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Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

Central corneal thickness (CCT) is a highly heritable trait associated with complex eye diseases such as keratoconus and glaucoma. We perform a genome-wide association meta-analysis of CCT and identify 19 novel regions. In addition to adding support for known connective tissue-related pathways, pathway analyses uncover previously unreported gene sets. Remarkably, >20% of the CCT-loci are near or within Mendelian disorder genes. These included FBN1, ADAMTS2 and TGFB2 which associate with connective tissue disorders (Marfan, Ehlers-Danlos and Loeys-Dietz syndromes), and the LUM-DCN-KERA gene complex involved in myopia, corneal dystrophies and cornea plana. Using index CCT-increasing variants, we find a significant inverse correlation in effect sizes between CCT and keratoconus (r = -0.62, P = 5.30 × 10-5) but not between CCT and primary open-angle glaucoma (r = -0.17, P = 0.2). Our findings provide evidence for shared genetic influences between CCT and keratoconus, and implicate candidate genes acting in collagen and extracellular matrix regulation.

The large yellow croaker (Pseudosciaena crocea) is an economically important marine fish in China. To understand the molecular basis for antiviral defense in this species, we used Illumia paired-end sequencing to characterize the spleen transcriptome of polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced large yellow croakers. The library produced 56,355,728 reads and assembled into 108,237 contigs. As a result, 15,192 unigenes were found from this transcriptome. Gene ontology analysis showed that 4,759 genes were involved in three major functional categories: biological process, cellular component, and molecular function. We further ascertained that numerous consensus sequences were homologous to known immune-relevant genes. Kyoto Encyclopedia of Genes and Genomes orthology mapping annotated 5,389 unigenes and identified numerous immune-relevant pathways. These immune-relevant genes and pathways revealed major antiviral immunity effectors, including but not limited to: pattern recognition receptors, adaptors and signal transducers, the interferons and interferon-stimulated genes, inflammatory cytokines and receptors, complement components, and B-cell and T-cell antigen activation molecules. Moreover, the partial genes of Toll-like receptor signaling pathway, RIG-I-like receptors signaling pathway, Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signaling pathway, and T-cell receptor (TCR) signaling pathway were found to be changed after poly(I:C) induction by real-time polymerase chain reaction (PCR) analysis, suggesting that these signaling pathways may be regulated by poly(I:C), a viral mimic. Overall, the antivirus-related genes and signaling pathways that were identified in response to poly(I:C) challenge provide valuable leads for further investigation of the antiviral defense mechanism in the large yellow croaker. © 2014 Mu et al.

Background Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. Objectives To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. Methods Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3 years and 9 months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0-II, n = 154) and metastatic melanoma (late stages, III-IV, n = 76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFβ2, PAX3d and MCAM. Results In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P = 0·001 and P = 0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. Conclusions Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status. See also the Commentary by Sullivan

[EN] To study genes involved in embryo developmental competence and implantation in rabbits, the expression of a panel of genes related to pluripotency, angiogenesis, proliferation, apoptosis and differentiation were evaluated in late rabbit blastocysts. Thirty nulliparous does were used to obtain a total of 184 in vivo-derived blastocysts on days 4, 5 and 6 of development. The relative transcript abundance of vascular endothelial growth factor (VEGF), epidermal growth factor receptor 3 (erbB3), transforming growth factor ß2 (TGF ß2) and transcription factor OCT-4 were analysed from eight pools of each stage of development, using quantitative real-time reverse transcriptase-polymerase chain reaction (qrtRT-PCR). mRNA expression was detected for all genes in 4-, 5- and 6-day-old blastocysts, according to blastocyst growth and implantation proximity. Significant differences in OCT-4, VEGF and TGF ß2 expression were observed between days of development. Results show a down-regulation of OCT-4 from the 4th day, contrasting with the up-regulation of VEGF and TGF ß2 at 6-day-old blastocyst. These findings corroborate the importance of VEGF and TGF ß2 in rabbit embryo development and implantation and suggest a possible regulator role of OCT-4 in embryonic angiogenetic factors. On the other hand, no differences were found for erbB3 expression. Therefore, the study of specific gene transcripts in rabbit blastocyst could provide novel embryo developmental competence markers and might be used as a new tool for further studies of embryo quality and in vitro development. © 2010 Blackwell Verlag GmbH.

BackgroundAirway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.MethodsAccordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.ResultsWe found that mechanical compression and scrape injury increase TGF-beta2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.ConclusionOur results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

BackgroundAirway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.MethodsAccordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.ResultsWe found that mechanical compression and scrape injury increase TGF-beta2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.ConclusionOur results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

TGF-betas have been reported to mediate the repression by progesterone of several matrix metalloproteinases in the human endometrium, thereby preventing menstrual breakdown. Because of conflicting reports on the expression profiles, source, and regulation of the TGF-beta system in this tissue, we investigated by real-time RT-PCR and ELISA the expression of the three TGF-betas (total and mature forms) and their two receptors throughout the menstrual cycle, and their regulation by ovarian steroids in cultured explants including their microdissected epithelial and stromal compartments. Regulation by cAMP and MAPK was further investigated. This comprehensive study on a large collection of endometrial samples evidenced a differential regulation of TGF-beta isoforms expression, both in vivo and in explant culture. In vivo, TGF-beta2 increased by about 5-fold at the mid-late secretory phase then declined after menstruation; TGF-beta3 increased at menstruation and remained high during the proliferative phase; TGF-beta1 was maximal at menstruation. In explants cultured without ovarian steroids both TGF-beta2 and -beta3 were preferentially expressed in the stroma. Ovarian steroids strongly repressed both TGF-beta2 and -beta3 in stroma but only TGF-beta2 in glands. cAMP prevented inhibition by ovarian steroids of TGF-beta2 but not -beta3. In presence of ovarian steroids, MAPK inhibitors (p38 and ERK pathways) stimulated TGF-beta3 but inhibited TGF-beta2 expression. In conclusion, TGF-beta2 and -beta3 are differentially expressed during the menstrual cycle and regulated by progesterone in epithelial vs stromal cells. The opposite regulation of TGF-beta2 and -beta3 by cAMP and MAPK could account for their distinct expression in vivo.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

The epithelium influences the mesenchyme during dynamic processes such as embryogenesis, wound healing, fibrosis, and carcinogenesis. Since transforming growth factor-beta (TGF-beta) modulates these processes, we hypothesized that epithelial-derived TGF-beta also plays a critical role in maintaining the extracellular matrix at basal conditions. We utilized an in vitro model of the epithelial-mesenchymal trophic unit in the human airways to determine the role of epithelial-derived TGF-beta in modulating the extracellular matrix under basal and wound-healing conditions. When differentiated at an air-liquid interface, the human bronchial epithelium produces active TGF-beta2 at a concentration of 50-70 pg/ml, whereas TGF-beta1 is undetectable. TGF-beta2 increases two- to threefold following scrape injury in a dose-dependent fashion and significantly enhances both alpha-smooth muscle actin expression in the underlying collagen-embedded fibroblasts and secretion of tenascin-C into the matrix. Multiphoton microscopy demonstrates substantially enhanced second harmonic generation from fibrillar collagen in the matrix. Pretreatment of the matrix with either sirolimus (2.5 nM) or paclitaxel (10 nM) abolishes the increases in both TGF-beta2 and second harmonic generation in response to epithelial injury. In the absence of the epithelium, exogenous active TGF-beta2 (0-400 pg/ml) produces a biphasic response in the second harmonic signal with a minimum occurring at the epithelial-derived basal level. We conclude that epithelial-derived TGF-beta2 is secreted in response to injury, significantly alters the bulk optical properties of the extracellular matrix, and its tight regulation may be required for normal collagen homeostasis.

Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity. In this study, we have investigated the mechanism by which TGF-beta(1), TGF-beta(2) and TGF-beta(3) stimulate biglycan mRNA expression in the osteoblastic cell line MG-63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5' DNA was found to respond to TGF-beta(1) with increased transcriptional activity in a dose-dependent manner. Also TGF-beta(2) and TGF-beta(3), two structurally highly related TGF-beta isoforms stimulated biglycan transcription. A TGF-beta responsive region was identified within the first 218 bp of the human biglycan promoter upstream from the transcriptional start site, which contained several binding sites for the transcription factor Sp1. Electrophoretic mobility shift assays with nuclear extracts from MG-63 cells showed binding of both Sp1 and Sp3 to a site at -216 to -208. When the biglycan promoter construct was co-transfected with Sp1 and Sp3 expression vectors in Sp1-deficient Drosophila Schneider-2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF-beta in MG-63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first time that TGF-beta(1) stimulation of human biglycan mRNA expression relies on increased transcription of the biglycan gene, and is mediated by members of the Sp1 family of transcription fact

Biglycan is a small leucine-rich proteoglycan which is localized in the extracellular matrix of bone and other specialized connective tissues. Both biglycan mRNA and protein are up-regulated by transforming growth factor-beta(1) (TGF-beta(1)) and biglycan appears to influence TGF-beta(1) activity. In this study, we have investigated the mechanism by which TGF-beta(1), TGF-beta(2) and TGF-beta(3) stimulate biglycan mRNA expression in the osteoblastic cell line MG-63. The cells were transfected with a series of deletional human biglycan promoter constructs and a region in the biglycan 5' DNA was found to respond to TGF-beta(1) with increased transcriptional activity in a dose-dependent manner. Also TGF-beta(2) and TGF-beta(3), two structurally highly related TGF-beta isoforms stimulated biglycan transcription. A TGF-beta responsive region was identified within the first 218 bp of the human biglycan promoter upstream from the transcriptional start site, which contained several binding sites for the transcription factor Sp1. Electrophoretic mobility shift assays with nuclear extracts from MG-63 cells showed binding of both Sp1 and Sp3 to a site at -216 to -208. When the biglycan promoter construct was co-transfected with Sp1 and Sp3 expression vectors in Sp1-deficient Drosophila Schneider-2 cells, Sp1 induced the transcriptional activity of biglycan. Addition of Sp3 augmented the effect of Sp1 on biglycan gene expression. Induction of biglycan mRNA expression in response to TGF-beta in MG-63 cells was abrogated by mithramycin, an inhibitor of Sp1 binding to GC-rich DNA sequences. A mutation in the Sp1 site at -216 to -208 within the -218 biglycan promoter construct substantially diminished the transcriptional up-regulation by TGF-beta(1). Taken together this data shows for the first time that TGF-beta(1) stimulation of human biglycan mRNA expression relies on increased transcription of the biglycan gene, and is mediated by members of the Sp1 family of transcription fact