the samples/stains using classic and various state-of-the-art approaches as well as isolate the genetic material for forensic DNA analysis. Specifically, samples were collected from the Hillsman House in Rice, VA that served as a Union field hospital during the last battle of the Civil War. Approximately 358 Union and 161 Confederate soldiers were treated over a twenty-four hour period during the battle at Sailor’s Creek. The prominent “bloodstains” on the floorboards under the single surgical table and two post-surgical beds provides evidence of the vast number of soldiers treated. These presumed bloodstains also found their way through the cracks in the wood floors onto the supporting floor joists. The presumed bloodstains were subjected to various presumptive blood tests (e.g., luminol and Bluestar®, leucomalachite green, phenolphthalein, and RSID™ Blood Competitive Analysis Kit), the DNA isolated, quantitated, and subjected to genetic analysis using capillary electrophoresis. The generation of partial or complete DNA profiles will confirm the presence of human DNA, as well as demonstrate the ability of DNA profiling to reveal a part of history from a battle fought over 150 years ago. Other presumed bloodstain samples from the Korean War era and tissue and hair samples collected from burial sites from a civilization long extinct have been analyzed in an attempt to generate DNA profiles and to corroborate historical documentations of accounts that occurred many decades ago.

Biography:

Junji Iwahara’s current research focuses on the dynamic processes whereby transcription factors scan DNA and recognize their target sites. His group developed some novel methods for investigating the dynamics and kinetics of the protein-DNA interactions at atomic and molecular levels. Using the biophysical and biochemical approaches, Prof. Iwahara’s group is trying to better understand how proteins scan and recognize DNA to regulate genes.

Abstract:

In eukaryotic genomes, there are numerous nonfunctional high-affinity sequences for transcription factors. These sequences potentially serve as natural decoys that sequester transcription factors. We have previously shown that the presence of sequences is like the target sequence could substantially impede association of the transcription factor Egr-1 with its targets. More recently, using a stopped-flow fluorescence method, we examined the kinetic impact of DNA methylation of decoys on the search process of the Egr-1 zinc-finger protein. We analyzed its association with an unmethylated target site on fluorescencelabeled DNA in the presence of competitor DNA duplexes, including Egr-1 decoys. DNA methylation of decoys alone did not affect target search kinetics. In the presence of the MeCP2 methyl-CpG-binding domain (MBD), however, DNA methylation of decoys substantially (~10-20-fold) accelerated the target search process of the Egr-1 zinc-finger protein. This acceleration did not occur when the target was also methylated. These results suggest that when decoys are methylated, MBD proteins can block them and thereby allow Egr-1 to avoid sequestration in nonfunctional locations. This effect may occur in vivo for DNA methylation outside CpG islands and could facilitate localization of some transcriptional activators within regulatory CpG islands, where DNA methylation is rare. Our recent studies to examine this model will be presented.

Biography:

Makkuni Jayaram is a Professor of Molecular Biosciences at the University of Texas at Austin. His primary research interest is in the biochemical mechanisms of site-specific DNA recombination. Over the past three decades, his research group has used the Flp site-specific recombinase as a template for understanding the chemistry, conformational dynamics and topological features of strand breakage/exchange reactions in nucleic acids. A second interest of the Jayaram laboratory concerns strategies devised by selfish DNA elements for moderating their selfishness so as to establish long-term peaceful coexistence with their host genomes.

Abstract:

Phosphoryl transfer reactions in RNA and DNA abound in living cells, and are central to biological information processing. A common feature of self-catalyzed or protein-catalyzed phosphoryl transfer in nucleic acids is the role of divalent metal ions in stabilizing the penta-coordinate phosphate transition state. Most systems appear to follow the classical ‘two-metal ion’ paradigm or its variations, while recent evidence suggests the potential involvement of a third metal ion, at least in some systems. By contrast, members of the serine- and tyrosine-family site-specific recombinases exemplify metal-free mechanisms for mediating phosphoryl transfer associated with the DNA strand cleavage and strand joining steps that they perform. In the tyrosine family, the positively charged side-chains of two highly conserved arginine residues appear to functionally bypass metal ion requirement. By using Flp and Cre recombinases as representatives of the tyrosine family, we probed the individual roles of this arginine duo (Arg-I and Arg-II) in transition state stabilization. We find that Flp or Cre variants lacking either Arg-I or Arg-II can be rescued by replacing the scissile phosphate with methylphosphonate, thereby eliminating the negative charge on one of the non-bridging oxygen atoms in the transition state. Stereochemically pure R_P and S_P forms of the methylphosphonate substrates in conjunction with recombinase variants lacking either Arg-I or Arg-II have enabled us to dissect the stereochemical contributions of the individual arginines to the recombination reaction. The general strategies employed by us are of broad utility in the analyses of other recombination systems.

Biography:

Robert 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.

Abstract:

Statement of the Problem: Fanconi Anemia (FA) is a rare genetic DNA repair disorder characterized by progressive bone marrow failure, congenital abnormalities, and cancer. Of the 21 genes linked to FA, the FA Group J (FANCJ) gene is unique that it encodes an ATP-dependent DNA helicase. Mutations in FANCJ are not only genetically linked to FA, but also associated with breast and ovarian cancer. Consistent with its known role in homologous recombination (HR) repair, FANCJ-/- cells are sensitive to DNA interstrand cross-linking (ICL) agents and are also hypersensitive to agents that induce replication stress.

Methodology & Theoretical Orientation: We characterized two FA patient-derived FANCJ mutations, R707C and H396D, which reside in the conserved helicase core domain. Genetic and biochemical analyses were performed to delineate the molecular defects underlying the genetic disease.

Findings: FANCJ-R707C retained partial (~30%) helicase activity, whereas FANCJ-H396D was nearly completely inactive. Single-turnover kinetic assays, ATPase measurements, and DNA binding determinations confirmed the differential effects of FANCJ missense mutations on helicase activity. Expression of either FANCJ-R707C or FANCJ-H396D in fancj-/- cells completely failed to rescue cisplatin sensitivity. In striking contrast, expression of FANCJ-R707C in fancj-/- cells restored resistance to the DNA polymerase inhibitor aphidicolin, whereas FANCJ-H396D completely failed. Single-molecule replication tract analysis confirmed that FANCJ-R707C, but not FANCJ-H396D, restored fork rates after cellular exposure to aphidicolin. Thus, a quantitatively lower threshold of FANCJ catalytic activity is required for the aphidicolin-induced replication stress response compared to cisplatin-induced damage.

Conclusion & Significance: The catalytic requirement of FANCJ to reconstruct broken replication forks after ICL-induced damage is distinct from that required to remodel stalled replication forks. These findings provide new insight to FANCJ’s role in DNA repair and molecular phenotypes of clinically relevant FANCJ missense mutations that are relevant to human disease and cancer.

Biography:

Yuliang Wu obtained his BSc and MSc from Zhejiang University, China in 1995 and 1998 respectively, and Ph.D. from International Centre of Genetic Engineering and Biotechnology (ICGEB), Delhi, India in 2002. In the following eight years, Dr. Wu did his postdoc training at the University of Alberta, Canada and the National Institute on Aging-NIH, where he studied the molecular and cellular basis of human genetic diseases characterized by genomic instability. Dr. Wu joined the Department of Biochemistry at the University of Saskatchewan, Canada in May 2011. Dr. Wu’s lab focuses on DNA repair proteins, including helicase, single strand DNA binding protein, and recombinase. Through structural and functional studies of these DNA repair proteins, we try to understand the molecular mechanisms underlying genomic instability. Ultimately the molecular information derived from these projects may be exploited to advance diagnosis, prognosis, and treatment of human diseases and cancers.

Abstract:

The K-homology (KH) domain is a nucleic acid–binding domain present in many proteins, but has not been reported in helicases. DDX43, also known as HAGE (helicase antigen gene), is a member of the DEAD-box protein family. It contains a helicase core domain in its C-terminus and a potential KH domain in its N-terminus. DDX43 is highly expressed in many tumors, and is therefore considered a potential target for immunotherapy. Despite its potential as a therapeutic target, little is known about its activities. Here, we purified recombinant DDX43 protein to near homogeneity and found that it exists as a monomer in solution. Biochemical assays demonstrated that it is an ATP-dependent RNA and DNA helicase. Although DDX43 was active on duplex RNA regardless of the orientation of the single-stranded RNA tail, it preferred a 5' to 3' polarity on RNA and a 3' to 5' direction on DNA. Truncation mutations and site-directed mutagenesis confirmed that the KH domain in DDX43 is responsible for nucleic acid binding. Compared with the activity of the full-length protein, the C-terminal helicase domain had no unwinding activity on RNA substrates and had significantly reduced unwinding activity on DNA. Moreover, the full length DDX43 protein, with single amino acid change in the KH domain, had reduced unwinding and binding activates on RNA and DNA substrates. Our results demonstrate that DDX43 is a dual helicase and the KH domain is required for its full unwinding activity.

Biography:

Patrick Arbuthnot is currently a Personal Professor and Director of the Wits/SAMRC Antiviral Gene Therapy Research Unit at the University of the Witwatersrand in South Africa. After graduating with a Medical degree, he has completed his PhD in 1992 then carried out his Post-Doctoral work at Necker Hospital in Paris, France. On returning to South Africa, he established the Antiviral Gene Therapy Research Unit, which has now published widely on HBV infection, liver cancer, HIV-1 infection and developing new methods of treating these diseases. His main research interest is in advancing use of biological and synthetic nanoparticles to carry potentially therapeutic nucleic acids (DNA or RNA) that are capable of permanently disabling HBV.

Abstract:

Chronic infection with hepatitis B virus (HBV) remains and is an important global health problem. Carriers of the virus are at high risk for cirrhosis and liver cancer. Available treatment only has modest curative efficacy and improved therapy is a priority to prevent the life-threatening complications that accompany the infection. The viral replication intermediate comprising covalently closed circular DNA (cccDNA) exists as a stable mini-chromosome in infected hepatocytes. Licensed treatment has no effect on cccDNA and devising methods based on gene therapy to disable this replication intermediate has considerable potential. Previous work from our laboratory demonstrated effective inhibition of HBV replication and targeted disruption of cccDNA by Transcription Activator-Like Effector Nucleases (TALENs). Although this approach is promising, unintended mutagenesis may occur in chronic carriers because of TALEN activity at HBV sequences that are integrated into the host genome. To circumvent this problem, we have produced repressor TALEs (rTALEs) that were designed to induce transcriptional repression at essential HBV transcriptional regulatory elements: the basic core promoter/enhancer II and preS2 promoter sequences. KRAB-encoding sequences were fused to the N-terminal regions of TALEs contain sequence-specific DNA binding domains derived from the AvrBs4 N1 Xanthomas TALE. Each rTALE was expressed from the CMV promoter and engineered to interact with an HBV-specific 18bp target. The repressors were incorporated into recombinant adenoassociated viral vectors which were used to deliver the antiviral elements. Inhibition of HBV replication was observed in cell culture models of HBV replication and in vivo. No evidence of toxicity was detected and inhibitory effects were sustained over a period of at least 2 months. Collectively these data indicate that rTALEs are effective against HBV and provide an efficient means of disabling HBV cccDNA without causing mutations that result from target DNA cleavage.

Biography:

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

Abstract:

Statement of the Problem: During last 30 years, the majority of basic genetic terms (mutation, recombination, genotype, gene, allele, etc.) became fuzzy due to discovery of multiple “non-canonical” phenomena like inheritance of acquired traits, protein inheritance, paramutations, and genotrophy. As a result, there is a significant gap between factual material and current genetic concepts, thus reflecting the need for a paradigm shift. Modern genetic concepts are required, which will be equally valid for all known canonical and “non-canonical” genetic phenomena.

Methodology & Theoretical Orientation: We accomplished a critical analysis of current genetic concepts (gene theory, mutation theory, the chromosome theory of inheritance, the DNA theory of inheritance) to find the key origins of the terminological fuzziness.

Findings: The current concepts stand on the idea that any genetic term simultaneously describes three following aspects: Phenomenology; the source of variation and the underlying mechanism. For instance, any mutation is considered as an alteration which is stable and hereditable, stochastically arising, and affecting DNA sequences. We name this idea “the integral concept of variability”. Meanwhile, the available factual material clearly demonstrates that the abovementioned aspects are autonomous from each other and thus cannot be covered by the same term. In particular, some hereditable alterations gradually decline, some are clearly predictable under certain environmental influences, some do not affect DNA sequences, and some alterations of DNA sequences are not heritable. We propose that each genetic term should describe only one aspect of variability. This idea (we name it “the differential concept of variability”) was already shown to be successful for a lot of genetic terms.

Conclusion & Significance: The way to resolve the fuzziness of genetic terminology and the crisis of current genetic concepts is a paradigm shift based on the differential concept of variability.

Biography:

Guang Zhu is a Professor of Division of Life Science in Hong Kong University of Science and Technology. He has obtained his BSc and MSc in Physics. He has completed his PhD degree from University of Maryland and National Institutes of Health, USA, specialized in Biomolecular NMR Spectroscopy. Currently, his research focuses on structure-functional study of human and viral proteins in DNA replication initiation. He has published more than 79 peer-reviewed reports. He has served on the editorial boards of International Journal of Spectroscopy, Chinese Journal of Magnetic Resonance and Scientific Report.

Abstract:

Oligonucleotides play many functional roles in cells. G-rich DNA and RNA sequences can form stable four-stranded structures termed G-quadruplexes, in which four guanine bases associate through Hoogsteen hydrogen bonding to form a square planar structure or guanine tetrad (G4). G4 motifs are evolutionarily conserved in certain regions and associated with a specific subset of the genome. Two or more guanine tetrads stack to form a G-quadruplex, which may differ in how the DNA strand(s) are folded. G4 DNA are found in telomeric sequences such as d(GGTTAG)n, and in the promoter regions of many other genes. Genome-wide search identify 370,000 potential quadruplex sequences in the human genome. It was suggested that G-quadruplex formation in a promoter may block transcription of the gene. It has also shown that RNAs also form G-quadruplex and play an important role in transcription and translation processes. We are interested in structure-functional study of G-quadruplex of DNA and RNA in human DNA replication initiation and related diseases. Our biochemical and structural study showed that human Cdc6 binds G4 DNA directly supporting a role for G4 DNA in the recruitment of Pre-RC to replication origins. In analyzing the structure of G4 DNA that Cdc6 binds, we revealed a novel structural fold of G-quadruplex of human telomeric DNA. We also investigated the role of G-rich RNA in latent DNA replication of Epstein-Barr virus. These mechanistic studies will provide insight on the molecular mechanism for origin selection in human and human viruses.

Biography:

Wancai Yang is the Dean of the Institute of Precision Medicine and School of Basic Medical Sciences, Jining Medical University, China, and a Professor of Pathology at University of Illinois at Chicago, USA. He is also an Adjunct Professor of Biological Sciences at University of Texas, El Paso, USA. He obtained his MD degree and was trained a Pathologist, and received Post-doctoral training from Rockefeller University and Albert Einstein Cancer Center. In 2006, he moved to the Department of Pathology, University of Illinois at Chicago. He is serving as Grant Reviewer for the National Institutes of Health (USA) and the National Nature Science Foundation of China. His research focuses on: (1) mechanisms of gastrointestinal carcinogenesis, (2) identification of biomarkers for cancer detection and patient selection for chemotherapy, (3) implication of precision medicine in cancers. He has published about 90 articles and has brought important impact in clinical significance.

Abstract:

Chronic colitis malignant transformation is one of major causes to colorectal cancer, but the mechanisms of colitis development and malignant transformation is largely unknown. Using a unique mouse model, we have demonstrated that the mice with targeted disruption of the intestinal mucin gene Muc2 spontaneously developed chronic inflammation at colon and rectum at early age, whose histopathology was similar to ulcerative colitis in human. In the aged mice, Muc2-/- mice developed colonic and rectal adenocarcinoma accompanying severe inflammation. To determine the mechanisms of the malignant transformation, we conducted miRNA array on the colonic epithelial cells from Muc2-/- and +/+ mice. MicroRNA profiling showed differential expression of miRNAs (i.e. lower or higher expression enrichments) in Muc2-/- mice. Based on relevance to cytokines and cancer, the miRNAs were validate and were found significantly downregulated or upregulated in human colitis and colorectal cancer tissues, respectively. The targets of the miRNAs were further characterized and their functions were investigated. More studies from the Muc2-/- mice showed disorder of gut microbiota. Moreover, a novel tumor suppressor PRSS8 also plays a critical role in colorectal carcinogenesis and progression, for instance, tissue-specific deletion of the PRSS8 gene resulted in intestinal inflammation and tumor formation in mice. Gene set enrichment analysis showed that the colitis and tumorigenesis were linked to the activation Wnt/beta-catenin, PI3K/AKT and EMT (epithelial-mesenchymal transition) signaling pathways. Taken above, the disorder of gut microbiota could result in genetic mutations, epigenetic alterations, leading to the activation of oncogenic signaling, in colorectal epithelial cells, and finally, to colitis development, promoting malignant transformation and mediating colorectal cancer metastasis.

Biography:

Feng Qu received her PhD degree of Analytical Chemistry from Chemistry and Molecular Engineering College, Peking University in 1997.  She has expertise in bioanalysis based on capillary electrophoresis. Her research focus on: capillary electrophoresis in biological and biomedical application; protein and nucleic acid interaction analysis; aptamers selection strategy and methodology for protein, cell, bacteria, small molecule targets .

Abstract:

Nucleic acid aptamers are short, single-stranded DNA (ssDNA) or RNA molecules that are selected for bind­ing to a specific target.  Aptamers can be used as recognition probes in biomedical, food and environment analysis. Moreover, they have great potential in disease diagnosis and treatment, drug discovery, medicine research, as well as bioimaging, which are expected to bring huge economic benefits. However, current aptamers application are far from satisfactory, and have not yet been fully developed. The complicated selection process with high cost and low efficiency is one of the bottlenecks of their application. There is still not universally accepted standard selection methods are accepted.  

Capillary electrophoresis (CE) is one of the most powerful methods for aptamers sieving (known as CE-SELEX), which has the advantages of fast, high resolution, low sample consumption and smart separation modes of capillary zone electrophoresis (CZE), affinity capillary electrophoresis (ACE) and capillary isoelectric focus (CIEF). Moreover, the binding of target and synthetic single stranded DNA (ss-DNA) occurs in free solution, which eliminates the biases caused by stationary support and linker. Some important protein aptamers have been successfully obtained based on CE, which greatly improves the selection efficiency.

Biography:

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

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

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.

Abstract:

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.

Biography:

Reham Abdel Haleem Aboelwafa, MD, a Lecturer of Clinical Pathology, Faculty of Medicine, Alexandria University, Egypt. She has expertise in research, teaching and administration both in hospital and education institution. She is expert in Molecular Techniques: PCR (conventional, and Real time PCR), microarray, Pyrosequencing and NGS in addition to FISH techniques including Prenatal Genetic Diagnosis (PGD) and Flow cytometric mmunophenotyping of different types of hematologic malignancies. She has several international publications in the field of molecular biology, genetics and epigenetics.

Abstract:

Overexpression of the Wilms tumor 1 gene (WT1) is implicated in the prognosis of acute myeloid leukemia (AML) with high expression predicting disease progression, as well as being intensively studied as a potential molecular marker for minimal residual disease (MRD) and treatment response. Many different isoforms for WT1 are generated by alternative transcription initiation, mRNA splicing and alternative translation initiation. Recently, an alternative promoter incorporating a unique first exon, alternative WT1 transcript (AWT1), has been described. The AWT1 expression and the underlying epigenetic alterations associated with its expression in AML are still unknown.

Biography:

Abstract:

Wnt signaling pathways are essential for bone formation. Previous studies showed that Wnt signaling pathways were regulated by miR-375. Thus, we aim to explore whether miR-375 could affect osteogenesis. In the present study, we investigated the roles of miR-375 and its downstream targets. Firstly, we revealed that miR-375-3p negatively modulated osteogenesis by suppressing positive regulators of osteogenesis and promoting negative regulators of osteogenesis. In addition, the results of TUNEL cell apoptosis assay showed that miR-375-3p induced MC3T3-E1 cell apoptosis. Secondly, miR-375-3p targeted low-density lipoprotein receptor-related protein 5 (LRP5), a co-receptor of the Wnt signaling pathways, and β-catenin as determined by luciferase activity assay, and it decreased the expression levels of LRP5 and β-catenin. Thirdly, the decline of protein levels of β-catenin was determined by immunocytochemistry and immunofluorescence. Finally, silence of LRP5 in osteoblast precursor cells resulted in diminished cell viability and cell proliferation as detected by WST-1-based colorimetric assay. Additionally, all the parameters including the relative bone volume from µCT measurement suggested that LRP5 knockout in mice resulted in a looser and worse-connected trabeculae. The mRNA levels of important negative modulators relating to osteogenesis increased after the functions of LRP5 were blocked in mice. Last but not least, the expression levels of LRP5 increased during the osteogenesis of MC3T3-E1, while the levels of β-catenin decreased in bone tissues from osteoporotic patients with vertebral compression fractures. In conclusion, we revealed miR-375-3p negatively regulated osteogenesis by targeting LRP5 and β-catenin. In addition, loss of functions of LRP5 damaged bone formation in vivo. Clinically, miR-375-3p and its targets might be used as diagnostic biomarkers for osteoporosis and might be also as novel therapeutic agents in osteoporosis treatment. The relevant products of miR-375-3p might be developed into molecular drugs in the future. These molecules could be used in translational medicine.

Biography:

Patrick Arbuthnot is currently personal professor and director of the Wits/SAMRC Antiviral Gene Therapy Research Unit at the University of the Witwatersrand in South Africa. After graduating with a medical degree he completed a PhD in 1992 then carried out postdoctoral work at Necker Hospital in Paris, France. It was during this time that Dr Arbuthnot developed an interest in advancing gene therapy for treatment of liver cancer and hepatitis B virus (HBV) infection. On returning to South Africa, he established the Antiviral Gene Therapy Research Unit, which has now published widely on HBV infection, liver cancer, HIV-1 infection and developing new methods of treating these diseases. Patrick Arbuthnot’s main research interest is in advancing use of biological and synthetic nanoparticles to carry potentially therapeutic nucleic acids (DNA or RNA) that are capable of permanently disabling HBV.

Abstract:

Chronic infection with hepatitis B virus (HBV) remains and important global health problem. Carriers of the virus are at high risk for cirrhosis and liver cancer. Available treatment only has modest curative efficacy and improved therapy is a priority to prevent the life-threatening complications that accompany the infection. The viral replication intermediate comprising covalently closed circular DNA (cccDNA) exists as a stable minichromosome in infected hepatocytes. Licensed treatment has no effect on cccDNA and devising methods based on gene therapy to disable this replication intermediate has considerable potential. Previous work from our laboratory demonstrated effective inhibition of HBV replication and targeted disruption of cccDNA by Transcription Activator-Like Effector Nucleases (TALENs). Although this approach is promising, unintended mutagenesis may occur in chronic carriers as a result of TALEN activity at HBV sequences that are integrated into the host genome. To circumvent this problem, we have produced repressor TALEs (rTALEs) that were designed to induce transcriptional repression at essential HBV transcriptional regulatory elements: the basic core promoter/enhancer II and preS2 promoter sequences. KRAB-encoding sequences were fused to the N-terminal regions of TALEs contain sequence-specific DNA binding domains derived from the AvrBs4 N1 Xanthomas TALE. Each rTALE was expressed from the CMV promoter and engineered to interact with an HBV-specific 18 bp target. The repressors were incorporated into recombinant adeno-associated viral vectors which were used to deliver the antiviral

Biography:

Imran Rashid has his expertise in cell culture research model development and working at Shantou University China. His expertise to develop animal cell culture model development bring him to the Shantou university to introduce new strategies to diagnose the stranding reasons and to find the possible reason to solve the problems. He is young dynamic and energetic rising scientist, his four years research experience in Zhejiang University China and later in Shantou University till now, proves that he is emerging scientist in the field of animal cell culture model development.

Abstract:

Statement of the Problem: Rapid declining population of cetacean is moving towards their extinctions, and protection department and researchers could not find the biological cause despite of physical injury and plastic materials in cetacean stomach. The sampling of cetaceans is restricted and field trails are likely to be impossible in the sea or oceans to diagnose cetaceans. Our presentation describes the state of art to know the unknow reason of cetacean death. Skin biopsy is only possible to some extent for sample collection, and we suggest that fibroblast cells culturing is possible to grow in the laboratory. These fibroblast cells can be genetically modified and converted into immortalized cell line. In the meantime, primary cells can also be converted into neuron and hepatocyte like cells by inducing some specific factors to conduct neurological and hepatological effects and response studies. These can be used to diagnose and estimate the molecular changes in the cetacean body. Several studies in the human has proven that fibroblast cells express about 100 genes related to neurodegenerative change and hepatic cells response related genes also found in fibroblast cells. This presentation demonstrates the new strategy to cetacean in the laboratory.

Biography:

Zhongwei Li has his expertise in RNA Biology and related biomedical problems. He studies applied biochemical, molecular and genomics approaches to understand how RNA is processed and degraded in various organisms, and to elucidate the role and significance of ribonucleases. He teaches Biomedical Science courses for Under-graduate medical program and Graduate programs. His research primarily focuses on RNA damage control and prevention of human diseases. His goals are to develop RNA damage biomarkers and identifying protective genes to cope with degenerative disorders. In addition, he is studying bacteria aiming to develop treatment of infectious diseases, including identification of bacterial genes involved in infection, different pathways for bacterial RNA metabolism, and methods for quick diagnosis of bacteria in clinical settings. More recently, he has taken the responsibility of faculty affairs administration to help the college grow after obtaining full LCME accreditation.

Abstract:

Statement of the Problem: Oxidation is probably the most common type of damage that occurs in cellular RNA. Oxidized RNA may be dysfunctional and is implicated in the pathogenesis of age-related human diseases. Cellular mechanisms controlling oxidized RNA have begun to be revealed. Currently, many ribonucleases and RNA-binding proteins have been shown to reduce oxidized RNA and to protect cells under oxidative stress. Although information about how these factors work is still very limited, we suggest mechanisms that can be used to minimize oxidized RNA in bacteria and human cells.

Methodology & Theoretical Orientation: RNA oxidation levels are determined by chromatographic separation of nucleosides and detection of 8-oxoG levels. Cell viability was determined by growth rate. Functions of ribonucleases and other proteins were studied by examining mutants lacking genes encoding these proteins, or over-expressing the genes.

Findings: Many ribonucleases and other proteins were found important for maintaining cell viability under oxidative stress. We have also identified proteins that specifically bind oxidized RNA at high affinity. In mutants lacking these activities, oxidized RNA accumulates. Over-expression of these activities reduces oxidized RNA and rescue cells under oxidative stress. Normalized RNA oxidation levels reduce overtime after pulse oxidative challenge.

Conclusion & Significance: The results demonstrate mechanisms for selective removal of oxidized RNA in bacteria and cultured human cells. Selective reduction of RNA oxidation depends on the activities of ribonucleases, suggesting that RNA degradation plays a major role in this process. The finding of specific proteins with high affinity to oxidized RNA implies that oxidized RNA is recognized by these proteins, targeting the RNA to effective degradation or repair. The findings demonstrate an important mechanism for maintaining RNA quality for normal function of the cell, and for prevention of related human diseases.

Biography:

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.

Abstract:

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.