Publications by authors named "Thomas Bair"

33 Publications

Early Suppression of Macrophage Gene Expression by .

Front Microbiol 2018 15;9:2464. Epub 2018 Oct 15.

Serviço de Imunologia, Hospital Universitário Professor Edgard Santos, Universidade Federal da Bahia, Salvador, Brazil.

is an intracellular parasite that resides mostly in macrophages. Both the parasite genome and the clinical disease manifestations show considerable polymorphism. Clinical syndromes caused by include localized cutaneous (CL), mucosal (ML), and disseminated leishmaniasis (DL). Our prior studies showed that genetically distinct clades associate with different clinical types. Herein, we hypothesized that: (1) induces changes in macrophage gene expression that facilitates infection; (2) infection of macrophages with strains associated with CL (clade B), ML (clade C), or DL (clade A) will differentially affect host cell gene expression, reflecting their different pathogenic mechanisms; and (3) differences between the strains will be reflected by differences in macrophage gene expression after initial exposure to the parasite. Human monocyte derived macrophages were infected with isolates from clades A, B, or C. Patterns of gene expression were compared using Affymetrix DNA microarrays. Many transcripts were significantly decreased by infection with all isolates. The most dramatically decreased transcripts encoded proteins involved in signaling pathways, apoptosis, or mitochondrial oxidative phosphorylation. Some transcripts encoding stress response proteins were up-regulated. Differences between clades were observed in the magnitude of change, rather than the identity of transcripts. Isolates from subjects with metastatic disease (ML and DL) induced a greater magnitude of change than isolates from CL. We conclude that enhances its intracellular survival by inhibiting macrophage pathways leading to microbicidal activity. Parasite strains destined for dissemination may exert a more profound suppression than less invasive strains that remain near the cutaneous site of inoculation.
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http://dx.doi.org/10.3389/fmicb.2018.02464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6196312PMC
October 2018

The vagal ganglia transcriptome identifies candidate therapeutics for airway hyperreactivity.

Am J Physiol Lung Cell Mol Physiol 2018 08 5;315(2):L133-L148. Epub 2018 Apr 5.

Department of Internal Medicine, University of Iowa , Iowa City, Iowa.

Mainstay therapeutics are ineffective in some people with asthma, suggesting a need for additional agents. In the current study, we used vagal ganglia transcriptome profiling and connectivity mapping to identify compounds beneficial for alleviating airway hyperreactivity (AHR). As a comparison, we also used previously published transcriptome data from sensitized mouse lungs and human asthmatic endobronchial biopsies. All transcriptomes revealed agents beneficial for mitigating AHR; however, only the vagal ganglia transcriptome identified agents used clinically to treat asthma (flunisolide, isoetarine). We also tested one compound identified by vagal ganglia transcriptome profiling that had not previously been linked to asthma and found that it had bronchodilator effects in both mouse and pig airways. These data suggest that transcriptome profiling of the vagal ganglia might be a novel strategy to identify potential asthma therapeutics.
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http://dx.doi.org/10.1152/ajplung.00557.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139658PMC
August 2018

Cutting Edge: β-Catenin-Interacting Tcf1 Isoforms Are Essential for Thymocyte Survival but Dispensable for Thymic Maturation Transitions.

J Immunol 2017 05 27;198(9):3404-3409. Epub 2017 Mar 27.

Department of Microbiology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242;

T cell factor 1 (Tcf1) is essential for T cell development; however, it remains controversial whether β-catenin, a known coactivator of Tcf1, has a role. Tcf1 is expressed in multiple isoforms in T lineage cells, with the long isoforms interacting with β-catenin through an N-terminal domain. In this study, we specifically ablated Tcf1 long isoforms in mice (p45mice) to abrogate β-catenin interaction. Although thymic cellularity was diminished in p45 mice, transition of thymocytes through the maturation stages was unaffected, with no overt signs of developmental blocks. p45 thymocytes showed increased apoptosis and alterations in transcriptome, but these changes were substantially more modest than in thymocytes lacking all Tcf1 isoforms. These data indicate that Tcf1-β-catenin interaction is necessary for promoting thymocyte survival to maintain thymic output. Rather than being dominant-negative regulators, Tcf1 short isoforms are adequate in supporting developing thymocytes to traverse through maturation steps and in regulating the expression of most Tcf1 target genes.
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http://dx.doi.org/10.4049/jimmunol.1602139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423537PMC
May 2017

Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice.

Proc Natl Acad Sci U S A 2017 04 27;114(15):E3119-E3128. Epub 2017 Mar 27.

Department of Pediatrics, Pappajohn Biomedical Institute, University of Iowa, Iowa City, IA 52242;

The Middle East respiratory syndrome (MERS) emerged in Saudi Arabia in 2012, caused by a zoonotically transmitted coronavirus (CoV). Over 1,900 cases have been reported to date, with ∼36% fatality rate. Lack of autopsies from MERS cases has hindered understanding of MERS-CoV pathogenesis. A small animal model that develops progressive pulmonary manifestations when infected with MERS-CoV would advance the field. As mice are restricted to infection at the level of DPP4, the MERS-CoV receptor, we generated mice with humanized exons 10-12 of the mouse locus. Upon inoculation with MERS-CoV, human DPP4 knockin (KI) mice supported virus replication in the lungs, but developed no illness. After 30 serial passages through the lungs of KI mice, a mouse-adapted virus emerged (MERS) that grew in lungs to over 100 times higher titers than the starting virus. A plaque-purified MERS clone caused weight loss and fatal infection. Virus antigen was observed in airway epithelia, pneumocytes, and macrophages. Pathologic findings included diffuse alveolar damage with pulmonary edema and hyaline membrane formation associated with accumulation of activated inflammatory monocyte-macrophages and neutrophils in the lungs. Relative to the parental MERS-CoV, MERS viruses contained 13-22 mutations, including several within the spike (S) glycoprotein gene. S-protein mutations sensitized viruses to entry-activating serine proteases and conferred more rapid entry kinetics. Recombinant MERS bearing mutant S proteins were more virulent than the parental virus in hDPP4 KI mice. The hDPP4 KI mouse and the MERS provide tools to investigate disease causes and develop new therapies.
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http://dx.doi.org/10.1073/pnas.1619109114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5393213PMC
April 2017

Histone demethylase PHF8 promotes epithelial to mesenchymal transition and breast tumorigenesis.

Nucleic Acids Res 2017 02;45(4):1687-1702

Department of Anatomy and Cell Biology, Carver College of Medicine, The University of Iowa, IA 52242, USA.

Histone demethylase PHF8 is upregulated and plays oncogenic roles in various cancers; however, the mechanisms underlying its dysregulation and functions in carcinogenesis remain obscure. Here, we report the novel functions of PHF8 in EMT (epithelial to mesenchymal transition) and breast cancer development. Genome-wide gene expression analysis revealed that PHF8 overexpression induces an EMT-like process, including the upregulation of SNAI1 and ZEB1. PHF8 demethylates H3K9me1, H3K9me2 and sustains H3K4me3 to prime the transcriptional activation of SNAI1 by TGF-β signaling. We show that PHF8 is upregulated and positively correlated with MYC at protein levels in breast cancer. MYC post-transcriptionally regulates the expression of PHF8 via the repression of microRNAs. Specifically, miR-22 directly targets and inhibits PHF8 expression, and mediates the regulation of PHF8 by MYC and TGF-β signaling. This novel MYC/microRNAs/PHF8 regulatory axis thus places PHF8 as an important downstream effector of MYC. Indeed, PHF8 contributes to MYC-induced cell proliferation and the expression of EMT-related genes. We also report that PHF8 plays important roles in breast cancer cell migration and tumor growth. These oncogenic functions of PHF8 in breast cancer confer its candidacy as a promising therapeutic target for this disease.
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http://dx.doi.org/10.1093/nar/gkw1093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389682PMC
February 2017

High throughput genomic sequencing of bioaerosols in broiler chicken production facilities.

Microb Biotechnol 2016 11 28;9(6):782-791. Epub 2016 Jul 28.

Department of Occupational and Environmental Health, University of Iowa, Iowa City, IA, USA.

Chronic inhalation exposure to agricultural dust promotes the development of chronic respiratory diseases among poultry workers. Poultry dust is composed of dander, chicken feed, litter bedding and microbes. However, the microbial composition and abundance has not been fully elucidated. Genomic DNA was extracted from settled dust and personal inhalable dust collected while performing litter sampling or mortality collection tasks. DNA libraries were sequenced using a paired-end sequencing-by-synthesis approach on an Illumina HiSeq 2500. Sequencing data showed that poultry dust is predominantly composed of bacteria (64-67%) with a small quantity of avian, human and feed DNA (< 2% of total reads). Staphylococcus sp. AL1, Salinicoccus carnicancri and Lactobacillus crispatus were the most abundant bacterial species in personal exposure samples of inhalable dust. Settled dust had a moderate relative abundance of these species as well as Staphylococcus lentus and Lactobacillus salivarius. There was a statistical difference between the microbial composition of aerosolized and settled dust. Unlike settled dust composition, aerosolized dust composition had little variance between samples. These data provide an extensive analysis of the microbial composition and relative abundance in personal inhalable poultry dust and settled poultry dust.
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http://dx.doi.org/10.1111/1751-7915.12380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072194PMC
November 2016

Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF.

J Bacteriol 2016 09 25;198(18):2458-69. Epub 2016 Aug 25.

Department of Microbiology, University of Iowa, Iowa City, Iowa, USA

Unlabelled: CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent.

Importance: The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence.
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http://dx.doi.org/10.1128/JB.00343-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4999922PMC
September 2016

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications.

Infect Immun 2016 Jan 4;84(3):765-74. Epub 2016 Jan 4.

Department of Microbiology, The University of Iowa, Iowa City, Iowa, USA

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.
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http://dx.doi.org/10.1128/IAI.01185-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771351PMC
January 2016

Interferon-γ Inhibits Ebola Virus Infection.

PLoS Pathog 2015 12;11(11):e1005263. Epub 2015 Nov 12.

Department of Microbiology, The University of Iowa, Iowa City, Iowa, United States of America.

Ebola virus outbreaks, such as the 2014 Makona epidemic in West Africa, are episodic and deadly. Filovirus antivirals are currently not clinically available. Our findings suggest interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option. Using mouse-adapted Ebola virus, we found that murine interferon gamma administered 24 hours before or after infection robustly protects lethally-challenged mice and reduces morbidity and serum viral titers. Furthermore, we demonstrated that interferon gamma profoundly inhibits Ebola virus infection of macrophages, an early cellular target of infection. As early as six hours following in vitro infection, Ebola virus RNA levels in interferon gamma-treated macrophages were lower than in infected, untreated cells. Addition of the protein synthesis inhibitor, cycloheximide, to interferon gamma-treated macrophages did not further reduce viral RNA levels, suggesting that interferon gamma blocks life cycle events that require protein synthesis such as virus replication. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited Ebola virus infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit negative strand RNA viruses and specifically Ebola virus infection. As treatment of interferon gamma robustly protects mice from lethal Ebola virus infection, we propose that interferon gamma should be further evaluated for its efficacy as a prophylactic and/or therapeutic strategy against filoviruses. Use of this FDA-approved drug could rapidly be deployed during future outbreaks.
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http://dx.doi.org/10.1371/journal.ppat.1005263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4643030PMC
April 2016

Calcium/calmodulin-dependent kinase II inhibition in smooth muscle reduces angiotensin II-induced hypertension by controlling aortic remodeling and baroreceptor function.

J Am Heart Assoc 2015 Jun 15;4(6):e001949. Epub 2015 Jun 15.

Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA (A.M.P., D.W.N., A.N.V., M.E.D., W.J.K., R.M.W., K.G.L., M.W.C., C.D.S., K.R., I.M.G.) The Iowa City VA Healthcare System, Iowa City, IA (A.N.V., K.G.L., M.W.C., I.M.G.).

Background: Multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMCs), but its function in experimental hypertension has not been explored. The aim of this study was to determine the impact of CaMKII inhibition selectively in VSMCs on Ang II hypertension.

Methods And Results: Transgenic expression of a CaMKII peptide inhibitor in VSMCs (TG SM-CaMKIIN model) reduced the blood pressure response to chronic Ang II infusion. The aortic depressor nerve activity was reset in hypertensive versus normotensive wild-type animals but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor activity account for the blood pressure difference between genotypes. Accordingly, aortic pulse wave velocity, a measure of arterial wall stiffness and a determinant of baroreceptor activity, increased in hypertensive versus normotensive wild-type animals but did not change in TG SM-CaMKIIN mice. Moreover, examination of blood pressure and heart rate under ganglionic blockade revealed that VSMC CaMKII inhibition abolished the augmented efferent sympathetic outflow and renal and splanchnic nerve activity in Ang II hypertension. Consequently, we hypothesized that VSMC CaMKII controls baroreceptor activity by modifying arterial wall remodeling in Ang II hypertension. Gene expression analysis in aortas from normotensive and Ang II-infused mice revealed that TG SM-CaMKIIN aortas were protected from Ang II-induced upregulation of genes that control extracellular matrix production, including collagen. VSMC CaMKII inhibition also strongly altered the expression of muscle contractile genes under Ang II.

Conclusions: CaMKII in VSMCs regulates blood pressure under Ang II hypertension by controlling structural gene expression, wall stiffness, and baroreceptor activity.
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http://dx.doi.org/10.1161/JAHA.115.001949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599535PMC
June 2015

Transcriptional changes that characterize the immune reactions of leprosy.

J Infect Dis 2015 May 14;211(10):1658-76. Epub 2014 Nov 14.

Department of Biochemistry Institute of Tropical Medicine of Rio Grande do Norte, Universidade Federal do Rio Grande do Norte National Institute of Science and Technology of Tropical Diseases, Salvador, Bahia, Brazil.

Background: Leprosy morbidity is increased by 2 pathologic immune reactions, reversal reaction (RR) and erythema nodosum leprosum (ENL).

Methods: To discover host factors related to immune reactions, global transcriptional profiles of peripheral blood mononuclear cells were compared between 11 RR, 11 ENL, and 19 matched control patients, with confirmation by quantitative polymerase chain reaction. Encoded proteins were investigated in skin biopsy specimens by means of immunohistochemistry.

Results: There were 275 genes differentially expressed in RR and 517 differentially expressed in ENL on the microarray. Pathway analysis showed immunity-related pathways represented in RR and ENL transcriptional profiles, with the "complement and coagulation" pathway common to both. Interferon γ was identified as a significant upstream regulator of the expression changes for RR and ENL. Immunohistochemical staining of skin lesions showed increased C1q in both RR and ENL.

Conclusions: These data suggest a previously underrecognized role for complement in the pathogenesis of both RR and ENL, and we propose new hypotheses for reaction pathogenesis.
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http://dx.doi.org/10.1093/infdis/jiu612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425823PMC
May 2015

A novel animal model for locally advanced breast cancer.

Ann Surg Oncol 2015 Mar 18;22(3):866-73. Epub 2014 Oct 18.

Department of Surgery, University of Iowa, Iowa City, IA, USA.

Background: Locally advanced breast cancer (LABC) poses complex management issues due to failure of response to chemotherapy and progression to local complications such as skin erosion, superinfection, and lymphedema. Most cell line and animal models are not adequate to study LABC.

Methods: A patient-derived xenograft (IOWA-1T) from a patient with LABC was characterized for expression profile, short tandem repeat profile, oncogenic mutations, xenograft growth, and response to therapy.

Results: Short tandem repeat profile authenticated the cell line as derived from a human woman. The primary tumor and derived xenografts were weakly estrogen receptor alpha positive (<5%), progesterone receptor negative, and HER2 nonamplified. Expression array profile compared to MCF-7 and BT-549 cell lines indicate that IOWA-1T was more closely related to basal breast cancer. IOWA-1T harbors a homozygous R248Q mutation of the TP53 gene; in vitro invasion assay was comparable to BT-549 and greater than MCF-7. IOWA-1T xenografts developed palpable tumors in 9.6 ± 1.6 days, compared to 49 ± 13 days for parallel experiments with BT-20 cells (p < 0.002). Tumor xenografts became locally advanced, growing to >2 cm in 21.6 ± 2 days, characterized by skin erosion necessitating euthanasia. The SUMO inhibitor anacardic acid inhibited the outgrowth of IOWA-1T xenografts, while doxorubicin had no effect on tumorigenesis.

Conclusions: IOWA-1T is a novel cell line with an expression pattern consistent with basal breast cancer. Xenografts recapitulated LABC and provide a novel model for testing therapeutic drugs that may be effective in cases resistant to conventional chemotherapy.
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http://dx.doi.org/10.1245/s10434-014-4174-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425290PMC
March 2015

Identification of genomic binding sites for Candida glabrata Pdr1 transcription factor in wild-type and ρ0 cells.

Antimicrob Agents Chemother 2014 Nov 8;58(11):6904-12. Epub 2014 Sep 8.

Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, Iowa USA

The fungal pathogen Candida glabrata is an emerging cause of candidiasis in part owing to its robust ability to acquire tolerance to the major clinical antifungal drug fluconazole. Similar to the related species Candida albicans, C. glabrata most typically gains azole tolerance via transcriptional induction of a suite of resistance genes, including a locus encoding an ABCG-type ATP-binding cassette (ABC) transporter that is referred to as CDR1 in Candida species. In C. glabrata, CDR1 expression is controlled primarily by the activity of a transcriptional activator protein called Pdr1. Strains exhibiting reduced azole susceptibility often contain substitution mutations in PDR1 that in turn lead to elevated mRNA levels of target genes with associated azole resistance. Pdr1 activity is also induced upon loss of the mitochondrial genome status and upon challenge by azole drugs. While extensive analyses of the transcriptional effects of Pdr1 have identified a number of genes that are regulated by this factor, we cannot yet separate direct from indirect target genes. Here we used chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) to identify the promoters and associated genes directly regulated by Pdr1. These genes include many that are shared with the yeast Saccharomyces cerevisiae but others that are unique to C. glabrata, including the ABC transporter-encoding locus YBT1, genes involved in DNA repair, and several others. These data provide the outline for understanding the primary response genes involved in production of Pdr1-dependent azole resistance in C. glabrata.
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http://dx.doi.org/10.1128/AAC.03921-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249425PMC
November 2014

Effects of vitamin D supplementation on alveolar macrophage gene expression: preliminary results of a randomized, controlled trial.

Multidiscip Respir Med 2014 Mar 26;9(1):18. Epub 2014 Mar 26.

Department of Internal Medicine, University of Iowa, 200 Hawkins Dr, Iowa City 52242, IA, USA.

Background: Vitamin D deficiency has been implicated as a factor in a number of infectious and inflammatory lung diseases. In the lung, alveolar macrophages play a key role in inflammation and defense of infection, but there are little data exploring the immunomodulatory effects of vitamin D on innate lung immunity in humans. The objective of this study was to determine the effects of vitamin D supplementation on gene expression of alveolar macrophages.

Methods: We performed a parallel, double-blind, placebo-controlled, randomized trial to determine the effects of vitamin D on alveolar macrophage gene expression. Vitamin D3 (1000 international units/day) or placebo was administered to adults for three months. Bronchoscopy was performed pre- and post-intervention to obtain alveolar macrophages. Messenger RNA was isolated from the macrophages and subjected to whole genome exon array analysis. The primary outcome was differential gene expression of the alveolar macrophage in response to vitamin D supplementation. Specific genes underwent validation by polymerase chain reaction methods.

Results: Fifty-eight subjects were randomized to vitamin D (n = 28) or placebo (n = 30). There was a marginal overall difference between treatment group and placebo group in the change of 25-hydroxyvitaminD levels (4.43 ng/ml vs. 0.2 ng/ml, p = 0.10). Whole genome exon array analysis revealed differential gene expression associated with change in serum vitamin D levels in the treated group. CCL8/MCP-2 was the top-regulated cytokine gene and was further validated.

Conclusions: Although only a non-significant increased trend was seen in serum vitamin D levels, subjects treated with vitamin D supplementation had immune-related differential gene expression in alveolar macrophages.

Trial Registration: ClinicalTrials.org: NCT01967628.
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http://dx.doi.org/10.1186/2049-6958-9-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986866PMC
March 2014

An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge.

Genome Biol 2014 Mar 25;15(3):R53. Epub 2014 Mar 25.

Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance.

Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization.

Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.
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http://dx.doi.org/10.1186/gb-2014-15-3-r53DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4073084PMC
March 2014

Identification of candidate B-lymphoma genes by cross-species gene expression profiling.

PLoS One 2013 9;8(10):e76889. Epub 2013 Oct 9.

Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America.

Comparative genome-wide expression profiling of malignant tumor counterparts across the human-mouse species barrier has a successful track record as a gene discovery tool in liver, breast, lung, prostate and other cancers, but has been largely neglected in studies on neoplasms of mature B-lymphocytes such as diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). We used global gene expression profiles of DLBCL-like tumors that arose spontaneously in Myc-transgenic C57BL/6 mice as a phylogenetically conserved filter for analyzing the human DLBCL transcriptome. The human and mouse lymphomas were found to have 60 concordantly deregulated genes in common, including 8 genes that Cox hazard regression analysis associated with overall survival in a published landmark dataset of DLBCL. Genetic network analysis of the 60 genes followed by biological validation studies indicate FOXM1 as a candidate DLBCL and BL gene, supporting a number of studies contending that FOXM1 is a therapeutic target in mature B cell tumors. Our findings demonstrate the value of the "mouse filter" for genomic studies of human B-lineage neoplasms for which a vast knowledge base already exists.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076889PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793908PMC
June 2014

Human Melanoma cells over-express extracellular matrix 1 (ECM1) which is regulated by TFAP2C.

PLoS One 2013 2;8(9):e73953. Epub 2013 Sep 2.

Department of Surgery, University of Iowa, Iowa City, Iowa, United States of America.

Extracellular matrix 1 (ECM1) is over-expressed in multiple epithelial malignancies. However, knowledge regarding the expression of ECM1 in melanomas and the mechanisms of ECM1 regulation is limited. In this study, we found that ECM1 is over-expressed in several melanoma cell lines, when compared to primary melanocytes, and furthermore, that ECM1 expression paralleled that of TFAP2C levels in multiple cell lines. Knockdown of TFAP2C in the A375 cell line with siRNA led to a reduction in ECM1 expression, and upregulation of TFAP2C with adenoviral vectors in the WM793 cell line resulted in ECM1 upregulation. Utilizing 5' RACE to identify transcription start sites (TSS) and luciferase reporter assays in the ECM1-overexpressing A375 cell line, we identified the minimal promoter region of human ECM1 and demonstrate that an approximately 100bp fragment upstream of the TSS containing a TATA box and binding sites for AP1, SP1 and Ets is sufficient for promoter activity. Chromatin immunoprecipitation and direct sequencing (ChIP-seq) for TFAP2C in the A375 cell line identified an AP2 regulatory region in the promoter of the ECM1 gene. Gelshift assays further confirmed binding of TFAP2C to this site. ECM1 knockdown reduces melanoma cell attachment and is consistent with findings that ECM1 overexpression has been associated with a poor prognosis. Our investigations show an as yet unrecognized role for TFAP2C in melanoma via its regulation of ECM1.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0073953PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759440PMC
April 2014

Analysis of nontypeable haemophilus influenzae phase-variable genes during experimental human nasopharyngeal colonization.

J Infect Dis 2013 Sep 28;208(5):720-7. Epub 2013 May 28.

Department of Microbiology, University of Iowa, Iowa City, IA 52242, USA.

Background: Studies of nontypeable Haemophilus influenzae (NTHi) have demonstrated that a number of genes associated with infectivity have long repeat regions associated with phase variation in expression of the respective gene. The purpose of this study was to determine the genes that underwent phase variation during a 6-day period of experimental human nasopharyngeal colonization.

Methods: Strain NTHi 2019Str(R)1 was used to colonize the nasopharynx of human subjects in a study of experimental colonization. Thirteen phase-variable genes were analyzed in NTHi 2019Str(R)1. Samples of NTHi 2019Str(R)1 were cultured from subjects during the 6-day colonization period. We used capillary electrophoresis and Roche 454 pyrosequencing to determine the number of repeats in each gene from each sample.

Results: A significant number of samples switched licA and igaB from phase off in the inoculated strain to phase on during the 4-day period of observation. lex2A also showed variability as compared to baseline, but the differences were not significant. The remaining genes showed no evidence of phase variation.

Conclusions: Our studies suggest that the phase-on genotypes of licA and igaB are important for early human nasopharynx colonization. lex2A showed a trend from phase off to phase on, suggesting a potentially important role in the colonization process.
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http://dx.doi.org/10.1093/infdis/jit240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3733508PMC
September 2013

Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cell-based aptamer selection.

PLoS One 2012 4;7(9):e43836. Epub 2012 Sep 4.

Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States of America.

Background: The broad applicability of RNA aptamers as cell-specific delivery tools for therapeutic reagents depends on the ability to identify aptamer sequences that selectively access the cytoplasm of distinct cell types. Towards this end, we have developed a novel approach that combines a cell-based selection method (cell-internalization SELEX) with high-throughput sequencing (HTS) and bioinformatics analyses to rapidly identify cell-specific, internalization-competent RNA aptamers.

Methodology/principal Findings: We demonstrate the utility of this approach by enriching for RNA aptamers capable of selective internalization into vascular smooth muscle cells (VSMCs). Several rounds of positive (VSMCs) and negative (endothelial cells; ECs) selection were performed to enrich for aptamer sequences that preferentially internalize into VSMCs. To identify candidate RNA aptamer sequences, HTS data from each round of selection were analyzed using bioinformatics methods: (1) metrics of selection enrichment; and (2) pairwise comparisons of sequence and structural similarity, termed edit and tree distance, respectively. Correlation analyses of experimentally validated aptamers or rounds revealed that the best cell-specific, internalizing aptamers are enriched as a result of the negative selection step performed against ECs.

Conclusions And Significance: We describe a novel approach that combines cell-internalization SELEX with HTS and bioinformatics analysis to identify cell-specific, cell-internalizing RNA aptamers. Our data highlight the importance of performing a pre-clear step against a non-target cell in order to select for cell-specific aptamers. We expect the extended use of this approach to enable the identification of aptamers to a multitude of different cell types, thereby facilitating the broad development of targeted cell therapies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043836PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433472PMC
January 2013

Expression of the RET proto-oncogene is regulated by TFAP2C in breast cancer independent of the estrogen receptor.

Ann Surg Oncol 2013 Jul 10;20(7):2204-12. Epub 2012 Aug 10.

Department of Surgery, University of Iowa, Iowa City, IA, USA.

Background: The RET proto-oncogene is expressed as part of the estrogen receptor (ER) cluster in breast cancer. We sought to determine if TFAP2C regulates Ret expression directly or indirectly through ER.

Methods: Chromatin immunoprecipitation sequencing (ChIP-Seq) and gel-shift assay were used to identify TFAP2C binding sites in the RET promoter in four breast cancer cell lines. Ret mRNA and protein levels were evaluated in ER-positive and ER-negative breast cancer cell lines after knockdown of TFAP2C. Luciferase expression assay was performed to assess expression from two of the identified sites.

Results: ChIP-Seq identified five main binding peaks for TFAP2C in the RET promoter at -101.5 kb, -50.7 kb, -32.5 kb, +5.0 kb, and +33.6 from the RET transcriptional start site. Binding at three of the AP-2 sites was conserved across all four cell lines, whereas the RET -101.5 and RET +33.6 sites were each found to be unbound by TFAP2C in one cell line. A TFAP2C consensus element was confirmed for all five sites. Knockdown of TFAP2C by siRNA in ER-positive MCF-7 cells resulted in significant down regulation of Ret mRNA compared to nontargeting (NT) siRNA (0.09 vs. 1.0, P < 0.001). Knockdown of TFAP2C in ER-negative MDA-MB-453 cells also led to a significant reduction in Ret mRNA compared to NT siRNA (0.16 vs. 1.0, P < 0.001). In MCF-7 cells, knockdown of TFAP2C abrogated Ret protein expression (0.02 vs. 1.0, P < 0.001) before reduction in ER.

Conclusions: TFAP2C regulates expression of the RET proto-oncogene through five AP-2 regulatory sites in the RET promoter. Regulation of Ret by TFAP2C occurs independently of ER expression in breast carcinoma.
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http://dx.doi.org/10.1245/s10434-012-2570-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697477PMC
July 2013

RNA aptamer-based functional ligands of the neurotrophin receptor, TrkB.

Mol Pharmacol 2012 Oct 29;82(4):623-35. Epub 2012 Jun 29.

Department of Neurobiology, Duke University Medical Center, Duke University, Durham, North Carolina, USA.

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (K(D) ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors.
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http://dx.doi.org/10.1124/mol.112.078220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463223PMC
October 2012

Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

PLoS One 2012 6;7(6):e38303. Epub 2012 Jun 6.

Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America.

Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0038303PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368942PMC
November 2012

Coordinated DNA methylation and gene expression changes in smoker alveolar macrophages: specific effects on VEGF receptor 1 expression.

J Leukoc Biol 2012 Sep 16;92(3):621-31. Epub 2012 Mar 16.

Departments of Psychiatry, The University of Iowa, Iowa City, IA, USA.

Cigarette smoking is implicated in numerous diseases, including emphysema and lung cancer. The clinical expression of lung disease in smokers is not well explained by currently defined variations in gene expression or simple differences in smoking exposure. Alveolar macrophages play a critical role in the inflammation and remodeling of the lung parenchyma in smoking-related lung disease. Significant gene expression changes in alveolar macrophages from smokers have been identified. However, the mechanism for these changes remains unknown. One potential mechanism for smoking-altered gene expression is via changes in cytosine methylation in DNA regions proximal to gene-coding sequences. In this study, alveolar macrophage DNA from heavy smokers and never smokers was isolated and methylation status at 25,000 loci determined. We found differential methylation in genes from immune-system and inflammatory pathways. Analysis of matching gene expression data demonstrated a parallel enrichment for changes in immune-system and inflammatory pathways. A significant number of genes with smoking-altered mRNA expression had inverse changes in methylation status. One gene highlighted by this data was the FLT1, and further studies found particular up-regulation of a splice variant encoding a soluble inhibitory form of the receptor. In conclusion, chronic cigarette smoke exposure altered DNA methylation in specific gene promoter regions in human alveolar macrophages.
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http://dx.doi.org/10.1189/jlb.1211632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427615PMC
September 2012

Inhibition of p53 expression modifies the specificity of chromatin binding by the androgen receptor.

Oncotarget 2012 Feb;3(2):183-94

Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa, USA.

The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326648PMC
http://dx.doi.org/10.18632/oncotarget.449DOI Listing
February 2012

Global gene expression profiling in mouse plasma cell tumor precursor and bystander cells reveals potential intervention targets for plasma cell neoplasia.

Blood 2012 Jan 6;119(4):1018-28. Epub 2011 Dec 6.

Molecular Genetics Section, Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA.

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eμ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.
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http://dx.doi.org/10.1182/blood-2011-06-363887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3271714PMC
January 2012

Nucleotide bias observed with a short SELEX RNA aptamer library.

Nucleic Acid Ther 2011 Aug 28;21(4):253-63. Epub 2011 Jun 28.

Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process.
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http://dx.doi.org/10.1089/nat.2011.0288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198618PMC
August 2011

Homozygosity mapping with SNP arrays confirms 3p21 as a recessive locus for gray platelet syndrome and narrows the interval significantly.

Blood 2011 Mar 24;117(12):3430-4. Epub 2011 Jan 24.

Department of Pediatrics and Genetics, University of Colorado, Denver, CO, USA.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by thrombocytopenia and the absence of α-granules in platelets. Patients with GPS present with mild to moderate bleeding and many develop myelofibrosis. The genetic cause of GPS is unknown. We present 2 Native American families with a total of 5 affected persons and a single affected patient of Pakistani origin in which GPS appears to be inherited in an autosomal recessive manner. Homozygosity mapping using the Affymetrix 6.0 chips demonstrates that all 6 GPS-affected persons studied are homozygous for a 1.7-Mb region in 3p21. Linkage analysis confirmed the region with a logarithm of the odds score of 2.7. Data from our families enabled us to significantly decrease the size of the critical region for GPS from the previously reported 9.4-Mb region at 3p21.
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http://dx.doi.org/10.1182/blood-2010-12-322990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3069679PMC
March 2011

Identification of primary gene targets of TFAP2C in hormone responsive breast carcinoma cells.

Genes Chromosomes Cancer 2010 Oct;49(10):948-62

Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.

The TFAP2C transcription factor is involved in mammary development, differentiation, and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERalpha) and ERBB2 (Her2) in breast carcinomas. However, the role of TFAP2C in different breast cancer phenotypes has not been examined in detail. To develop a more complete characterization of TFAP2C target genes, ChIP-seq with anti-TFAP2C antibody and expression arrays with TFAP2C knock down were analyzed in MCF-7 breast carcinoma cells. Genomic sequences common to the ChIP-seq data set defined the consensus sequence for TFAP2C chromatin binding as the nine base sequence SCCTSRGGS (S = G/C, r = A/G), which closely matches the previously defined optimal in vitro binding site. Comparing expression arrays before and after knock down of TFAP2C with ChIP-seq data demonstrated a conservative estimate that 8% of genes altered by TFAP2C expression are primary target genes and includes genes that are both induced and repressed by TFAP2C. A set of 447 primary target genes of TFAP2C was identified, which included ESR1 (ERalpha), FREM2, RET, FOXA1, WWOX, GREB1, MYC, and members of the retinoic acid response pathway. The identification of ESR1, WWOX, GREB1, and FOXA1 as primary targets confirmed the role of TFAP2C in hormone response. TFAP2C plays a critical role in gene regulation in hormone responsive breast cancer and its target genes are different than for the Her2 breast cancer phenotype.
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http://dx.doi.org/10.1002/gcc.20807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2928401PMC
October 2010

Transcriptional profiling identifies the metabolic phenotype of gonococcal biofilms.

Infect Immun 2009 Sep 15;77(9):3522-32. Epub 2009 Jun 15.

Department of Microbiology, The University of Iowa, Iowa City, Iowa 52242, USA.

Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is frequently asymptomatic in women, often leading to chronic infections. One factor contributing to this may be biofilm formation. N. gonorrhoeae can form biofilms on glass and plastic surfaces. There is also evidence that biofilm formation may occur during natural cervical infection. To further study the mechanism of gonococcal biofilm formation, we compared transcriptional profiles of N. gonorrhoeae biofilms to planktonic profiles. Biofilm RNA was extracted from N. gonorrhoeae 1291 grown for 48 h in continuous-flow chambers over glass. Planktonic RNA was extracted from the biofilm runoff. In comparing biofilm with planktonic growth, 3.8% of the genome was differentially regulated. Genes that were highly upregulated in biofilms included aniA, norB, and ccp. These genes encode enzymes that are central to anaerobic respiratory metabolism and stress tolerance. Downregulated genes included members of the nuo gene cluster, which encodes the proton-translocating NADH dehydrogenase. Furthermore, it was observed that aniA, ccp, and norB insertional mutants were attenuated for biofilm formation on glass and transformed human cervical epithelial cells. These data suggest that biofilm formation by the gonococcus may represent a response that is linked to the control of nitric oxide steady-state levels during infection of cervical epithelial cells.
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http://dx.doi.org/10.1128/IAI.00036-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737991PMC
September 2009

ZEB1 enhances transendothelial migration and represses the epithelial phenotype of prostate cancer cells.

Mol Biol Cell 2009 Apr 18;20(8):2207-17. Epub 2009 Feb 18.

Department of Molecular Physiology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, 52242, USA.

Metastatic colonization involves cancer cell lodgment or adherence in the microvasculature and subsequent migration of those cells across the endothelium into a secondary organ site. To study this process further, we analyzed transendothelial migration of human PC-3 prostate cancer cells in vitro. We isolated a subpopulation of cells, TEM4-18, that crossed an endothelial barrier more efficiently, but surprisingly, were less invasive than parental PC-3 cells in other contexts in vitro. Importantly, TEM4-18 cells were more aggressive than PC-3 cells in a murine metastatic colonization model. Microarray and FACS analysis of these cells showed that the expression of many genes previously associated with leukocyte trafficking and cancer cell extravasation were either unchanged or down-regulated. Instead, TEM4-18 cells exhibited characteristic molecular markers of an epithelial-to-mesenchymal transition (EMT), including frank loss of E-cadherin expression and up-regulation of the E-cadherin repressor ZEB1. Silencing ZEB1 in TEM4-18 cells resulted in increased E-cadherin and reduced transendothelial migration. TEM4-18 cells also express N-cadherin, which was found to be necessary, but not sufficient for increased transendothelial migration. Our results extend the role of EMT in metastasis to transendothelial migration and implicate ZEB1 and N-cadherin in this process in prostate cancer cells.
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http://dx.doi.org/10.1091/mbc.e08-10-1076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669028PMC
April 2009