Molecular biology

I am interested in exploring innovative and useful chemical tools and small molecules as probes to study biology and as therapeutics for disease treatment. My laboratory has been particularly interested in exploring chemical tools to address the important biological questions. I am a well-established investigator with over 20 years research experience and more than 240 peer reviewed publications (H-index: 72) in the fields of organic and medicinal chemistry and chemical biology. The small molecule-based fluorescence probes developed from my laboratory have been widely used by biomedical researchers as tools to study the cellular and molecular mechanisms. One of the small molecules discovered by my laboratory has been licensed to the Andaman Therapeutics for clinical trials as a new class of anticancer therapy.

We are a plant genomics lab who specialize in whole genome sequencing and assembly; with analyses of structural variation, gene modeling and transcriptomes. Our work on major projects of rice, corn, barley, etc, allows us to share our technical expertise with other researchers. Our research in plant and animal genomes, at the whole genome and transcriptome levels, will impact successful genetic selections toward the goal of feeding the 9 billion people toward the year 2050. Keywords: "Genome Sequencing", "PacBio", "Structural Genomics", "Plant Genetics", "DNA Extraction"

Mechanistic studies of the Nrf2/Keap1 signaling pathway Oxidative stress, an imbalance between production and removal of reactive oxygen species, can damage biological macromolecules including DNA, proteins and lipids ( Oxidative damage to biological macromolecules can have profound effects on cellular functions and has been implicated in cancer, inflammation, neurodegenerative diseases, cardiovascular diseases and aging. Eukaryotic cells have evolved anti-oxidant defense mechanisms to neutralize reactive oxygen species (ROS) and maintain cellular redox homeostasis. One of the most important cellular defense mechanisms against ROS and electrophilic intermediates is mediated through the ARE (antioxidant responsive element, or electrophile responsive element) sequence in the promoter regions of phase II and antioxidant genes. The ARE-dependent cellular defense system is controlled by the transcription factor Nrf2. Recent advances in the mechanistic studies of this pathway have provided the following models for Nrf2 regulation: Keap1, a key player in the activation of this pathway, has been identified to function as a molecular switch to turn on and off the Nrf2-mediated antioxidant response. Under basal condition, Keap1 is in the off position and functions as an E3 ubiquitin ligase, constantly targeting Nrf2 for ubiquitination and degradation. As a consequence, the constitutive levels of Nrf2 are very low. The switch is turned on when oxidative stress or chemopreventive compounds inhibit the activity of the Keap1-Cul3-Rbx1 E3 ubiquitin ligase, resulting in increased levels of Nrf2 and activation of its downstream target genes. The switch is turned off again upon recovery of cellular redox homeostasis; Keap1 travels into the nucleus to remove Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the nuclear export signal (NES) in Keap1. In the cytosol, the Nrf2-Keap1 complex associates with the Cul3-Rbx1 core ubiquitin machinery, resulting in degradation of Nrf2. We are currently working on the detailed steps of the Nrf2-Keap1-ARE pathway in response to oxidative stress and to chemopreventive compounds The protective role of Nrf2 in arsenic-induced toxicity and carcinogenicity Another direction of our research is to understand the molecular mechanisms of toxicity/carcinogenicity of environmental pollutants and the endogenous cellular defense systems to cope with pollutants. Drinking water contaminated with arsenic is a worldwide public health issue. Arsenic has been classified as a human carcinogen that induces tumors in the skin, lung, and bladder. Arsenic damages biological systems through multiple mechanisms, one of them being reactive oxygen species. The ARE-Nrf2-Keap1 signaling pathway, activated by compounds possessing anti-cancer properties, has been clearly demonstrated to have profound effects on tumorigenesis. More significantly, Nrf2 knockout mice display increased sensitivity to chemical toxicants and carcinogens and are refractory to the protective actions of chemopreventive compounds. Therefore, we hypothesize that activation of the ARE-Nrf2-Keap1 pathway acts as an endogenous protective system against arsenic-induced toxicity and carcinogenicity. The following Specific Aims are intended to further elucidate the mechanism of Nrf2-activation in protection from arsenic-induced toxicity/tumorigenicity. We will (1) determine the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and cell transformation using a model cell line UROtsa, (2) define the molecular mechanisms of activation of the ARE-Nrf2-Keap1 pathway by arsenic, sulforaphane, and tBHQ, and (3) define the protective role of the ARE-Nrf2-Keap1 pathway in arsenic-induced toxicity and tumorigenicity using Nrf2 knockout mouse as a model. So far, we have demonstrated a protective role of Nrf2 against arsenic-induced toxicity using cell culture and Nrf2-/- mouse model. We have provided evidence demonstrating that Nrf2 protects against liver and bladder injury in response to six weeks of arsenic exposure in a mouse model. Nrf2−/− mice displayed more severe pathological changes in the liver and bladder, compared to Nrf2+/+ mice. Furthermore, Nrf2−/− mice were more sensitive to arsenic-induced DNA hypomethylation, oxidative DNA damage, and apoptotic cell death. Recently, we submitted another manuscript to Toxicological and Applied Pharmacology, reporting our long-term study of the effect of Nrf2 on arsenic-mediated cell transformation and tumor formation. In this study, we provide evidence demonstrating the importance of Nrf2 activation in preventing the carcinogenetic effects induced by long-term exposure to low-dose arsenic both in vitro and in vivo. The UROtsa cell line was used to show that daily exposure to the Nrf2 inducer, tBHQ, alleviated arsenic-induced hypomethylation and cell transformation. Moreover, tBHQ treatment reduced tumorigenicity of arsenic-transformed cells in SCID mice. Chronic treatment with arsenic also compromised the Nrf2-dependent defense response in the bladder epithelium in Nrf2+/+ implicating the important role of Nrf2 in protecting against arsenic-induced carcinogenicity. This study supports the advantages of using dietary supplements specifically targeting Nrf2 as a chemopreventive strategy to protect humans from various environmental insults that may occur on a daily basis. Identification and development of Nrf2 activators into dietary supplements/ for disease preventio Identification and development of Nrf2 inhibitors into therapeutic drugs to enhance the efficacy of cancer treatment High-throughput screening of Nrf2 activators or inhibitors: we are screening a chemical library and a natural product library to identify compounds that are able to activate or inhibit ARE-luciferase activity using a stable cell line established in our laboratory, MDA-MB-231-ARE-Luc. Based on the critical role of Nrf2 in disease prevention, using Nrf2 activators to boost our antioxidant response represents an innovative strategy to enhance resistance to environmental insults. Once Nrf2 activators are identified and the specificity of these compounds in activating Nrf2 is validated, the compounds will then be tested for the mechanism by which they confer cellular protection and the feasibility of using these compounds for disease prevention using various disease models. On the other hand, recent findings point to the “dark side” of Nrf2, as studies have shown that Nrf2 promotes cancer formation and contributes to chemoresistance. Using a genetic approach, we have provided evidence that the level of Nrf2 correlates well with cancer cell resistance to several therapeutic drugs, demonstrating that Nrf2 is likely responsible for chemoresistance. More recently, we have reported a study on Nrf2 expression in endometrial cancer patients (117 cases). We found no detectable Nrf2 expression in complex hyperplasia, 28% Nrf2 positive cases in endometrial endometrioid carcinoma (type I), and 89% Nrf2 positive cases in endometrial serous carcinoma (type II). Please note that type II endometrial cancer is the most malignant and recurrent carcinoma among various female genital malignancies. Furthermore, inhibition of Nrf2 by overexpressing Keap1 sensitized SPEC-2 cells, which are derived from type II endometrial cancer, or SPEC-2 xenografts to cisplatin using both cultured cells and SCID mouse models. These studies demonstrate that Nrf2 contributes to chemoresistance in many cancers originating from different organs and illustrate the urgent need for identification of Nrf2 inhibitors and for the development of Nrf2 inhibitors into druggable compounds to enhance the efficacy of cancer treatment. We have identified the very first Nrf2 inhibitor and characterized its use to sensitize cancer cells to chemotherapy. We just submitted a manuscript to Science reporting our discovery. The following is the abstract of the manuscript: “The major obstacle in cancer treatment is the resistance of cancer cells to chemotherapy. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive to cancer cell survival. We have identified brusatol as a selective Nrf2 inhibitor that is able to sensitize cancer cells and xenografts to chemotherapeutic drugs by enhancing the degradation of Nrf2 and inhibiting the Nrf2-dependent antioxidant response. These results suggest that brusatol can be developed into an adjuvant drug to enhance the efficacy of cancer treatments”. The importance of this project: (i) the use of an Nrf2 inhibitor to enhance the efficacy of cancer therapeutics represents a novel approach to caner treatment. Nrf2 inhibitors may be used in a broad spectrum across many types of cancers and chemotherapeutic drugs to increase the effectiveness of cancer treatment. Development of brusatol into an adjuvant for clinical use to sensitizer many cancer types to treatment will have an enormous impact on human health worldwide. (ii) Brusatol will be extremely useful for the mechanistic investigation of Nrf2 regulation by complex cellular networks. Cross talk between the Nrf2 signaling pathway and others During the last couple of years, crosstalk between the Nrf2 pathway and other important pathways has emerged. Our group has identified two separate branches that converge with the Nrf2 pathway, the p53-p21(Cip1/WAF1) pathway and the autophagy pathway. Crosstalk is mediated by the direct interaction between p21 and Nrf2, and Keap1 with p62, respectively. In the p53-p21 study, we provide molecular and genetic evidence suggesting that the previously suggested antioxidant function of p53 or p21 is mediated through activation of the Nrf2 pathway. Mechanistically, p21 is able to stabilize Nrf2 by competing away Keap1, thus, activating the Nrf2-mediated antioxidant response. Therefore, the interaction between Nrf2 and p21 represents a fine-tuning mechanism between life and death according to the level of stress. In the study with p62 and Nrf2, we reported a novel mechanism of Nrf2 activation by autophagy deficiency through a direct interaction between Keap1 and p62. In response to stress, cells can utilize several cellular processes, such as autophagy, a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1-dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a non-canonical cysteine-independent mechanism. These work was published in Molecular Cell and Molecular and Cellular Biology, respectively, both are high profile journals. Furthermore, both articles were highlighted in Molecular Cell and Science Signaling, respectively, indicating the importance and high impact of the projects.

Michael Worobey, PhD, uses the genomes of viruses to trace the evolution of major communicable diseases, such as HIV/AIDS and influenza. He employs an evolutionary approach to understand the origins, emergence and control of pathogens, in particular RNA viruses and retroviruses such as HIV and influenza virus. The research program integrates fieldwork, theory and methodology, molecular biology, and molecular evolutionary analysis of gene sequences in a phylogenetic framework.Current wet-lab projects in Dr. Worobey’s Biosafety Level 3 facility involve recovery of damaged and/or ancient DNA from a variety of sources including paraffin-embedded human tissue specimens, blood smears, and museum specimens. The two main efforts are: 1) reconstructing the emergence of HIV-1 group M in central Africa and North America using fossil HIV-1 sequences, and 2) investigating the evolution of AIDS-related viruses in wild-living African primates using non-invasively-collected samples.

Rod Wing, PhD, and his lab, The Arizona Genomics Institute, specialize in building what geneticists call a physical map of a genome- a crucial foundation of any genome sequencing effort. AGI has earned a reputation for providing extremely high-quality maps, as documented in previous sequencing efforts leading to the genome sequences of rice and corn. The genome sequence will allow scientists to locate and identify genes that can improve and strengthen crops and increase yield in order to help solve the Earth’s looming food crisis by creating new strains of the cereal crops that make up 60% of humankind’s diet. Keywords: Genome Biology, Genome Sequencing/Assembly/Annotation, Food Security, Rice

Josef Vagner, PhD, is an Associate Research Professor at the University of BIO5 Research Institute and the Director of the Ligand Discovery Laboratory. Dr. Vagner is expert in the field of drug discovery and development, and he is focused on the design, synthesis, purification, characterization and screening of compound arrays.He has published over 70 original research papers and 31 patents. He is a frequent presenter at national and international meetings (American Chemical Society and Peptide Societies).Dr. Vagner designed and developed of compounds for in vivo pharmacologic applications and translation programs. He has over 25 years experience in synthesis and structural analysis of de novo ligands for various biological targets, including a recent focus on ligands targeting GPCRs and multivalent ligands. These experiences include 10 years of work in the pharmaceutical industry (Sanofi/ Selectide, Novo Nordisk, Discovery Partner International) where he specialized in combinatorial chemistry and array synthesis of small molecules. During his time in industry, he supervised teams of workers who successfully accomplished the synthesis of more than ten large libraries (with >10,000 compounds each).

Carol Soderlund, PhD, is an Associate Research Professor at the BIO5 Institute at the University of Arizona. While working on her PhD in Computer science in 1988, she collaborated with a biologist to develop one of the first gene prediction programs. She received a DOE Human Genome Distinguished Postdoctoral Fellowship hosted by Los Alamos National Laboratory, where she became involved with mapping the human genome. Her work continued at the Sanger Centre in the UK, which was on the forefront of sequencing the human genome. She developed the FPC software, which was used for mapping the human genome, and has since been the primary software package for mapping large genomes.Her primary research objective is to provide environments for biologists to run algorithms (both her own and existing software), with highly interactive graphics for query and display of the data and results. Towards this end, she has published seven software packages for various genomic problems, where the three most important are: (1) The FPC program mentioned above, which is still being used after its initial release 15 years ago and has been extended for next generation sequencing. (2) The SyMAP software for the computation, query and display for synteny for the comparison of plant genomes. (3) The Transcriptome Computational Workbench (TCW) for the analysis of the transcriptome across tissues or conditions, and across the species for finding shared and unique genes.Dr. Soderlund has published over 60 original research papers and 20 book chapters on a range of genomic problems. She has collaborated with a range of scientists on a variety of organisms and genomic problems, where the majority of the collaborations have been on mapping genomes and transcriptome analysis, but she has also been involved in metagenomics, sequencing, and host-pathogen interactions.

Catharine Smith, PhD, focuses on epigenetic mechanisms of gene expression, particularly their regulation through signaling pathways and their modulation by anti-cancer drugs. Epigenetic mechanisms play a very important role in transcriptional regulation of genes but the specifics of these mechanisms require ongoing study. A very exciting new area of research focuses on how these mechanisms are disrupted during tumorigenesis but may also be harnessed to treat cancer. Signaling pathways control the expression of key genes in non-cancerous cells but are often misregulated during the process of oncogenesis. Chromatin proteins and transcription factors that interact with chromatin are often targets of these pathways. Two projects in the lab are directed at the interface of signaling pathways and chromatin. First, Dr. Smith is interested in the mechanism by which the female reproductive steroid, progesterone, regulates target genes in the physiological context of chromatin. Chronic progestin exposure has been linked to increased incidence of breast cancer in post-menopausal women on hormone-replacement therapy. However, the function of the progesterone receptor in mammary tissue and its role in oncogenesis are not well understood. Current studies in this area are directed at the role of chaperone proteins in determining how the progesterone receptor functions at target genes in chromatin and how it is impacted by other signaling pathways.Second, her lab has discovered a novel cAMP signaling pathway that regulates cell cycle progression and are focused on identifying specific components and targets of this pathway.Third, histone deacetylases (HDACs) are key transcriptional regulatory proteins. Inhibitors that target these enzymes have shown great promise as anti-cancer drugs and are currently in clinical trials. However, a lack of knowledge of HDAC biology has made it difficult to predict which tumors will respond to these drugs. HDACs are known to participate in gene repression, but recent work indicates that they are also transcriptional coactivators. Further studies on the mechanism of gene repression through HDAC inhibitors will provide insight into the role of these enzymes as coactivators.

Every investigation that they have pursued, even investigating novel disease models, has produced profound discoveries in basic biology and biochemistry. They are currently working in collaborations with labs to exploit three system to explore the basic function of the RNA-binding protein FUS. First, they are collaborating with the lab of Rob Batey (UC Boulder) to investigate the role of RGG-rich domains in mediating RNA recognition. Next they are collaborating with lab of Kate Fitzgerald (U Mass Med) to investigate the role of FUS in transcriptional pause release and initiation as macrophage cells respond to stimulation of Toll-like receptor 4. Lastly, they are collaborating with the lab of Ran Taube (Ben-Gurion U) to investigate the role of FUS as a scaffold protein to promote the formation of the Super Elongation Complex (SEC) both genome-wide and for the Tat gene in HIV. They are also pursuing the role of FUS and noncoding RNAs in DNA damage repair. They believe that the function of FUS in affecting transcription is also crucial to the repair of DNA damage in cells.

Marek Romanowski, PhD, and his work on translating physics into medical products have huge implications for the evolution of personalized medicine. On cue, a tiny pillbox of gold floating in your bloodstream can deliver its medicine exactly to the right cell, one that is sick with cancer, avoiding all of your healthy cells. A gold capsule – about 50 to 200 nanometers in diameter, large enough to do the work of transporting a few molecules of medicine and respond to light signals – is too large to pass out through the kidneys. But on command by an enzyme, it can fall apart into pieces smaller than 10 nanometers, just a few molecules. The new size can easily leave our bodies at no risk. The gold pillbox has many other possible applications. In addition to delivering a drug, it can become a part of a diagnostic test, or deliver genetic material to a cell to permanently modify the cells’ DNA—a key step in gene therapy.