Proteinase-activated receptor (PAR)-2, a G-protein-coupled receptor for trypsin and mast cell tryptase, is highly expressed in the intestine. Luminal trypsin and tryptase are elevated in the colon of inflammatory bowel disease patients. We hypothesized that luminal proteinases activate PAR-2 and induce colonic inflammation. Mice received intracolonically PAR-2 agonists (trypsin, tryptase, and a selective PAR-2-activating peptide) or control drugs (boiled enzymes, inactive peptide) and inflammatory parameters were followed at various times after this treatment. Colonic administration of PAR-2 agonists up-regulated PAR-2 expression and induced an inflammatory reaction characterized by granulocyte infiltration, increased wall thickness, tissue damage, and elevated T-helper cell type 1 cytokine. The inflammation was maximal between 4 and 6 hours and was resolved 48 hours after the intracolonic administration. PAR-2 activation also increased paracellular permeability of the colon and induced bacterial trans-location into peritoneal organs. These proinflammatory and pathophysiological changes observed in wild-type mice were not detected in PAR-2-deficient mice. Luminal proteinases activate PAR-2 in the mouse colon to induce inflammation and disrupt the integrity of the intestinal barrier. Because trypsin and tryptase are found at high levels in the colon lumen of patients with Crohn's disease or ulcerative colitis, our data may bear directly on the pathophysiology of human inflammatory bowel diseases.

The endocrine system dynamically controls tissue differentiation and homeostasis, but has not been studied using dynamic tissue culture paradigms. Here we show that a microfluidic system supports murine ovarian follicles to produce the human 28-day menstrual cycle hormone profile, which controls human female reproductive tract and peripheral tissue dynamics in single, dual and multiple unit microfluidic platforms (Solo-MFP, Duet-MFP and Quintet-MPF, respectively). These systems simulate the in vivo female reproductive tract and the endocrine loops between organ modules for the ovary, fallopian tube, uterus, cervix and liver, with a sustained circulating flow between all tissues. The reproductive tract tissues and peripheral organs integrated into a microfluidic platform, termed EVATAR, represents a powerful new in vitro tool that allows organ–organ integration of hormonal signalling as a phenocopy of menstrual cycle and pregnancy-like endocrine loops and has great potential to be used in drug discovery and toxicology studies.

Hedgehog (Hh) signaling is essential for multiple aspects of embryogenesis1 , 2. In Drosophila, Hh transduction is mediated by a cytoplasmic signaling complex3 -5 that includes the putative serinethreonine kinase Fused (Fu) and the kinesin Costal 2 (Cos2), yet Fu does not play a conserved role in Hh signaling in mammals6 , 7. Mouse Fu mutants are viable and appear to respond normally to Hh signaling. Here we show that mouse Fu is essential for construction of the central pair (CP) apparatus of motile, 9+2 cilia and offers a novel model of human primary ciliary dyskinesia. We found that mouse Fu physically interacts with Kif27, a mammalian Cos2 ortholog8, and linked Fu to known structural components of the CP apparatus, providing evidence for the first regulatory component involved in CP construction. We also demonstrated that zebrafish Fu is required both for Hh signaling and cilia biogenesis in Kupffer's vesicle. Mouse Fu rescued both Hh-dependent and independent defects in zebrafish. Our results delineate a novel pathway for CP apparatus assembly, identify common regulators of Hh signaling and motile ciliogenesis, and add insight into evolution of the Hh cascade.To further investigate the role of Fu in mammalian Hh signaling, we asked whether Hhdependent Smo localization to the primary cilium is affected in the absence of Fu. Primary cilia, which have a "9+0" arrangement of 9 outer doublet microtubules (MTs), are required for Hh responses and contain several Hh pathway components2 , 9. We found that Fu -/-mouse embryonic fibroblasts (MEFs) formed primary cilia normally, trafficked Smo to the primary cilium in response to Hh ligand, and exhibited a typical Gli transcriptional response (Supplementary Fig. 1 , 2; data not shown). This suggests that the single mammalian Fu ortholog is dispensable for Hh signaling. To explore the function of Fu in mice, we examined its expression in postnatal tissues by in situ hybridization. Fu transcript was expressed strongly in the respiratory epithelium, the ependymal lining of the ventricles in the brain, and in oviduct and testis ( Fig. 1a-c; data not shown). These expression patterns are reminiscent of genes involved in biogenesis of motile cilia, which function in these tissues to propel mucus, fluid and cells. In contrast to the primary cilium, the classical "9+2" motile cilium consists of 9 outer doublet MTs and two singlet central pair (CP) MTs10. The CP apparatus plays a key role in regulating ciliary motility but its formation is poorly understood since the centriole-derived basal body, from which the cilia axoneme extends, does not provide a template for CP outgrowth. Disruption of human motile cilia function leads to primary ciliary dyskinesia (PCD), which is associated with recurrent respiratory infection, hydrocephalus and infertility11 -13. To determine whether motile cilia function is * These authors contributed equally to this work.

Transcription enhancer factor 1 is essential for cardiac, skeletal, and smooth muscle development and uses its N-terminal TEA domain (TEAD) to bind M-CAT elements. Here, we present the first structure of TEAD and show that it is a three-helix bundle with a homeodomain fold. Structural data reveal how TEAD binds DNA. Using structure-function correlations, we find that the L1 loop is essential for cooperative loading of TEAD molecules on to tandemly duplicated M-CAT sites. Furthermore, using a microarray chip-based assay, we establish that known binding sites of the full-length protein are only a subset of DNA elements recognized by TEAD. Our results provide a model for understanding the regulation of genome-wide gene expression during development by TEA͞ATTS family of transcription factors.DNA-binding protein ͉ gene regulation ͉ NMR structure ͉ development ͉ Scalloped

Background and purpose: Protease-activated receptor-4 (PAR 4 ), the most recently discovered member of the PARs family, is activated by thrombin, trypsin and cathepsin G, but can also be selectively activated by small synthetic peptides (PAR 4 -activating peptide, PAR 4 -AP). PAR 4 is considered a potent mediator of platelet activation and inflammation. As both PAR 1 and PAR 2 have been implicated in the modulation of nociceptive mechanisms, we investigated the expression of PAR 4 in sensory neurons and the effects of its selective activation on nociception. Experimental approach and key results:We demonstrated the expression of PAR 4 in sensory neurons isolated from rat dorsal root ganglia by reverse transcription-polymerase chain reaction and immunofluorescence. We found that PAR 4 colocalized with calcitonin gene-related peptide and substance P. We also showed that a selective PAR 4 -AP was able to inhibit calcium mobilization evoked by KCl and capsaicin in rat sensory neurons. Moreover, the intraplantar injection of a PAR 4 -AP significantly increased nociceptive threshold in response to thermal and mechanical noxious stimuli, while a PAR 4 inactive control peptide had no effect. The anti-nociceptive effects of the PAR 4 -AP were dose-dependent and occurred at doses below the threshold needed to cause inflammation. Finally, co-injection of the PAR 4 -AP with carrageenan significantly reduced the carrageenaninduced inflammatory hyperalgesia and allodynia, but had no effect on inflammatory parameters such as oedema and granulocyte infiltration. Conclusions and implications: Taken together, these results identified PAR 4 as a novel potential endogenous analgesic factor, which can modulate nociceptive responses in normal and inflammatory conditions.

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The epidermal growth factor receptor is overexpressed in up to 60% of ovarian epithelial malignancies. EGFR regulates complex cellular events due to the large number of ligands, dimerization partners, and diverse signaling pathways engaged. In ovarian cancer, EGFR activation is associated with increased malignant tumor phenotype and poorer patient outcome. However, unlike some other EGFR-positive solid tumors, treatment of ovarian tumors with anti-EGFR agents has induced minimal response. While the amount of information regarding EGFR-mediated signaling is considerable, current data provides little insight for the lack of efficacy of anti-EGFR agents in ovarian cancer. More comprehensive, systematic, and well-defined approaches are needed to dissect the roles that EGFR plays in the complex signaling processes in ovarian cancer as well as to identify biomarkers that can accurately predict sensitivity toward EGFR-targeted therapeutic agents. This new knowledge could facilitate the development of rational combinatorial therapies to sensitize tumor cells toward EGFR-targeted therapies.

Longer eyes, greater scleral strain in some directions at baseline, and generalized scleral thinning after glaucoma were characteristic of CD1 mice that have greater tendency to retinal ganglion cell damage than B6 mice. Increased scleral stiffness after glaucoma exposure in mice mimics findings in monkey and human glaucoma eyes.