The Role of Fibrillin-1 in Eye Development and Disease

The ciliary zonule of the human eye consists of a circumferential array of fibers that connect the ocular lens to the nonpigmented ciliary epithelium (NPCE) located at the inner wall of the eye. Zonular fibers consist of bundles of beaded filaments called microfibrils. Microfibrils are major structural elements of the extracellular matrix and are present in pure form in the ciliary zonule. Microfibrils are composed principally of fibrillin-1 (FBN-1); a large extracellular matrix glycoprotein. In humans, mutations in FBN1 underlie Marfan syndrome; a pleiotropic connective tissue disorder that profoundly affects the eye. Ocular manifestations include ectopia lentis (dislocated lenses), cataracts, glaucoma and axial myopia. The ocular phenotypes in Marfan syndrome suggest an important role for FBN-1 in eye development. In this report, I used mice as a model system to test the role of FBN1 in eye development and disease with an emphasis on understanding the role of FBN-1 in the synthesis of the ciliary zonule. Our lab has shown that the organization and composition of the mouse ciliary zonule is similar to humans. A recent proteomic study identified FBN-1 as the main component of the ciliary zonule, accounting for 60% to 70% of total zonule protein. In order to understand the role of FBN-1 in eye development, I had to first identify the cells in the eye responsible for expressing FBN-1 and other core components of the zonule (FBN-2, MFAP2, LTBP2 and ADAMTSL4). I used in situ hybridization on wild type mouse eye tissue to show that some zonule components (Fbn1, Fbn2, Ltbp2, Mfap2) are expressed by cells of the NPCE while others (Adamtsl4, Mfap2) are expressed by the lens. This suggests that the ciliary zonule is synthesized by both tissues. Immunofluorescence experiments on adult wild type mouse eyes using antibodies against core zonule proteins showed that the ciliary zonule is heterogeneous in composition and nonuniform along the length of fibers suggesting that the spatial distribution of proteins in the ciliary zonule could reflect the temporal expression of zonule components during eye development. The identification of NPCE cells as the likely source of FBN-1 in the zonule allowed me to test the contribution of Fbn1 directly. I used a conditional knockout approach to delete Fbn1 in the nasal and temporal regions of the mouse NPCE. Three-dimensional reconstructions of conditional Fbn1 knockout mouse eyes (Fbn1-NPCE) demonstrated that the zonule was produced in the nominal absence of its most abundant component. However, the Fbn1-NPCE mice developed ectopia lentis; the major ocular manifestation and diagnostic criterion for MFS in humans. Given the clinical importance of the disease, I decided to investigate the structural and biomechanical changes leading to ectopia lentis in the Fbn1-NPCE mouse model. Ultrastructural studies on FBN-1 deficient mouse eyes using scanning electron microscopy revealed that Fbn1-NPCE mice had significant changes in zonular fiber density and thickness. Biomechanical testing on wild type and Fbn1-NPCE mouse zonules showed that FBN-1 deficient zonular fibers were significantly weaker (~50%) than their wild type counterparts. Conditional deletion of Fbn1 in the mouse lens (Fbn1-lens) had no discernible effect on the structure or function of the ciliary zonule, suggesting that FBN-1 protein synthesized by the lens is not required for the stability of the ciliary zonule. Together, these data show that the ectopia lentis phenotype in Fbn1-NPCE mice is secondary to changes in ciliary zonule ultrastructure and biomechanical properties and that these effects may give some insights to the mechanism of ectopia lentis in Marfan syndrome. In addition to ectopia lentis, Fbn1-NPCE mice express other ocular phenotypes commonly associated with Marfan syndrome including cataracts, increased axial length and, in rare cases, glaucoma. The data presented in this report provide insights into the role of FBN-1 in eye development and the biology of the ciliary zonule. In addition, the development of the Fbn1-NPCE mouse model will serve as a platform for understanding the role of FBN-1 in ocular disease associated with Marfan and related syndromes

Johnson County Food Education Program

If consumers are better informed about access to local foods as well as ways to prepare these foods, they may be more likely to seek out locally produced food

Establishment of a local food system in eastern Iowa

Several strategies to enhance local food production and marketing were employed by the Johnson County (IA) Soil and Water Conservation District. Among them were a pilot project on institutional buying practices, a directory of local food products, planning of locally sourced All-Iowa meals, and other educational and outreach activities

Community Guide to Agriculture (Johnson County)

More than 1,000 people have been reached though the various Johnson County Guide to Agriculture programs. Among the outreach efforts were workshops, displays, farm and farm business tours, and programs on Iowa City\u27s public cable channel

The Public Papers of Governor Wendell H. Ford, 1971-1974

This volume presents a record of the Ford administration. From among the many public speeches delivered by Wendell Ford during the three years he served as Governor, W. Landis Jones has chosen a representative sample that reflects the wide-ranging concerns of the Ford administration. Arranged topically, the volume covers subjects from government reorganization and party politics to health and welfare, education, highways, and energy and environment. This selection does not include executive orders or proclamations, since they are part of the preserved public record. The cross section of public speeches and press releases that are included provides an easily accessible source for historians to view the broad spectrum of issues that faced the people of the Commonwealth during the early years of the 1970s. Included also are appendixes that provide a complete listing of speeches delivered by Governor Ford during his term of office, a chart that shows the organization of Kentucky government at the end of the Ford administration, and a synopsis of the administration by Thomas L. Preston. W. Landis Jones is Professor of Political Science at the University of Louisville.https://uknowledge.uky.edu/upk_political_science_papers/1012/thumbnail.jp

Learning from microarray interlaboratory studies: measures of precision for gene expression

<p>Abstract</p> <p>Background</p> <p>The ability to demonstrate the reproducibility of gene expression microarray results is a critical consideration for the use of microarray technology in clinical applications. While studies have asserted that microarray data can be "highly reproducible" under given conditions, there is little ability to quantitatively compare amongst the various metrics and terminology used to characterize and express measurement performance. Use of standardized conceptual tools can greatly facilitate communication among the user, developer, and regulator stakeholders of the microarray community. While shaped by less highly multiplexed systems, measurement science (metrology) is devoted to establishing a coherent and internationally recognized vocabulary and quantitative practice for the characterization of measurement processes.</p> <p>Results</p> <p>The two independent aspects of the metrological concept of "accuracy" are "trueness" (closeness of a measurement to an accepted reference value) and "precision" (the closeness of measurement results to each other). A carefully designed collaborative study enables estimation of a variety of gene expression measurement precision metrics: repeatability, several flavors of intermediate precision, and reproducibility. The three 2004 Expression Analysis Pilot Proficiency Test collaborative studies, each with 13 to 16 participants, provide triplicate microarray measurements on each of two reference RNA pools. Using and modestly extending the consensus ISO 5725 documentary standard, we evaluate the metrological precision figures of merit for individual microarray signal measurement, building from calculations appropriate to single measurement processes, such as technical replicate expression values for individual probes on a microarray, to the estimation and display of precision functions representing all of the probes in a given platform.</p> <p>Conclusion</p> <p>With only modest extensions, the established metrological framework can be fruitfully used to characterize the measurement performance of microarray and other highly multiplexed systems. Precision functions, summarizing routine precision metrics estimated from appropriately repeated measurements of one or more reference materials as functions of signal level, are demonstrated and merit further development for characterizing measurement platforms, monitoring changes in measurement system performance, and comparing performance among laboratories or analysts.</p

DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

<p>Abstract</p> <p>Background</p> <p>DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.</p> <p>Results</p> <p>The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (<it>CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A</it>), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by <it>CDH1</it>, <it>CEACAM6, CST6, ESR1, LCN2</it>, and <it>SCNN1A</it>. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.</p> <p>Conclusion</p> <p>These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.</p

DNA-Based Genetic Markers for Rapid Cycling Brassica Rapa (Fast Plants Type) Designed for the Teaching Laboratory

We have developed DNA-based genetic markers for rapid cycling Brassica rapa (RCBr), also known as Fast Plants. Although markers for B. rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP) based markers and 14 variable number tandem repeat (VNTR)-type markers spread over four chromosomes. The DNA sequences of these markers represent variation in a wide range of genomic features. Among the VNTR-type markers, there are examples of variation in a non-genic region, variation within an intron, and variation in the coding sequence of a gene. Among the SNP-based markers there are examples of polymorphism in intronic DNA and synonymous substitution in a coding sequence. Thus these markers can serve laboratory exercises in both transmission genetics and molecular biology