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Click on any project title for a more detailed description of the project. For more information about any of these awards (e.g., PI contact information or associated publications), please use the corresponding project number to search for information at the NIH Reporter website.
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|Project #
of Award
|PI Name(s)
|Abstract Text (Official)
|Droplet Cell Array Assays
|Understanding the basic genetic and molecular markers of cancer at the cellular level is vital for preventing, diagnosing, and treating cancer. Recent work in our lab has led to new technologies for manipulating small aqueous drops containing biological molecules. We use laser light to induce surface tension gradients, which allows us to selectively move very small individual droplets (mu L-pL), and we have performed simple enzymatic assays with this approach. Because our technique uses laser heating as a basis for droplet control, we believe that it is well suited to genomic analysis methods, such as polymerase chain reaction (PCR), which rely on thermal cycling. We propose to combine our laser-based droplet control techniques with genomic analysis tools to develop a device for screening large numbers of individual cells. To test the capabilities of our apparatus, we propose an R21 project with the following specific aims: Aim 1: Optimize and automate the liquid handling system for genomic analysis. We will choose the optimal materials and reagents for use in our apparatus. We will automate both the droplet delivery and the droplet handling capabilities of our device. Aim 2: Perform a real-time PCR in our droplet-based system. We will test the overall sensitivity and quantify nonspecific amplification in our binary liquid system. Aim 3: Examine single cells within small droplets. This examination will include testing for short- and long-term cell viability and PCR amplification of single genomic material. This research is fully consistent with the goals outlined in RFA-CA-06-002. If this program is successful, we believe that the resulting technology will prove invaluable in helping doctors and scientists better understand the molecular basis of cancer while also providing tools to help diagnose specific variants of disease, plan and assess therapy, and monitor disease recurrence.
|Nanoparticles for Harvesting and Targeting Angiogenic Proteins
|This R21/R33 application entitled "Nanoparticles for Harvesting and Targeting Angiogenic Proteins" has as its hypothesis that development and refinement of surface characteristics of silica chips with nanocharacteristics can enhance sensitivity of mass spectrometry (MS) detection of the low molecular weight angiogenic proteins present in serum and tumors that produced at very early times of tumor development. In addition, refinement of conjugation methods of nanoporous particles will allow selective targeting of endothelial cells in vitro and tumor-associated blood vessels in vivo and in combination with refinement of loading strategies, cytotoxic agents loaded into nanoparticles can selectively destroy these vessels. Our experimental plan is based on our expertise in development and refinement of emerging nanotechnology approaches for protein capture, for selective targeting and loading of silicon nanoparticles. These studies also take advantage of our experience in identification of novel proteins within the vascular endothelial growth factor (VEGF) family of proteins that are essential in the process of tumor-associated angiogenesis. To achieve the goal of developing and refining tools for detection of angiogenic proteins and for selective targeting and destruction of tumor-associated blood vessels, the following Specific Aims are proposed: 1. Develop and refine silica chips with nanocharacteristics to enhance the sensitivity of LC-MS/MS identification VEGF proteins in serum and in skin tumors during skin tumor-associated angiogenesis in vivo; 2. Refine conjugation of silicon nanoparticles to anti-VEGFR-2 receptor antibodies for selective targeting of endothelial cells in vitro and targeting tumor-associated blood vessels in vivo; 3. Determine the ability of silicon nanoparticles conjugated with anti-VEGFR-2 antibodies to be loaded with and to deliver the cytotoxic agent melatin for destruction of endothelial cells in vitro and for destruction of tumor-associated blood vessels in vivo. These studies will provide sensitive nanotechnology tools that are critical in defining the proteome in serum and tumors related to tumor angiogenesis that is currently unexplored. These studies may also provide strategies to selectively target tumor vessels for destruction using nanotechnology approaches.
|Targeted antigen delivery for cancer immunotherapy
|Metastatic melanoma patients currently have a dismal diagnosis, and treatment at the metastatic stage is generally ineffective. The long-term objective of this research project is to develop a novel treatment and vaccination approach for cancer in general, and melanoma in particular, based on immunotherapy. Ex vivo antigen delivery for immunotherapy is laborious and expensive, and is thus not affordable to many of those in need. The investigators propose to develop an antigenic entity that can be applied on the skin, with direct antigen delivery to skin dendritic cells and without the need for in vitro cell manipulations. Thus, the major practical objective of this study is to establish the proof of principle that topically delivered tumor associated antigens can elicit effective anti-tumor responses, and can be used for cancer immunotherapy. Specific Alms The study will be based on two antigenic proteins derived from melanoma: the first is a hydrophilic recombinant gp100 protein, and the second is a multiepitope polypeptide that comprises 3', repeats of 4 HLA-A2 melanoma peptides derived from 3 different melanoma proteins. In order to allow and to improve topical transdermal delivery, the antigens will be genetically fused to potential carrier molecules. One of these is E. coli heat labile enterotoxin, a molecule recently shown to act as carrier and adjuvant. Another is a novel haptotactic C-terminal fibrinopeptide (Haptide). During the first phase of the project, R21, the new antigenic entities will be cloned, expressed, and purified. Novel in vitro models using human skin will be used to evaluate transcutaneous passage of molecules, Langerhans' cells activation and mobilization, and stimulation of specific cytotoxic T cells. The rationale for the milestones that will determine continuation to the second phase, R33, is based on the efficacy of antigen delivered transcutaneously to stimulate the immune system in human in vitro models, and will allow for the selection of the molecules that will be further evaluated in depth in vivo models. In the R33 phase, specific immune responses of splenic T cells from vaccinated mice will be evaluated, tumor models will be established in mice, and the response to vaccination will be determined. Finally, the most effective molecule/s will be produced under GMP or GMP-like conditions for phase I/II clinical trials in a subsequent study. Public Health The success of this project would allow topical application of an immunostimulant for treatment of melanoma and other cancers and would thus significantly simplify treatment, eliminating the need for hospitalization and even day-care and without the need for a specialized laboratory. As a result, one could treat a much larger number of patients, with the potential to clinically evaluate new antigens and immunotherapeutic modalities, improving the life quality and expectancy of metastatic melanoma patients
|Direct Detection of Hypermethylation in Cancer
|The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70-80 percent of the human genome, most often in repetitive genomic regions. On the other hand, CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60 percent) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer, leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called SEquence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and beta-lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach, if successful, will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by 1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs; and 2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTp1) implicated in cancer.
|Characterization of Methods for Preservation of Phosphoproteins in fixed tissues
|Protein phosphorylation is an important mechanism of regulating protein function and activity that depends on a competing system of kinases and phosphatases. It is a dynamic processes that is altered in many disease states. For example, activated tyrosine kinases are central to the pathogenesis of chronic myelogenous leukemia (BCR-ABL1) and gastrointestinal stromal tumors(KIT). Detection of phosphoproteins (PPs) in fixed tissues by in situ immunohistologic methods may have diagnostic, prognostic, and therapeutic implications for cancer patients. Initial studies have shown that PPs are quite labile. Little is known regarding methods to preserve phosphorylation status in tissues. The purpose of this application is to develop optimal tissue handling methods that will be suitable for detection of PPs in fixed tissues, keeping in mid practical limitations in the clinical setting. To this end we intend to 1) develop a quantitative immunofluorescence (IF) method using quantum dots to quantitate PP status in fixed cell blocks; 2) characterize optimal fixation conditions (time, fixative, requirement of phosphatase inhibitors) in murine xenografts of human cell lines as a controlled model of available control material that is assayed both by quantitative Western blot and IF; and 4) show proof of principle of in a murine model of BCR-ABL1 containing cell line xenograft treated with imatinib mesylate (IM) and bone marrow biopsies from patients suspected of chronic myeloproliferative disorder harboring the JAK2 V617F mutation. Phospho-STATS is known to be increased in both these systems. Decreased expression by phospho-STAT5 immunostaining in IM-treated xenografts and increased expression in JAC2 V617F+ bone marrow megakarycotyes is expected in optimally handled tissues. This application has relevance in the diagnosis, prognosis, and therapy of malignancies and other diseases that have altered PP levels as part of their pathogenic pathways. It will define tissue handling conditions that adequately preserve in vivo PP status for subsequent diagnostic and prognostic testing. Furthermore, control material with defined relative expression levels of many PPs will result from this application and allow laboratories to assess performance of their individual assays.
|Novel Method for Isolating Actively Translated mRNAs
|Transcriptional regulation has been the main focus for gene regulation in the past. However, a tremendous amount of evidence from recent studies also indicates that translational regulation plays a key role during development, cell cycle control, and mechanisms related to acute drug resistance. Gene expression analysis on actively translated mRNA transcripts provides a unique approach to study post transcriptional regulation. Previous studies have relied on a traditional sucrose gradient ultracentrifugation procedure to isolate polysome complexes and requires a large amount of cells (up to 500 million cells). As a result, this still remains a major bottleneck for the investigation of post transcriptional regulation with limited quantities of clinical samples. Therefore, there is an urgent need to develop a novel approach to isolate actively translated polysomes from a small number of cells (10 to 500 cells). The new approach will allow us to systematically study potential translational regulation with limited clinical samples. It has been shown that actively translated mRNAs are associated with multiple units of ribosomes and the newly synthesized polypeptides are closely associated with molecular chaperones such as hsp73. These molecular chaperones assist in the proper folding of nascent polypeptides into higher ordered structures. These chaperones will provide the anchor to separate actively translated mRNAs associated with polysomes from free mRNAs. Affinity antibody capture beads will be developed to capture hsp73 chaperones associated with the polysome complexes so that all polysomes can be separated from monosomes and free mRNAs. The isolated actively translated mRNAs will be used for high throughput gene expression analysis. The specific aims of the proposed project are: 1.) Develop antibody conjugated affinity capture magnetic beads and conditions to capture actively translated mRNAs associated with the polysome complex from a small number of cells. 2.) Validate the antibody affinity capture approach for polysome isolation by comparing with traditional polysome isolation protocols via quantitative RT-PCR gene expression analysis. 3.) Identify potential translationally regulated genes that are responsible for determining chemosensitivity during 5- fluorouracial (5-FU) treatment from human colon cancer samples.
|Transposon-based somatic mutagenesis/prostate cancer genetics
|Prostate cancer is the second leading cause of male cancer death in the United States and often results in a reduced quality of life for those living with or treated for this disease. Prostate cancer is commonly treated by androgen ablation therapy and although many tumors initially respond to this treatment, many eventually progress to hormone refractory prostate cancer (HRPC). The genetic basis for the transition to hormone insensitivity is poorly understood. We propose to use a mouse model for invasive prostate cancer that results from prostate specific loss of the tumor suppressor gene Pten. This mouse model is relevant to human disease as PTEN expression is lost in many human prostate tumors and the tumors that form in the mice remain partially sensitive to hormone withdrawal. We will use a novel method for cancer gene discovery in mice, the Sleeping Beauty (SB) transposon system, to promote aggressive tumor formation in this model. The SB transposon is a DMA element that is capable of mobilizing and inserting in a different location in the genome. If a mobilized transposon reinserts near a cancer gene, it can promote changes in expression of that gene that promote the transition from a normal cell to a transformed cancer cell. We have previously generated mice engineered with all the components necessary for mobilizing SB transposons in various tissues in the adult mouse. In unpublished experiments, we have successfully used the SB system to identify genes involved in sarcoma and lymphoma formation in mice, and we believe that SB will prove to be equally as successful in prostate tumor models. By using SB to promote HRPC formation we can both identify the genetic changes that cause a tumor to become insensitive to hormone withdrawal and also generate a useful mouse model of HRPC that will be useful for discovery and testing of novel chemotherapeutic agents for advanced prostate cancer. Finally, this approach represents a novel method for the unbiased molecular/genetic analysis of cancer development and could be used widely in the study of important clinical cancer problems.
|Recovery of RNA from Formalin-Fixed Tissues
|State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of the project. Describe concisely the research design and methods for achieving these goals. Avoid summaries of past accomplishments and the use of the first person. This abstract is meant to serve as a succinct and accurate description of the proposed work when separated from the application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. High-throughput molecular biologic and proteomic methods provide several promising approaches for relating genetic changes, such as mutation or altered gene expression, to metastasis, to treatment outcomes, and to survival. In cancers where the interval between initial diagnosis and treatment and the appearance of metastases is long, clinical correlations would be more readily obtained if formalin-fixed paraffin-embedded (FFPE) tissues could be used instead of fresh or frozen specimens. Large-scale multiplex techniques, such as serial analysis of gene expression (SAGE), and gene chip methods yield experimental results that are somewhat different for FFPE tissue and unfixed tissue. The long-term goal of our research program is to use high-throughput molecular biologic screening methods to identify the molecular and genetic basis of cancer origins and behavior. The objective of this proposal is to identify the formaldehyde-induced chemical modifications that occur to nucleic acids during histologic tissue processing and to develop methods to reverse these modifications. Our centralhypothesis is that formaldehyde adducts and cross-links formed during tissue processing can be sequentially reversed by a series of heating and dialysis steps, carried out under appropriate solvation conditions. We formulated this hypothesis on the basis of preliminary data which show that the reversal of formaldehyde-induced chemical changes in proteins and nucleic acids is relatively facile in aqueous solutions, but less so following dehydration in the presence of organic solvents. The rationale for these studies is that their successful completion will provide a foundation for applying high-throughput screening methods to FFPE tissues. This will lead to improved practical interventions for the diagnosis, evaluation, treatment, and prevention of cancer and facilitate the development of therapeutic agents. Our studies are innovative in that we have pioneered a novel model system (tissue surrogates) ideally suited to identify the formaldehyde-induced modifications to proteins and nucleic acids that occur during tissue processing. At the completion of this project it is our expectation to have established a comprehensive understanding of the formaldehyde-induced chemical modifications to mRNA that occur during tissue histology, and methods for optimally reversing these modifications. This knowledge should result in an ability to carry out genomic analysis on FFPE tissue, significantly expanding our capability to conduct genomic research and opening important new areas to practical investigation. PERFORMANCE SITE(S) (organization, city, state) American Registry of Pathology UNIVERSITY of Maryland, Baltimore County (UMBC) 1413 Research Boulevard, Building #102 Department of Chemistry and Biochemistry Rockville, MD 20850 1000 Hilltop Circle (application organization site) Baltimore, MD 21250 (contractual arrangement site) KEY PERSONNEL. See instructions. Use continuation pages as neededio provide the required information in the format shown below. Start with Principal Investigator. List all other key personnel in alphabetical order, last name first. Name Organization Role on Project O'Leary, Timothy J. Armed Forces Institute of Pathology Principal Investigator Cunningham, Robert E. Armed Forces Institute of Pathology Research Associate Fabris, Daniele UNIVERSITY of Maryland, Bait. County Co-Investigator Mason, Jeffrey T. Armed Forces Institute of Pathology Co-Investigator Rait, Vladimir K. American Registry of Pathology Research Associate Sheng, Zongmei American Registry of Pathology Research Associate Disclosure Permission Statement. Applicable to SBIR/STTR Only. See instructions.' l~l Yes I No PHS 398 (Rev. 05/01) Page 2 Form Page 2 Principal Investigator/Program Director (Last, first, middle): O'Leary, Timothy Joseph [The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page.] RESEARCH GRANT
|Development of gene targeting in C. elegans and D. rerio using zinc finger
|The goal of this research is to develop methods for the precise modification of specific target genes in two important genetic model organisms, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. Both nematodes and fish are powerful experimental systems that combine elegant developmental biology with large scale genetics. Both systems have contributed to our understanding of fundamental problems in cancer biology, including programmed cell death, the control of organogenesis, the interaction of cancer susceptibility genes with the environment, and the genetics of melanoma. An important limitation of these model systems is that techniques for site-specific manipulation of the genome are not currently available in either nematodes or fish. Thus, in contrast to murine embryonic stem cells and the yeast S. cerevisiae, it is not possible to knock out specific genes or to precisely control the time and place of gene expression. In the last two years, a powerful new approach to gene-targeting has been developed and successfully used in flies and in mammalian somatic cells. This technique uses chimeric zinc finger nucleases to stimulate precise targeting of specific genes in their native genomic context. The aim of this proposal is to induce targeted, heritable genetic changes via zinc finger nuclease-mediated homologous recombination in C. elegans and D. rerio. Initially we will employ a well-characterized zinc finger nuclease that recognizes the green fluorescent protein (GFP) gene. We will introduce the nuclease into transgenic nematodes and zebrafish that express GFP. We expect the resulting double-strand DNA breaks to stimulate mutagenic non-homologous end joining (NHEJ), leading to the loss of GFP signal. In the second phase, we will simultaneously introduce the nuclease and a repair template that will allow us to create precise mutations in the target locus by homologous recombination. Based on the success of this work we will then target native genes in the worm and the fish by designing novel nucleases and testing them in vitro and in vivo for activity against the targeted gene. We expect that, if successful, this novel approach would be a practical, flexible, and powerful technique that would find wide application, significantly increasing the power of these systems to illuminate human cancer biology.
|Epigenetic Targeting in Non-Hodgkin's Lymphoma
|Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis.
|Development of Dynamic Isoelectric Focusing for Cancer Proteomics
|Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200. An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.
|Novel Probe for Quantitative Imaging of Gene Expression
|We propose to develop a molecular imaging probe that will provide quantitative information on the expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe will be designed to form a stem-loop structure and will be labeled with a 'reporter' fluorophore at one end and a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled with a second optically distinct 'reference' dye/nanoparticle, which will be selected such that it is unquenched regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2) The ability to remove via ratiometric imaging (i.e. reporter fluorscence/reference fluorescence) the impact of instrumental and experimental variability. (3) The ability to quantitatively compare variations in gene expression levels between samples, between cells within individual samples, and even between subular compartments by using the reference dye as a point of reference (4) The ability to quantify gene expression with spatial and temporal resolution since the covalent linkage between the reporter and reference dye ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo imaging. (6) The possibility of an improved signal-to-background due to quenching of the 'reporter' dye by both the quencher molecule and the 'reference' dye. To evaluate these features we will pursue two major aims during the proposed research: 1) We will design, synthesize and characterize the 'quantitative' molecular beacon (QMB) in terms of its signal-to-background and lower detection limit (in vitro and in vivo) and 2) we will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer cells in real-time. It is envisioned that the approach proposed here will allow significant advancements in our understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.
|MRI contrast agents targeting carbohydrate biomarkers
|Malignant transformation is often associated with alteration of cell surface carbohydrates. The expression or over-expression of certain carbohydrates, such as sialyl Lewis X (sLex), sialyl Lewis a (sLea), Lewis X (Lex) and Lewis Y (Ley), has been correlated with the development of certain cancers. These cell surface carbohydrates can be used for cell-specific identification and targeting of carcinoma cells. Recently, we have developed boronic acid-based small molecule lectin mimics (named boronolectins) that can recognize certain carbohydrates with selectivity. The same or similar methods can be used for the preparation of lectin mimics for a wide variety of carbohydrates. The long-term goal of this project is the development of conjugates of boronolectin-MRI contrast agents as biomarker-directed cancer imaging agents. Specifically, such conjugates can be used for the delivery of MRI contrast agents based on cell-surface carbohydrate biomarkers. In the R21 phase of this application, we plan to study the feasibility of this approach by (1) synthesizing boronolectin-MRI contrast agent conjugates using a boronolectin which is known selectively bind to sialyl Lewis X, (2) studying their ability to bind to cells with the target carbohydrate biomarkers, and (3) examining their ability to image implanted tumors in both an ex vivo and in vivo models. If the R21 phase is successful, in the R33 phase we plan to expand our biological evaluation to include tumors implanted at different positions, and to search for other lectin mimics that can bind specifically for other important carbohydrate-based cancer biomarkers. In addition, we also plan to examine the cytotoxicity of the boronolectin-MRI contrast agent conjugates. These small molecule-based recognition/delivery systems may have the following advantages over antibody-based systems: (1) greater stability during storage and in vivo; (2) lower propensity to elicit undesirable immune responses, (3) easier conjugation chemistry, and (4) more desirable pharmaceutical properties.
|Cancer Vertical Arrays
|One of the experimental challenges in cancer molecular biology is assessing the validity and generality of biomarkers. This has become a critical bottleneck in the development of biomarkers from differential gene expression revealed by microarray studies. In this proposal, we develop the concept of using vertical arrays for exploration of differential gene expression in cancer. Vertical arrays explore the expression of a gene in many biological samples simultaneously, whereas standard microarrays explore the expression of many genes in response to one biological variable at a time. Vertical arrays are like dot blots in this regard, but vertical arrays are printed on glass slides, giving them better signal-to-noise behavior, and, rather than spotting the entire complexity of the RNA population in each spot, the RNA population is divided up among multiple spots. These low complexity representations have superb signal-to-noise performance. The work in this proposal will focus on establishing the feasibility of making a vertical array for studying gene regulation in many cancer samples simultaneously. Potential throughput is very high, such that multiple regions from each tumor can be studied simultaneously. This approach will be useful in confirming that a gene is indeed differentially regulated, in determining the distribution of expression of the gene in the transformed and surrounding normal tissue, and in determining whether the gene behaves in a similar manner in different cases of the same type of cancer and in different kinds of cancer. The goals require extensive and efficient microdissection, and we have built a novel instrument, the "tissue mill," to achieve these ends. Relevance: Biomarkers are useful for diagnosis, prognosis, and as potential therapeutic targets for cancer. There are hundreds of potential biomarkers, but further validation is needed before they can be exploited.
|DXMS-Facilitated Membrane Protein Construct Design/Cancer
|Many cancer-implicated proteins are integral membrane proteins (IMPs). There is a pressing need for improved methods for the production of IMP constructs for use in high-resolution structure determination efforts. Three years ago, we completed a fifteen- year effort to develop methods for the performance of peptide amide hydrogen/deuterium exchange- mass spectrometry (DXMS). In collaboration with the Joint Center for Structural Genomics (JCSG), we recently demonstrated that DXMS can provide precisely the information needed to guide the design of well-crystallizing constructs of otherwise poorly-crystallizing soluble proteins. The NCI IMAT program is now funding our efforts to optimize DXMS-guided construct design for soluble cancer-implicated proteins (R33 CA099835). Until recently, we thought it unlikely that successful DXMS analysis of membrane proteins would be possible, and this funded grant contains no reference to membrane proteins (IMPs), nor does it support work on them. However, insights and preliminary studies described in the present application now make it likely that, with intensive development work, we can devise highly modified methods that will allow the facile DXMS analysis of IMPs. Development of membrane protein DXMS will greatly impact the structural biology of cancerimplicated IMPs, which are particularly difficult to prepare in crystallizable form. Initial year 1 development efforts will focus on the integrin allbbS, with which I have had considerable experience. Integrins are widely implicated in cancer cell and cancer vasculature biology, and findings with the prototypic allbbS integrin have proven applicable to the understanding of all integrins. The resulting IMPDXMS methods will be further refined and validated in year 2 through study of additional cancer- relevant IMPs and daughter constructs provided by Dr. Raymond Stevens, P.I. of the newly NIH-funded JCSG Center for Innovative Membrane Protein Technologies (JCIMPT). Once IMP- DXMS has been fully developed and validated, it will be made available to investigators studying cancer-implicated IMPs, by integrating the methods with our soluble-protein DXMS resource now supported by the NCI IMAT program. Thus the NCI's investment in presently funded DXMS work will be greatly leveraged by the relatively modest support requested for the development of IMP-DXMS.
|Fluorescent Activity Sensors in Analysis of Oncogenic P13K/Akt Signaling
|Activation of phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine kinase Akt (also known as protein kinase B) triggers a cascade of responses that are critical for tumorigenesis, from cell growth and proliferation to survival and mobility. Aberrations of components in the PI3K/Akt pathway have been shown to be present in a majority of tumors. We hypothesize that aberrant PI3K/Akt activation could be characterized by combined activity profiles and used as a diagnostic marker in cellular activity-profiling. To test this hypothesis, we propose the following specific aims: 1) To analyze the activities of PI3K and Akt in breast cancer cell lines and to further develop fluorescent activity sensors for various components in the PI3K/Akt pathway; 2) To develop cellular assay platforms for high throughput activity-profiling of oncogenic PI3K/Akt signaling. These studies will take advantage of a series of fluorescence resonance energy transfer (FRET)-based reporters we have recently developed for measuring the activities of Akt and PI3K in living mammalian cells. Fluorescent activity sensors and cellular assay platforms developed in this study can be used in systematic analysis of the critical components in PI3K/Akt pathway in various cancers to generate activity profiles. Correlation of genetic alterations with activity profiles and phenotypes should provide new insights into the molecular mechanisms of cancer development. On the other hand, molecular diagnostics based on such activity-profiling could identify the molecular defects and the malfunctioned key nodes in the signaling network for a given cancer, and guide appropriate molecular therapeutics as well as facilitate their development and evaluation.
|Integrated Microdevice to Capture and Detect Circulating Tumor Cells
|Metastasis is probably the most important event for determining outcome in cancer patients. The detection of occult metastases in the bone marrow, while known to be clinically important, has not become routine clinical practice. This is due to the technical difficulties and costs involved in the current methods for their collection and detection. Detection of circulating tumor cells (CTC) in the blood is less sensitive than in bone marrow and suffers from the same technical barriers as the detection of tumor cells in the bone marrow, but offers the distinct advantage of being less invasive and better for patient compliance. Therefore, sensitive detection of earliest metastatic spread of tumor in a minimally invasive and user-friendly manner will have a great impact on the clinical management of cancer patients. The currently available methodologies for CTC capture and identification face significant barriers including multiple procedural steps, substantial human intervention, extremely high cost, and importantly, lack of reliability and standardization for the detection methods. We have demonstrated the potential for sized-based tumor cell capture using a parylene-based micropore membrane. We propose to develop this into a microchip device for processing blood, and eventually bone marrow and other fluids like pleural effusions or ascites. This microdevice, coupled with microfluidics, has the potential to revolutionize the approach to tumor cell capture and identification. Further, we propose to develop methods for on-chip characterization of the captured cells. First, in R21 Phase, we will develop and optimize the capture device using a model system to isolate and molecularly characterize cultured cancer cells admixed in blood, followed by a pilot study to examine blood from 45 actual cancer patients with metastatic disease for breast, prostate or bladder cancer. In R33 Phase, we will extend the application of microdevice to assess about 310 patient samples from the same three malignancies, and we will also assess the molecular characteristics of the CTC using the Quantum Dots to understand the biological features of these otherwise rare cells (such as existence of putative stem cell sub-population which may be more malignant). At completion, studies in this project will develop a cost effective on-chip system for capture, identification, and characterization of CTC, easily usable in the clinical setting.
|Analysis of Tumorigenic Signaling Pathways with PROTACS
|A key part of determining the course of treatment for a specific cancer is the identification of the specific activated signaling pathways, which are causing the malignant growth. In fact the treatment for a given cancer can be dependent upon the activated signaling pathway; for example HER2/neu positive vs. negative breast cancers are treated differently. This personalized medicine approach is best exemplified by the development of the Abl tyrosine kinase inhibitor Gleevec, which has revolutionized the treatment of CML. As more drugs targeting specific signaling pathways are developed, it will be important to identify those oncogenic signaling pathways activated in a given tumor biopsy. Towards this end, our long-term goal is the development of a library of small molecules to be used as diagnostic tools for assessing primary cancerous tissue samples. We have recently developed a new technology known as PROteolysis TArgeting Chimera molecules (PROTACs) that can selectively knock down a specific protein in vivo. These cell permeable hetero- bifunctional molecules utilize the cells own ubiquitin/proteasome protein degradation pathway to selectively destroy a target protein of our choosing. We propose to adapt this technology so that proteins required for continued tumor growth are degraded only in those cells with a particular activated tyrosine kinase pathway. In this way, it will be possible to identify those signaling pathways upregulated in a particular tumor cell and which are required for its growth. Towards the goal of novel tumor diagnostic technology development, in the subsequent R33 application, we propose to develop a panel of PROTACs that can be used in identifying the activated cancerous cell signaling pathways. This panel will be tested for use as a diagnostic tool for determining the best course of drug treatment.
|Defining Secreted Glycan Alterations in Pancreatic Cancer
|The development of methods to accurately detect early pancreatic cancer and to better differentiate benign from malignant disease could greatly improve the outcomes for pancreatic cancer patients. It is known that malignant transformation of epithelial cells of the pancreas results in alterations in the carbohydrate chains of certain proteins secreted or released by these cells. Glycosylated proteins form the basis for current biomarkers for detecting pancreatic cancer and other adenocarcinomas, and refinement of these tests are predicted to enable detection of early pancreatic cancer. Our preliminary data has shown that a novel antibody-microarray technology allows the efficient detection of glycans on distinct proteins and the identification of specific glycan structures associated with pancreatic cancer. The method uses antibody microarrays to capture specific proteins from serum samples, followed by the incubation of a glycan-binding protein (such as a lectin) to quantify specific glycans on the captured proteins. Two classes of glycoproteins, mucins and carcinoembryonic-antigen-related proteins, are particularly associated with cancer, both in altered expression patterns and in altered glycan structures on the proteins. In the R21 phase, we will determine the levels of multiple specific glycans on members of those protein classes to test the hypothesis that the measurement of specific cancer-associated glycans on specific proteins, as opposed to measuring just protein or just glycan levels, will yield improved sensitivities and specificities for cancer detection. The R33 phase of the project will expand and thoroughly test the approach. The sensitivity and specificity of detecting pancreatic cancer using measurements of glycans on mucins, CEA proteins, and proteins identified in the R33 phase will be characterized in a large set of serum samples from subjects with pancreatic cancer, benign pancreatic disease, other cancers, and no disease. We expect to characterize the value of these measurements for disease diagnostics and to gain insights into the generality and frequency of specific glycan alterations on secreted proteins. Relevance to public health: The ability to more accurately diagnose cancers at earlier stages could lead to improved outcomes for many patients. This research could lead to significantly improved blood tests for the detection of cancer, as well as a powerful, generally- applicable platform for studying carbohydrate alterations on multiple proteins.
|Dissection of the Modular Structure of Cancer Signaling Systems
|The development of human cancer is a multistep process in which future cancer cells acquire mutant alleles of proto-oncogenes, tumor-suppressor genes, and other regulatory genes. Many or most of these genes are signaling related proteins and we are focusing here on the design principles of signaling networks that control the cancer related processes of proliferation, migration and endocytosis. We will test the key questions of 1) whether these cancer related signaling networks have a modular structure and 2) whether cancer cells have missing or added signaling modules that cannot be observed in normal cells. We have made significant advances to answer these questions by developing a method to create 2304 in vitro Dicer generated siRNAs against a core set of human signaling proteins. Using these siRNAs, we have already discovered the function of STIM1, a Ca2+ sensor in the ER lumen that controls Ca2+ influx into cells, and which also acts as a tumor suppressor. We have also developed quantitative microscopy- based measurement tools to track signaling processes and cell functions. Phase 1 of the proposal will demonstrate the overall feasibility of using a microscopy-based siRNA strategy to investigate multiple cancer-related cell functions. Phase 2 will address the questions posed above using an expanded set of 6000 siRNAs and a focus on six cell-types, 3 non-transformed and three breast cancer epithelial cell lines. We will screen to identify signaling siRNAs that alter proliferation, cell migration or endocytosis and then utilize follow-up studies with live cell biosensors that we developed to measure the duration of different cell cycle phases, as well as migration velocity and other kinetic parameters. We will then link genes that alter these cell functions to a subset of cancer-relevant signaling pathways using secondary siRNA screens. Based on these functional and signaling datasets, we will create a modular map of signaling systems using clustering methods. We will experimentally test the predictive power of modular maps using perturbations with pairs of effective siRNAs. We will show if and how modularity in a signaling system can be used to predict how cell functions can be manipulated using combinations of siRNAs and learn if and what distinguishing features exist that define modularity of signaling systems in cancer versus non-cancer cells. This will likely lead to the identification of new cancer drug targets and new therapeutic strategies.
|Real-time PCR expression profiling of microRNA
|microRNA is a newly discovered class of endogenous, small interfering RNA. MicroRNA binds to messenger RNA and translationally represses protein levels. While over 300 microRNAs have been discovered in humans alone, their biological function, targets, expression levels and role in disease remain largely unknown. A role between microRNA expression and carcinogenesis has been proposed. There is a lack of sensitive, high-throughput methodologies to monitor the expression of microRNAs. microRNA are challenging molecules to quantify because the microRNA precursors consists as a stable hairpin and the mature microRNA is only 22 nucleotides in length. We propose to evaluate sensitive and specific real-time PCR assays to quantify the expression of the mature and microRNA precursors. The microRNA expression will be analyzed in a number of important biological conditions relating to human cancer. The microRNA expression will be determined in specific sections of cancer and normal tissue isolated by laser-capture microdissection. The expression of mature and precursor microRNAs will be compared to using real-time PCR and a cDNA micro array. microRNA expression will be studied in clinical samples of human pancreatic cancer. The unparallel sensitivity and specificity of real-time PCR as applied to this new and exciting class of regulatory RNAs should propel the field into new directions not only in cancer but in other areas of human health.
|Tadpole Assays for the Molecular Assessment of Cancer
|Biologists have long known that cancer cells sometimes announce their presence by shedding certain molecules into the blood. More recently, many have come to believe that some cancers might be detectable by "signatures", patterns of sets of molecules, perhaps normally present in the blood, but, in for certain cancers, present in higher or lower amounts than normal. Recently, we learned to make new kinds of molecules, which we call "tadpoles". We have demonstrated that we can use them to detect and count small numbers of molecules. These assays are relatively simple and relatively inexpensive, and they should be applicable to both kinds of cancer detection. During the next 3 years, we seek funding to develop and "harden" these assays to the point that they can be tested in clinical cancer diagnosis.
|An RNA Sensor for Detection of Circulating Tumor Cells
|This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon an hybridization "sandwich". Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-,) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a 2nd library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (R??) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R??) will initially be detected optically). We have successfully measured R? of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable. Preliminary data related to all aspects of RNA Sensor development have been obtained. These include: library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity "off-chip" synthesis and derivatization of nanowires, R?. measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.
|Microarray Platform for Profiling Cancer Proteases
|Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered question. Indeed, proteases are regulated by a complex series of posttranslational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes. To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody-based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33 application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.
|Quantitative proteomic analysis of lymphoma transformation
|Non-Hodgkin's lymphoma accounts for approximately 50,000 new cases of cancer annually. This figure represents an increase beyond that seen for most other forms of cancer. Among the non-Hodgkin's lymphomas, follicular lymphoma represents the most common subtype of low-grade B lymphoma in adults, and typically pursues an indolent clinical course. In a significant proportion of cases there is histologic transformation from a low-grade neoplasm to an aggressive diffuse large B lymphoma with significantly decreased median survival. The recent advent of sophisticated mass spectrometry technology coupled with software algorithms that permit instantaneous protein identification, makes it feasible to study the pattern of protein deregulation that distinguish two biologic states. We propose to employ a combination of chromatographic techniques and tandem mass spectrometry in the identification of the alterations in protein expression that accompany histologic transformation. We shall be analyzing a cohort of matched pairs of follicular lymphoma and their transformed diffuse large B lymphoma counterparts occurring in the same individual. Relevance: Comprehensive identification of the qualitative and quantitative changes in protein expression that are involved in follicular lymphoma transformation will permit the delineation of deregulated pathways, identify distinct prognostic subgroups of transformed lymphoma, and facilitate the development of novel therapies that target susceptible elements in the deregulated pathways.
|Applications of "Recombomice" for Cancer Research
|Applications of "Recombomice" for Cancer Research Every time a cell divides, billions of base pairs of information must be accurately copied in the face of an onslaught of DNA damage. Homology directed repair (HDR) provides one of the most important mechanisms for coping with damaged DNA. If coding information is missing or corrupted, HDR can extract sequence information available elsewhere in the genome. Although HDR is generally beneficial, transfer of genetic information is risky, since misalignments can lead to tumorigenic rearrangements. To investigate the process of HDR in vivo, we have engineered the first mouse model in which HDR can be detected in somatic cells by the appearance of a fluorescent signal. In the fluorescent yellow direct repeat (FYDR) recombomice, recombination at an engineered substrate yields fluorescence. Recombination assays are simple and rapid, making it possible to do in days what used to take weeks. In addition, the FYDR mice overcome limitations of previous systems. For example, although APRT mice can be used to detect loss of heterozygosity, technically demanding assays are necessary to identify HDR events; in the pun mice, only embryonic recombination events can be detected. In contrast, FYDR mice yield a fluorescent signal that is specific to HDR events, and the recombination rate can be readily measured in cells from both embryonic and adult tissues. Furthermore, fluorescence makes it possible to capture in situ images of recombined cells, making it possible to discern independent lineages of recombinant cells in vivo. Our Specific Aims are to I) Evaluate the frequency of recombinant cells in multiple tissues; II) Develop methodology for quantification of recombinant pancreatic cells in situ and reveal the relative frequency of recombinant cells among two different cell types within a normal tissue for the first time; III) Measure the effects of environmental factors on recombination in vivo; and IV) Reveal how specific genes (Blm and p53) affect recombination susceptibility in vivo. The broad long term objectives of this work are to demonstrate the utility of this newly developed technology for studying recombination in mammals, to substantially expand the capabilities of the existing system, and to elucidate environmental and genetic factors that influence a person's susceptibility to spontaneous, environmentally-induced, and cancer therapy-induced DNA rearrangements.
|Novel Image-Based Screening of Mammary Tumors
|Breast tumors are detected by self exam, clinical exam, and mammogram and suspicious lesions are biopsied. The ensuing histological classification plays a determining role in the treatment decision but the associated risk of malignancy, the appropriate treatment, and the risk of reoccurrence are difficult to determine. As a consequence, patient treatment is based on epidemiological findings rather than individual needs. Non- invasive imaging provides some information but early breast cancer detection by routine screening is not currently feasible. However, once a tumor has been detected and biopsied, there is urgent need for novel methods to aid in the detection and classification of sub-classes of lesions. One key epigenetic marker of cell phenotype is the organization of nuclear proteins which direct and reflect normal cell function. Based on the hypothesis that the redistribution of chromatin-related proteins reflects changes in gene expression that accompany alterations in cell phenotype, we have developed image analysis methodologies to quantify the nuclear distribution of specific chromatin-associated proteins from three-dimensional, high-resolution, fluorescently immunostained images. By applying these methods to culture models that mimic normal and malignant breast epithelial tissue, we have demonstrated that the distribution of nuclear mitotic apparatus protein (NuMA) and heterochromatin related protein histone-4 methylated on lysine-20 (H4-K20m) are biomarkers capable of clearly distinguishing non-neoplastic and malignant human mammary epithelial cells. The goal of this project is to quantify the distribution of specific nuclear proteins in culture models that mimic premalignant and malignant tissue to uncover epigenetic characteristics of premalignant disease. Our image-based methodologies will be expanded to produce a novel technique, the phenotype tissue-map, capable of resolving local tissue phenotype at cellular resolution and uncovering subtle differences in tissue morphology and behavior. The technology, which will work alongside the usual H&E staining and histological techniques, will be tested on needle-core biopsies of a variety of premalignant tumors with the aim of defining sub-classes of graded lesions. The results will be correlated with the histopathology of the initial needle-core and the follow-up surgical biopsy with the hope of predicting more aggressive phenotypes missed by the initial screen. The future public health benefit of this research is to aid the treatment decision process of breast cancer patients. By better understanding the organization of molecular components within the cell and how the organization of these components is altered during the progression to cancer, we can develop and provide pathologists with novel image analysis tools to aid and support the histological classification of biopsied breast tissue.
|Microdevice for Direct DNA Purification
|With an ever-increasing interest in the molecular typing of cells from histologic tissue sections, the ability rapidly and efficiently extract DNA from the selected cells will be of paramount importance. The overall goal of this project is to develop a microchip-based sample preparation method for high efficiency, low-cost extraction of DNA from tissue samples - this microdevice will easily accommodate blood or other cells sources. The microdevices will be created using state-of-the-art microfabrication techniques coupled with fluidically controlled on-chip cell lysis and solid phase extraction chemistries. This project couples the industrial capabilities of HT Micro for facile fabrication of complex, high surface area microstructures, with surface modification chemistries developed at the UNIVERSITY of Virginia that enable efficient and high capacity DNA extraction. The microdevices will be tested using a variety of sample varying in type and quality, and extraction efficiency will be determined using real-time PCR. As a demonstration of integration with current laser-capture microdissection instruments, the microdevice will be fabricated to directly accept the cap from the Arcturus Pixcell IIe which will contain the selected cells bound to an ethylene vinyl acetate polymer membrane on its bottom surface. The tissue samples will be collected and laser microdissected by our surgical pathology collaborator here at UVa - samples of normal and malignant cells will be analyzed. The final device will offer the rapid analysis, high extraction efficiency, and high-throughput advantages of microdevices and, in addition, is expected to offer higher capacity, and lower cost-per-device than current conventional or microchip techniques.
|Technology/Map Endothelial Targets/Human Renal Tumors
|The molecular complexity and in vivo inaccessibility of most tumor cells within solid tumors can greatly limit genomic- and proteomic-based discovery of useful targets for tumor-specific imaging and therapeutic agents in vivo. To overcome endothelial cell (EC) barriers and achieve more effective targeting and penetration into solid tumors, we shift analytical focus from the tumor cell to the vascular EC surface and its caveolae in direct contact with the circulating blood. To reduce data complexity to a meaningful subset of targetable proteins expressed on the EC surface, we will use tissue subular fractionation, novel multimodal mass spectrometric analysis, in silico subtraction, and bioinformatics interrogation of structure and function to unmask, from the >100,000 proteins in the tissue, those few intravenously accessible proteins differentially expressed on vascular endothelium in human renal tumors. This technology and overall approach has been validated in rodent solid tumors whereby new vascular targets have been uncovered permitting tumor-specific imaging, penetration, and effective radio immunotherapy (Nature, 429:629-35, 2004). But, currently very little is known about the expression of proteins in tumor neovascular endothelium, especially in human tissue. We now wish to apply our new technology to map comprehensively the proteome of luminal EC surfaces and caveolae in human renal tumors in vivo. It is likely that human tumors will express a different constellation of proteins on tumor neovasculature not yet uncovered or induced in animal models. To this end, we propose the following specific aims: 1) To use novel tissue sub fractionation and proteomic analytical approaches to map comprehensively vascular EC surfaces and caveolae in human renal tumors vs. matched normal renal tissue to unmask candidate tumor-induced/associated vascular proteins. 2) To create new antibodies to newly discovered human renal tumor EC targets and to use antibodies as probes to validate the expression of tumor-induced/associated proteins at the EC surface and its caveolae in human tissues and thereby to assess the degree of target specificity for the neovasculature of human solid tumors. Such mapping may also elucidate the effects of the tumor on the developing vascular endothelium and yield important tumor-specific vascular targets for improving noninvasive diagnostic imaging and therapy as well as yield new diagnostic and prognostic markers for the molecular classification of tumor biopsies


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of the Alphabet. Many centuries later, these were superseded by a species of Hieratic Charac- ter, the components of which were known to the Monks of the Middle Ages under the name of Neumae. The final stage of perfection was reached, when these last were developed into the characters now called Notes, and written upon the Lines and Spaces of the Stave.
The Greeks made use of Uncial Letters, inter- mixed occasionally with a few Minusculae, and written in an endless variety of different posi- tions upright, inverted, lying on the right or left side, divided in half, placed side by side, and otherwise grouped into some hundred and twenty well-marked combinations, which, with more than a thousand minor variations, have been so clearly described by Alypius, Aristides Quintilianus, and other Hellenic writers, that, could we but obtain authentic copies of the Hymns of Pindar, or the Choruses of Sophocles, we should probably find them easier to decypher than many mediaeval MSS. 1
When Greece succumbed beneath the power of Western Europe, Roman Letters took the place of the more archaic forms, but with a different application ; for, while the details of Greek No- tation were designed with special reference to the division of the system into those peculiar Tetrachords which formed its most prominent characteristic, the Roman Letters were, at a very early period, applied, in alphabetical order, to the Degrees of the Scale a much more simple arrangement, the value of which is too well known to need comment. Boethius, writing in the 6th century, sanctioned the use of the first fifteen Letters of the Roman Alphabet, for certain special purposes. This number was afterwards reduced to seven it is not easy to say by whom. 8 Tra- dition ascribes the first use of the lesser number to S. Gregory, but on very insufficient grounds ; though the reactionary idea that he was unac- quainted with the Alphabetical System, cannot for a moment be entertained. 3 It is certain that Letters were used, for many centuries, in the Notation of Plain Chaunt, in the West ; just as the use of the Greek Characters was retained in the Ofiice-Books of the Eastern Church. After the 8th century, though they rarely appeared in writing, the Degrees of the Scale were still named after them. AB symbols of these Degrees, they have never been discarded. Guido used them, in the 1 1 th century, in connection with the Solmisa- tion of the Hexachords ; though their presence, as written characters, was then no longer needed. The first eight, indeed, lived on, in a certain way, until quite recent times, in the Tablature for the Lute, which always claimed a special method
1 The authenticity of the three Hymns, printed. In 1581, by Yln- eenzo Galilei, rests on such slender grounds, that it would be ex- tremely unsafe to accept them as genuine.
2 The teptem ditcrimina vocum of Virgil (.En. vl. 645) have been supposed to allude to these seven letters ; and the context certainly suggests some possible connection with the subject.
3 Though discussion of Individual authorities is quite foreign to the purpose of the present article, it may be well to observe, that, within the last five years, a well-known Belgian writer F. A. Gevaert has advanced certain opinions connected with thfi subject of antient Notation, very much at variance with those of most earlier Historians. The reader will find Mons. Gevaert's Tlews fully explained in his Hlttoire et TMorie dc la Musique dans l-Antlquite/ Paris. 1876.
��of its own. This, however, was an exceptional case. Long before the invention of the Stave, the system came virtually to an end : and, in our own day, it survives only in the nomenclature of our notes, and the employment of the F, C, and G Clefs. [See HEXACHORD, TABLATURE.]
Though wanting neither in clearness nor in cer- tainty, this primitive system was marred, through- out all its changes, by one very serious defect. A mere collection of arbitrary signs, arranged in straight lines above the poetical text, it made no attempt to imitate, by means of symmetrical forms, the undulations of the Melody it repre- sented. To supply this deficiency, a new system was invented, based upon an entirely different principle, and bringing into use an entirely new series of characters, of which we first find well- formed examples in the MSS. of the 8th cen- tury, though similar figures are believed to have been traced back as far as the 6th. These cha- racters consisted of Points, Lines, Accents, Hooks, Curves, Angles, Retorted Figures, and a multi- tude of other signs, or l Neumae, placed, more or less exactly, over the syllables to which they were intended to be sung, in such a manner as to indicate, by their proportionate distances above the text, the places in which the Melody was to rise or fall. Joannes de Muris mentions seven different species of Neumae. A MS. preserved at Kloster Murbach describes seventeen. A still more valuable Codex, once belonging to the Monastery of S. Blasien, in the Black Forest, gives the names and figures of forty : and many curious forms are noticed in Era Angelico Ottobi's Calliopea leghale (written in the latter half of the 1 4th century), and other similar works. The following were the forms most commonly used ; though, of course, mediaeval caligraphy varied greatly at different periods.
��1. The Virgo, indicated a long single note, which was understood to be a high or a low one, according to the height of the sign above the text. A group of two was called a Bivirga, and one of three, a Trivirga representing two and three notes respectively.
2. The Punctus indicated a shorter note, sub- ject to the same rule of position, and of multipli- cation into the Bipunctus, and Tnpunctus.
3. The Podatus represented a group of two
1 From i'<i'ij.a, a nod, or sign ; or, as some have supposed, from irvevnia, the long succession of notes sung after a 1'lalu Chaunt Alleluia.'
�� �

subset: commoncrawl


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James Zabiela
BiographyTechnical wunderkind James Zabiela is the sort of DJ that makes other DJs nervous. He also serves as a reminder that there is more to the art of deejaying than mixing records, but more on that later. His musical foundations are solid -- working from a young age in his father's record shop, he had access to everything and time to mess with the equipment. By the age of 18 he was already the chief buyer in the house department, and had taken to handing out mixes to any names that happened to drop in. Legend has it that his big break came when he handed a mix to Lee Burridge, who then gave it to Sasha, who asked him to join his Excession agency. This however is only part of the story, since the now- defunct Muzik magazine also claimed him as their discovery when he won their "Bedroom Bedlam" amateur mix competition. Either way, his rise up through the ranks was rapid and impressive and he was soon to be seen playing all over the world, writing about his experiences in the "Zabiela we feel ya" Muzik magazine column and being called in for remixing duties for Virgin Souls and RÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂÃÂöyksopp, amongst others.
His rapid success was well deserved -- he clearly loves doing this. The ability to strip down tracks to bar-long loops, reverse them, tweak them, pitch bend and create something completely new obviously thrills him and his joy at the endless malleability of this music generates an infectious enthusiasm within his audience. His technical skill is virtually unmatched anywhere else in the DJ world, with one famous global name claiming to "want to throw up" when he first saw Zabiela rip into his set. To this day, the trainspotters will crowd the decks just to see his hands fly around the equipment.
Mixing abilities are only part of the story however, and as any seasoned DJ or clubber knows, it's all about the programming. The relative lack of time spent on the dancing side of the decks showed in Zabiela's early performances, but it wasn't a problem as he was usually opening for Sasha and didn't really need to seriously bang it out. This was a distant memory by 2005, where his breathtaking performance at the BBC Radio1 party during the annual Winter Music Conference showcased his ability to send a dancefloor into a frenzy. The bravura display was also helped by the inclusion of his track "Robophobia" (2005) -- a seductive blend of driving house, complex beats and catchy vocals -- which he placed before the Buick Project remix of an old KLF classic, a masterful piece of mixing and programming that confirmed his status as one of the great all-rounders in the DJ world. Other DJs should remain nervous -- Zabiela is only just getting started.


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|age (eɪdʒ)
|the period of time that a person, animal, or plant has lived or is expected to live: the age of a tree; what age was he when he died?; the age of a horse is up to thirty years
|the period of existence of an object, material, group, etc: the age of this table is 200 years
|a. a period or state of human life: he should know better at his age; she had got beyond the giggly age
|b. (as modifier): age group
|the latter part of life
|a. a period of history marked by some feature or characteristic; era
|b. (capital when part of a name): the Middle Ages; the Space Age
|generation: the Edwardian age
|geology, palaeontol
|a. a period of the earth's history distinguished by special characteristics: the age of reptiles
|b. the period during which a stage of rock strata is formed; a subdivision of an epoch
|myth any of the successive periods in the legendary history of man, which were, according to Hesiod, the golden, silver, bronze, heroic, and iron ages
|informal (often plural) a relatively long time: she was an age washing her hair; I've been waiting ages
|psychol achievement age See also mental age the level in years that a person has reached in any area of development, such as mental or emotional, compared with the normal level for his chronological age
|age before beauty (often said humorously when yielding precedence) older people take precedence over younger people
|of age adult and legally responsible for one's actions (usually at 18 or, formerly, 21 years)
|—vb , ages, ageing, aging, aged
|to grow or make old or apparently old; become or cause to become old or aged
|to begin to seem older: to have aged a lot in the past year
|brewing to mature or cause to mature
|[C13: via Old French from Vulgar Latin aetatīcum (unattested), from Latin aetās, ultimately from aevum lifetime; compare
|—suffix forming nouns
|indicating a collection, set, or group: acreage; baggage
|indicating a process or action or the result of an action: haulage; passage; breakage
|indicating a state, condition, or relationship: bondage; parentage
|indicating a house or place: orphanage
|indicating a charge or fee: postage
|indicating a rate: dosage; mileage
|[from Old French, from Late Latin -āticum, noun suffix, neuter of -āticus, adjectival suffix, from -ātus
age (āj)
The length of time that one has existed; duration of life. v.
To become old.
To manifest traits associated with old age.
acute gastroenteritis
used to denote the period of a man's life (Gen. 47:28), the maturity of life (John 9:21), the latter end of life (Job 11:17), a generation of the human race (Job 8:8), and an indefinite period (Eph. 2:7; 3:5, 21; Col. 1:26). Respect to be shown to the aged (Lev. 19:32). It is a blessing to communities when they have old men among them (Isa. 65:20; Zech. 8:4). The aged supposed to excel in understanding (Job 12:20; 15:10; 32:4, 9; 1 Kings 12:6, 8). A full age the reward of piety (Job 5:26; Gen. 15:15).
see act one's age; coon's age; golden age; in this day and age; of age; ripe old age; under age.


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LuxorPro Style Stix 7/8" Purple
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Carolann F.Asking About Hair RodsI purchased the LexorPro style stix, in two different sizes,, what I want to know is,,Can I use them for perm rods also?? which is a question,, I should have asked in the first place, , but did'nt !~~~~ please respond,,~thank you~~~1 out of 1 shoppers thought this post wasn't helpful.
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Patrice E.Hazleton,paDid You Get A Response To Your Question?Can you use the style stix for perming? Did you? And if so how did it come? Did you like the results?
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One might ask what is a computer check? A computer check is simply are printable checks for your business. They are functional and very convenient to use. You simply print them on your computer. You can also have custom computer checks by printing the company's logo into the check. This allows the receiver to have all the information needed to process the payment. One of the main advantages of having computer checks is that you will no longer have to handwrite the checks you have. These checks are easily readable. There is also no hassle in printing these customer computer checks since software is already provided.
Are Computer Checks Safe?
Computer checks work hand in hand with business software. This software is basically a package that will help you handle your business better. There is no need for you to worry about the security for your checks as they are created with all the security measures as one would do in a bank. There is no way anyone can copy these computer checks. For custom computer checks, you can easily modify your check to make it look extravagant or you can simply print out the basic design.
Where and How To Buy Computer Checks?
In buying computer checks, you need to find the cheapest prices without compromising the quality of both the security and the product itself. When looking for a good computer check, make sure that you have found a trustworthy website. They often have testimonials of past clients posted in front of their website. You can research these clients and call them if you want to. Another thing you need to know is that they should offer a lot of payment options. The more options the customer has, the more likely they are accommodating to you. This means that they handle their customers well and that they provide convenience to any type of customer who have different payment methods. Make sure to choose a website that offers the maximum customer service. Ask them if they have an overnight shipment of the computer checks or if they have emergency options for those who want to have custom computer checks right away. Make sure that each transaction that you have is smooth and easy to do as well.
How Computer Checks Affect Your Business
Every piece of printed paper that comes out from your business tells your customer about the kind of business you have. If you have a good custom computer check with intricate design, chances are your customers will most likely be amazed at how much detail and how much effort you put into such design. You should also help them realize that computer checks are safe and effective methods of financial transactions.
In business, image and reputation is everything. When you have a good image, you are most likely to be trusted by a lot. This is why you need to maintain a good image with your business by having custom computer checks to let your customers know that you treat them well as well as their finances.
Author Bio:
Victor Evans works at Techchecks. TechChecks is the easiest, fastest and quickest way to order checks online whether for your home or office. They provide Computer Checks, Business Checks, Intuit Checks and many more at the Lowest Cheap Checks Prices.


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Botox injections in Atherton, Los Gatos and Palo Alto offered by cosmetic surgery specialist Dr Struck. Information on non-surgical treatments, dermal fillers, injectables, skin rejuvenation and more.
Botox can be used to treat facial wrinkles such as crow's feet and forehead creases. It works by weakening the muscles that cause large wrinkles. Botox® takes effect several days after treatment and lasts approximately four months. We can administer Botox to patients in Atherton, Los Gatos, Morgan Hill, Palo Alto, Los Altos, Cupertino, Saratoga, San Jose, and the larger Bay area. We have three offices to serve you.
Botox® treats areas of the face that often do not respond well to other therapy. It can be combined with different treatments for maximal rejuvenation, such as using laser therapy to minimize finer wrinkles. An advantage of botox therapy is that you can have your treatment and return immediately to your regular schedule.
Frequently Asked Questions about Botox®
Q: How long does it take for botox to take effect?
Q: How long does botox last?


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Roast Chicken with Pomegranate Glaze
From EatingWell.com
This sweet-tart pomegranate molasses glaze is delicious combined with the citrusy sumac spice rub, and gives the roasted chicken a dark, ruby luster. Rub the chicken earlier in the day, or overnight, for the best flavor.
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Nutritional Information
(per serving)
|Total Fat
|Saturated Fat
|Total Carbohydrate
|Dietary Fiber
Ken Burris
Serves: 6
Yields: 6 servings
Total Time: 1 hr 30 min
Prep Time: 30 min
Oven Temp: 425
|Conversion chart
- 1 tablespoon(s) ground sumac (see Tips)
- 1 tablespoon(s) kosher salt
- 1 (4-pound) chicken
- 6 cup(s) (2-3 large bulbs) sliced cored fennel
- 1 large yellow onion, chopped
- 2 teaspoon(s) extra-virgin olive oil
- 1/2 cup(s) pomegranate molasses (see Note)
- 2 tablespoon(s) honey
- 1 teaspoon(s) freshly ground pepper
- Pomegranate seeds for garnish (see Tips)
- Preheat oven to 425 degrees F.
- Combine sumac and salt in a small bowl. Remove giblets from chicken (if included) and trim any excess skin; pat dry. Loosen the skin over the breast and thigh meat and rub the spice mixture under the skin plus a little on the skin. Tuck the wings under and tie the legs together with kitchen string, if desired.
- Combine fennel and onion in a large roasting pan and toss with oil to coat. Place the chicken, breast-side up, on the vegetables.
- Combine pomegranate molasses, honey, and pepper in a small bowl. Transfer half the mixture to a small saucepan and set aside to serve with the chicken.
- Roast the chicken and vegetables for 20 minutes. Turn the chicken over, stir the vegetables and cook for 20 minutes more.
- Turn the chicken over one more time (so it is breast-side up) and stir the vegetables again. Reduce oven temperature to 400 degrees F. Brush the chicken all over with the remaining pomegranate mixture, and continue to roast until an instant-read thermometer inserted into a thigh without touching bone reaches 165 degrees F, 20 to 30 minutes more.
- Transfer the chicken to a clean cutting board and let rest for 10 minutes. Meanwhile, heat the pomegranate glaze in the saucepan over low heat. Remove the string from the chicken, if necessary, and carve the chicken. Serve with the fennel and onion, drizzled with the warm glaze. Sprinkle with pomegranate seeds, if desired.
Exchanges: 1 vegetable, 1 carbohydrate (other), 5 lean meat Carbohydrate Servings: 1 1/2 Nutrition Bonus: Vitamin C (22% daily value), Potassium (20% dv), Zinc (18% dv).
Tips & Techniques
Tips: The tart red berries of the Mediterranean sumac bush add fruity, sour flavor to many regional dishes. Find ground sumac in Middle Eastern markets, specialty food shops, and online at penzeys.com. Or use 2 teaspoons freshly grated lemon zest in place of the sumac. To seed a pomegranate, fill a large bowl with water. Lightly score the fruit into quarters from crown to stem end, cutting through the skin but not into the interior of the fruit. Hold the fruit under water, break it apart, and use your hands to gently separate the plump seeds (arils) from the outer skin and white pith. The seeds will drop to the bottom of the bowl and the pith will float to the surface. Discard the pith. Pour the seeds into a colander. Rinse and pat dry. Seeds can be frozen for up to 3 months.
Note: Pomegranate molasses has a bright, tangy flavor. (Don't confuse it with grenadine syrup, which contains little or no pomegranate juice.) Find it in Middle Eastern markets and some large supermarkets near the vinegar or molasses. To make your own: Simmer 4 cups pomegranate juice, uncovered, in a medium nonreactive saucepan over medium heat until thick enough to coat the back of a spoon, 45 to 50 minutes. (Do not let the syrup reduce too much or it will darken and become very sticky.) Makes about 1/2 cup. Refrigerate in an airtight container for up to 3 months.
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|Dividend Growth Is A Differentiating Factor In Long-Term Stock Performance
Dividend Growth Is A Differentiating Factor In Long-Term Stock Performance
Dividends have made a comeback from a period during the 1990s when investors prized capital growth more than dividends. Several factors will significantly affect dividend growth going forward, explained Judith Saryan, portfolio manager of Eaton Vance Utilities Fund.
The significant impact of dividends on the total return of stocks over time is another factor. From 1926 to 2005, over 40 percent of the total return of stocks came from dividends, according to Ibbotson Associates and Standard & Poor's(r) index service 2005.
by Tom Johanson


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The Sun might seem like a strange place to hunt for dark matter. The Sun is a ball of superhot gas that produces energy galore. Dark matter, on the other hand, produces no energy at all. We know it's there only because it exerts a gravitational pull on the matter that we can see. And there's a lot of it -- several times more dark matter than visible matter.
So far, no one's sure just what dark matter is. But one idea says it may consist of axions -- subatomic particles with almost no mass. If the idea's correct, enormous amounts of axions were created shortly after the Big Bang, and today they fill the universe.
Axions may also be created in the hearts of stars -- and that's where the Sun comes in.
The theory says that nuclear reactions in the Sun's core convert X-rays into axions. As they zip out into space, some of the axions may be converted back into X-rays by the Sun's powerful magnetic field. A team of astronomers is using orbiting X-ray telescopes to look for that energy, which would look different from other X-rays produced by the Sun.
Some of the axions may make it all the way to Earth's surface. A couple of teams are searching for those axions with special telescopes that use superconducting magnets to convert the axions to X-rays.
So far, the searches have turned up empty, but they're still going on. At the same time, other astronomers are searching for axions in other stars. We'll have more about that tomorrow.
Script by Damond Benningfield, Copyright 2009
For more skywatching tips, astronomy news, and much more, read StarDate magazine.[3]


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Discover new entertainment. For your family.
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- The Greenbacks Gal - http://thegreenbacksgal.com -
Seventh Generation Dish Soap Just $2.32 Shipped
Posted By Andrea Green On February 12, 2013 @ 3:27 pm In Daily Deals and Green Steals | No Comments
Are you in need of dish soap? This is a great deal for Seventh Generation Dish Soap [1].
First, you need to clip the coupon for $3.00 off. That will drop your price from $19.14 to just $16.14.
Next, select Subscribe & Save to deduct another 5%. Your total will then be $13.93 for a pack of 6. That makes each bottle just $2.32 each – lower than the Target price cut where bottles are $2.66.
Per Amazon's own policy, you can cancel Subscribe & Save at any time. Simply log-in to your account and select Manage Subscriptions to cancel.
Be sure to check out the rest of the Amazon Coupons. [2]
Thanks Maven of Savin [3].
Article printed from The Greenbacks Gal: http://thegreenbacksgal.com
URL to article: http://thegreenbacksgal.com/seventh-generation-dish-soap-just-2-32-shipped/
URLs in this post:
[1] Image: http://bit.ly/Y7qqet
[2] Amazon Coupons.: http://bit.ly/12cUBZn
[3] Maven of Savin: http://www.mavenofsavin.com/2013/02/seventh-generation-dish-liquid-25oz-2-32-shipped.html
Click here to print.
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