Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Bone turnover in vivo is regulated by mechanical forces such as shear stress originating from interstitial oscillatory fluid flow (OFF), and bone cells in vitro respond to mechanical loading. However, the mechanisms by which bone cells sense mechanical forces, resulting in increased mineral deposition, are not well understood. The aim of this study was to investigate the role of the primary cilium in mechanosensing by osteoblasts. MLO-A5 murine osteoblasts were cultured in monolayer and subjected to two different OFF regimens: 5 short (2 h daily) bouts of OFF followed by morphological analysis of primary cilia; or exposure to chloral hydrate to damage or remove primary cilia and 2 short bouts (2 h on consecutive days) of OFF. Primary cilia were shorter and there were fewer cilia per cell after exposure to periods of OFF compared with static controls. Damage or removal of primary cilia inhibited OFF-induced PGE2 release into the medium and mineral deposition, assayed by Alizarin red staining. We conclude that primary cilia are important mediators of OFF-induced mineral deposition, which has relevance for the design of bone tissue engineering strategies and may inform clinical treatments of bone disorders causes by load-deficiency.—Delaine-Smith, R. M., Sittichokechaiwut, A., Reilly, G. C. Primary cilia respond to fluid shear stress and mediate flow-induced calcium deposition in osteoblasts

Treewilder

Treewilder is an exhibition of site responsive interventions, sculptures and video work set in Barnwell Country park aiming to encourage visitors to explore the woodland space throughout autumn and winter aspart of the two year Xylophobia season

Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3

Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a beta-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies. Objectives: To examine the role of galectin-3 in pulmonary fibrosis. Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF. Measurements and Main Results: Transforming growth factor (TGF)-beta and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-beta 1 induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of beta-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin -3, TD139, blocked TGF-beta-induced beta-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that. galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF. Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF

Understanding the mechanism of insulin and insulin-like growth factor (IGF) receptor activation by IGF-II

Extent: 9p.Background: Insulin-like growth factor-II (IGF-II) promotes cell proliferation and survival and plays an important role in normal fetal development and placental function. IGF-II binds both the insulin-like growth factor receptor (IGF-1R) and insulin receptor isoform A (IR-A) with high affinity. Interestingly both IGF-II and the IR-A are often upregulated in cancer and IGF-II acts via both receptors to promote cancer proliferation. There is relatively little known about the mechanism of ligand induced activation of the insulin (IR) and IGF-1R. The recently solved IR structure reveals a folded over dimer with two potential ligand binding pockets arising from residues on each receptor half. Site-directed mutagenesis has mapped receptor residues important for ligand binding to two separate sites within the ligand binding pocket and we have recently shown that the IGFs have two separate binding surfaces which interact with the receptor sites 1 and 2. Methodology/Principal Findings: In this study we describe a series of partial IGF-1R and IR agonists generated by mutating Glu12 of IGF-II. By comparing receptor binding affinities, abilities to induce negative cooperativity and potencies in receptor activation, we provide evidence that residue Glu12 bridges the two receptor halves leading to receptor activation. Conclusions/Significance: This study provides novel insight into the mechanism of receptor binding and activation by IGFII,which may be important for the future development of inhibitors of its action for the treatment of cancer.Clair L. Alvino, Shee Chee Ong, Kerrie A. McNeil, Carlie Delaine, Grant W. Booker, John C. Wallace and Briony E. Forbe

Preconditioning of mesenchymal stromal cells with low-intensity ultrasound: influence on chondrogenesis and directed SOX9 signaling pathways

Background: Continuous low-intensity ultrasound (cLIUS) facilitates the chondrogenic differentiation of human mesenchymal stromal cells (MSCs) in the absence of exogenously added transforming growth factor-beta (TGFβ) by upregulating the expression of transcription factor SOX9, a master regulator of chondrogenesis. The present study evaluated the molecular events associated with the signaling pathways impacting SOX9 gene and protein expression under cLIUS. Methods: Human bone marrow-derived MSCs were exposed to cLIUS stimulation at 14 kPa (5 MHz, 2.5 Vpp) for 5 min. The gene and protein expression of SOX9 was evaluated. The specificity of SOX9 upregulation under cLIUS was determined by treating the MSCs with small molecule inhibitors of select signaling molecules, followed by cLIUS treatment. Signaling events regulating SOX9 expression under cLIUS were analyzed by gene expression, immunofluorescence staining, and western blotting. Results: cLIUS upregulated the gene expression of SOX9 and enhanced the nuclear localization of SOX9 protein when compared to non-cLIUS-stimulated control. cLIUS was noted to enhance the phosphorylation of the signaling molecule ERK1/2. Inhibition of MEK/ERK1/2 by PD98059 resulted in the effective abrogation of cLIUS-induced SOX9 expression, indicating that cLIUS-induced SOX9 upregulation was dependent on the phosphorylation of ERK1/2. Inhibition of integrin and TRPV4, the upstream cell-surface effectors of ERK1/2, did not inhibit the phosphorylation of ERK1/2 and therefore did not abrogate cLIUS-induced SOX9 expression, thereby suggesting the involvement of other mechanoreceptors. Consequently, the effect of cLIUS on the actin cytoskeleton, a mechanosensitive receptor regulating SOX9, was evaluated. Diffused and disrupted actin fibers observed in MSCs under cLIUS closely resembled actin disruption by treatment with cytoskeletal drug Y27632, which is known to increase the gene expression of SOX9. The upregulation of SOX9 under cLIUS was, therefore, related to cLIUS-induced actin reorganization. SOX9 upregulation induced by actin reorganization was also found to be dependent on the phosphorylation of ERK1/2. Conclusions: Collectively, preconditioning of MSCs by cLIUS resulted in the nuclear localization of SOX9, phosphorylation of ERK1/2 and disruption of actin filaments, and the expression of SOX9 was dependent on the phosphorylation of ERK1/2 under cLIUS

Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin’s ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues

Archaeology, Architecture, and the Postcolonial Critique

Peer reviewe

Let’s not forget tautomers

A compound exhibits tautomerism if it can be represented by two structures that are related by an intramolecular movement of hydrogen from one atom to another. The different tautomers of a molecule usually have different molecular fingerprints, hydrophobicities and pKa’s as well as different 3D shape and electrostatic properties; additionally, proteins frequently preferentially bind a tautomer that is present in low abundance in water. As a result, the proper treatment of molecules that can tautomerize, ~25% of a database, is a challenge for every aspect of computer-aided molecular design. Library design that focuses on molecular similarity or diversity might inadvertently include similar molecules that happen to be encoded as different tautomers. Physical property measurements might not establish the properties of individual tautomers with the result that algorithms based on these measurements may be less accurate for molecules that can tautomerize—this problem influences the accuracy of filtering for library design and also traditional QSAR. Any 2D or 3D QSAR analysis must involve the decision of if or how to adjust the observed Ki or IC50 for the tautomerization equilibria. QSARs and recursive partitioning methods also involve the decision as to which tautomer(s) to use to calculate the molecular descriptors. Docking virtual screening must involve the decision as to which tautomers to include in the docking and how to account for tautomerization in the scoring. All of these decisions are more difficult because there is no extensive database of measured tautomeric ratios in both water and non-aqueous solvents and there is no consensus as to the best computational method to calculate tautomeric ratios in different environments