Thomas B. Bair

ZEB1 Enhances Transendothelial Migration and Represses the Epithelial Phenotype of Prostate Cancer Cells

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.

Rapid Identification of Cell-Specific, Internalizing RNA Aptamers with Bioinformatics Analyses of a Cell-Based Aptamer Selection

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.

Use of Transposon Tn5367 Mutagenesis and a Nitroimidazopyran- Based Selection System To Demonstrate a Requirement for fbiA and fbiB in Coenzyme F 420 Biosynthesis by Mycobacterium bovis BCG

Three transposon Tn5367 mutagenesis vectors (phAE94, pPR28, and pPR29) were used to create a collection of insertion mutants of Mycobacterium bovis strain BCG. A strategy to select for transposon-generated mutants that cannot make coenzyme F(420) was developed using the nitroimidazopyran-based antituberculosis drug PA-824. One-third of 134 PA-824-resistant mutants were defective in F(420) accumulation. Two mutants that could not make F(420)-5,6 but which made the biosynthesis intermediate FO were examined more closely. These mutants contained transposons inserted in two adjacent homologues of Mycobacterium tuberculosis genes, which we have named fbiA and fbiB for F(420) biosynthesis. Homologues of fbiA were found in all seven microorganisms that have been fully sequenced and annotated and that are known to make F(420). fbiB homologues were found in all but one such organism. Complementation of the fbiA mutant with fbiAB and complementation of the fbiB mutant with fbiB both restored the F(420)-5,6 phenotype. Complementation of the fbiA mutant with fbiA or fbiB alone did not restore the F(420)-5,6 phenotype, but the fbiA mutant complemented with fbiA produced F(420)-2,3,4 at levels similar to F(420)-5,6 made by the wild-type strain, but produced much less F(420)-5. These data demonstrate that both genes are essential for normal F(420)-5,6 production and suggest that the fbiA mutation has a partial polar effect on fbiB. Reverse transcription-PCR data demonstrated that fbiA and fbiB constitute an operon. However, very low levels of fbiB mRNA are produced by the fbiA mutant, suggesting that a low-level alternative start site is located upstream of fbiB. The specific reactions catalyzed by FbiA and FbiB are unknown, but both function between FO and F(420)-5,6, since FO is made by both mutants.

Identification of Primary Gene Targets of TFAP2C in Hormone Responsive Breast Carcinoma Cells

The TFAP2C transcription factor is involved in mammary development, differentiation, and oncogenesis. Previous studies established a role for TFAP2C in the regulation of ESR1 (ERα) 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 (ERα), 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.

Structures of coenzyme F420 in Mycobacterium species

Abstract. The structure of coenzyme F420 in Mycobacterium smegmatis was examined using proton NMR, amino acid analysis, and HPLC. The two major F420 structures were shown to be composed of a chromophore identical to that of F420 from Methanobacterium thermoautotrophicum, with a side chain of a ribityl residue, a lactyl residue and five or six glutamate groups (F420–5 and F420–6). Peptidase treatment studies suggested that L-glutamate groups are linked by γ-glutamyl bonds in the side chain. HPLC analysis indicated that Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium fortuitum have F420–5 and F420–6 as the predominant structures, whereas Mycobacterium avium contains F420–5, F420–6 and F420–7 in significant amounts. 7,8-Didemethyl 8-hydroxy 5-deazariboflavin (FO), an intermediate in F420 biosynthesis, accounted for about 1–7% of the total deazaflavin in cells. Peptidase treatment of F420 created F420 derivatives that may be useful for the assay of enzymes involved in F420 biosynthesis.

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

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 13C6-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.

Transcriptional profiling identifies the metabolic phenotype of gonococcal biofilms.

ABSTRACT 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.

Analysis of Nontypeable Haemophilus influenzae Phase Variable Genes during Experimental Human Nasopharyngeal Colonization

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.

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

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.

RNA Aptamer-Based Functional Ligands of the Neurotrophin Receptor, TrkB

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 (KD ∼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.