Kevin L. Knudtson

Regulation of gene expression in the mammalian eye and its relevance to eye disease

We used expression quantitative trait locus mapping in the laboratory rat (Rattus norvegicus) to gain a broad perspective of gene regulation in the mammalian eye and to identify genetic variation relevant to human eye disease. Of >31,000 gene probes represented on an Affymetrix expression microarray, 18,976 exhibited sufficient signal for reliable analysis and at least 2-fold variation in expression among 120 F2 rats generated from an SR/JrHsd × SHRSP intercross. Genome-wide linkage analysis with 399 genetic markers revealed significant linkage with at least one marker for 1,300 probes (α = 0.001; estimated empirical false discovery rate = 2%). Both contiguous and noncontiguous loci were found to be important in regulating mammalian eye gene expression. We investigated one locus of each type in greater detail and identified putative transcription-altering variations in both cases. We found an inserted cREL binding sequence in the 5′ flanking sequence of the Abca4 gene associated with an increased expression level of that gene, and we found a mutation of the gene encoding thyroid hormone receptor β2 associated with a decreased expression level of the gene encoding short-wavelength sensitive opsin (Opn1sw). In addition to these positional studies, we performed a pairwise analysis of gene expression to identify genes that are regulated in a coordinated manner and used this approach to validate two previously undescribed genes involved in the human disease Bardet–Biedl syndrome. These data and analytical approaches can be used to facilitate the discovery of additional genes and regulatory elements involved in human eye disease.

Quantification of mRNA for Endothelial NO Synthase in Mouse Blood Vessels by Real-Time Polymerase Chain Reaction

The mouse is useful in studies of vascular biology because of its well-defined genetics and because the mouse genome can be manipulated. However, because only small amounts of mRNA can be extracted from blood vessels, the quantification of gene expression in individual mice is difficult. Endothelial NO synthase (eNOS) plays a major role in the regulation of vascular tone and growth. In addition, there appear to be sex differences in the production of NO under basal conditions in mouse aortas. The goals of this study were to develop a real-time polymerase chain reaction (PCR) method to quantify eNOS mRNA in blood vessels from mice and to examine eNOS mRNA levels in vessels from male and female mice. Blood vessels were isolated from C57BL/6 mice. Total RNA from individual mice was isolated and reverse-transcribed. The number of molecules of eNOS mRNA (after reverse transcription) was determined against cDNA standards, with 18S rRNA used as a control for RNA input and reverse-transcription efficiency. When expressed as copy numbers per nanogram of total RNA or as the ratio of eNOS to 18S rRNA, eNOS mRNA was lower in the aortas of female mice than in those of male mice at 7 to 9 months of age. In contrast, no difference in eNOS mRNA was found in the aortas of 2-month-old mice. In addition, eNOS mRNA levels were similar in the carotid, cerebral, and coronary arteries. These findings provide the first quantitative measurements of eNOS mRNA by using real-time PCR in the vessels of mice and suggest age- and sex-related differences in the basal levels of eNOS mRNA in mice. In addition, the eNOS region that was used for real-time PCR was amplified and sequenced for monkeys and other species. With modifications, this region may be used to design real-time PCR for eNOS in other species.

A novel rat 523 amino acid phosphophoryn: nucleotide sequence and genomic organization

Phosphophoryns (PP), the major noncollagenous proteins (NCPs) in dentin, are believed to play a crucial role in mineral nucleation and hydroxyapatite growth during dentin mineralization. Previously we identified two mature rat PP transcripts, one coding for a 240 amino acid protein (designated as PP(240)) (H.H. Ritchie, L.-H. Wang, J. Biol. Chem. 271 (1996) 21695-21698), and another coding for a 171 amino acid protein (PP(171)) (H. Ritchie, L. Wang, Biochim. Biophys. Acta 1493 (2000) 27-32). We now have identified a third novel dentin sialoprotein (DSP)-PP cDNA transcript that encodes a 523 amino acid protein (PP(523)) with typical PP characteristics including DSS and DS motifs suitable as potential casein kinase I and II phosphorylation sites. Based on amino acid composition, the PP(523) protein product is identical to native rat HP2. We also show that the PP(523) sequence is identical to the corresponding genomic DNA sequence. Taken together, the existence of multiple DSP-PP transcripts, each significantly different from the other in net negative charge, suggests that dentin mineralization processes may be under fine-tune control by these PP protein isoforms.

A Genome-Wide Association Study for Primary Open Angle Glaucoma and Macular Degeneration Reveals Novel Loci

Glaucoma and age-related macular degeneration (AMD) are the two leading causes of visual loss in the United States. We utilized a novel study design to perform a genome-wide association for both primary open angle glaucoma (POAG) and AMD. This study design utilized a two-stage process for hypothesis generation and validation, in which each disease cohort was utilized as a control for the other. A total of 400 POAG patients and 400 AMD patients were ascertained and genotyped at 500,000 loci. This study identified a novel association of complement component 7 (C7) to POAG. Additionally, an association of central corneal thickness, a known risk factor for POAG, was found to be associated with ribophorin II (RPN2). Linked monogenic loci for POAG and AMD were also evaluated for evidence of association, none of which were found to be significantly associated. However, several yielded putative associations requiring validation. Our data suggest that POAG is more genetically complex than AMD, with no common risk alleles of large effect.

Fibrotic Aortic Valve Stenosis in Hypercholesterolemic/Hypertensive Mice

Objective— Hypercholesterolemia and hypertension are associated with aortic valve stenosis (AVS) in humans. We have examined aortic valve function, structure, and gene expression in hypercholesterolemic/hypertensive mice. Approach and Results— Control, hypertensive, hypercholesterolemic (Apoe −/− ), and hypercholesterolemic/hypertensive mice were studied. Severe aortic stenosis (echocardiography) occurred only in hypercholesterolemic/hypertensive mice. There was minimal calcification of the aortic valve. Several structural changes were identified at the base of the valve. The intercusp raphe (or seam between leaflets) was longer in hypercholesterolemic/hypertensive mice than in other mice, and collagen fibers at the base of the leaflets were reoriented to form a mesh. In hypercholesterolemic/hypertensive mice, the cusps were asymmetrical, which may contribute to changes that produce AVS. RNA sequencing was used to identify molecular targets during the developmental phase of stenosis. Genes related to the structure of the valve were identified, which differentially expressed before fibrotic AVS developed. Both RNA and protein of a profibrotic molecule, plasminogen activator inhibitor 1, were increased greatly in hypercholesterolemic/hypertensive mice. Conclusions— Hypercholesterolemic/hypertensive mice are the first model of fibrotic AVS. Hypercholesterolemic/hypertensive mice develop severe AVS in the absence of significant calcification, a feature that resembles AVS in children and some adults. Structural changes at the base of the valve leaflets include lengthening of the raphe, remodeling of collagen, and asymmetry of the leaflets. Genes were identified that may contribute to the development of fibrotic AVS.

Cellular Stress Response to Varicella-Zoster Virus Infection of Human Skin Includes Highly Elevated Interleukin-6 Expression

Abstract Background The infectious cycle of varicella-zoster virus (VZV) after reactivation from the dorsal root ganglia includes replication and assembly of complete enveloped virions in the human skin to cause the characteristic herpes zoster (shingles). Methods To pursue studies of innate immunity to VZV infection, we have adapted a fetal skin organ culture model to a human neonatal foreskin explant model. Results Abundant expression of VZV IE62, gE, and gC was visualized by confocal microscopy while numerous enveloped virions were observed by electron microscopy in infected skin organ cultures. Microarray experiments demonstrated that the patterns of upregulated transcripts differed between VZV-infected cells and VZV-infected skin explants. One result stood out, namely a >30-fold elevated interleukin (IL)-6 level in the infected skin explant that was not present in the infected monolayer culture. The IL-6 results in the polyermase chain reaction (PCR) assay were reproduced by quantitative PCR testing with newly designed primers. To determine if increased transcription was accompanied by increased IL-6 expression, we quantitated the levels of IL-6 protein in the explant media at increasing intervals after infection. We found a statistically significant increase in IL-6 protein levels secreted into the media from VZV-infected skin explants as compared with mock-infected explants. Conclusions The cellular stress response to VZV infection in neonatal skin explants included highly elevated levels of IL-6 transcription and expression. This skin organ model could be adapted to other viruses with a skin tropism, such as herpes simplex virus.

ABRF 2012: Best Poster Competition.

With the continuing mission of providing significant educational opportunities to the Association of Biomolecular Resource Facilities (ABRF) membership, the Education Committee (EdComm) has a proud heritage of overseeing the Waters Research Poster Competition for the annual meeting. The competition is open to all and includes scientific reports from academia, as well as cutting-edge research from core facilities and corporate R&D groups. The goal of offering the awards is to elicit the highest level of information on techniques, applications, and technologies and share knowledge pertinent to further excellence in core facility services. The process for judging the poster submissions begins with the author submitting an abstract on-line during the registration procedure. The author chooses a submission category and self-selects whether to be considered for competition. Each abstract is reviewed by at least three members of the EdComm, based on the following criteria adapted from the Annual BioMedical Research Conference for Minority Students: development of a hypothesis or statement of the problem, description of methods and controls, clarity in the presentation of results, a conclusion that summarizes the results, and impact of the results to the core facility mission. The entire committee comes to a consensus as to the semifinalists. All semifinalists must agree to give a short oral presentation during the poster review session for the EdComm and to give a 15-min talk at a regular meeting session if they are a winner. During the annual meeting, teams of EdComm members circulate through the poster session and evaluate the semifinalist presentations. The metrics used for evaluating abstracts also contain information about how to judge (rank) each of three additional aspects determined during the designated poster presentation: clarity of the poster presentation, originality and contribution to the core facility, and oral presentation. Four finalists are ultimately selected. Competition has provided the ABRF membership with excellent posters to enjoy during the annual meeting. The presentation by finalists during the annual meeting is an opportunity to obtain further information about the science behind the poster. We are fortunate to provide ABRF attendees with this opportunity and thank Waters Corporation for continuing its support of this award by providing a monetary honorarium to the winners. The EdComm proudly congratulates the winners of the ABRF 2012 Waters Best Poster Award: Alexandre Campos ProteoRed Multicenter Experiment for Long-term Quality Control Evaluation of Proteomics Core Facilities Proteomics Core Facility, Barcelona Science Park, Barcelona, Spain Toumy Guettouche An Improved Workflow for miRNA Expression Profiling Using Ion Semiconductor Sequencing Hussman Institute for Human Genomics and Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA Zhenjiu Liu Redox Protein Characterization and Quantification Using ICAT/Itraq-MS to Investigate Thiol-Based Regulatory Mechanisms Induced by Oxidative Stress in Plants Donald Danforth Plant Science Center, St. Louis, MO, USA Peter Tonner Transcriptomic Profiling of Ribosomal Protein Pseudogenes in Diverse Human Tissuesy Department of Electrical Engineering & Computer Science, University of Central Florida, Orlando, FL, USA