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Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions,wherein the regulation of Wnt/$\beta$-catenin pathway is often desirable. Here,we examined the effect of trolox,a vitamin E analog,on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/$\beta$-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/$\beta$-catenin regulator. View Publication

S-glutathionylation of reactive protein cysteines is a post-translational event that plays a critical role in transducing signals from oxidants into biological responses. S-glutathionylation can be reversed by the deglutathionylating enzyme glutaredoxin (GLRX). We have previously demonstrated that ablation of Glrx sensitizes mice to the development of parenchymal lung fibrosis(1). It remains unclear whether GLRX also controls airway fibrosis,a clinical feature relevant to asthma and chronic obstructive pulmonary disease,and whether GLRX controls the biology of airway epithelial cells,which have been implicated in the pathophysiology of these diseases. In the present study we utilized a house dust mite (HDM) model of allergic airway disease in wild type (WT) and Glrx-/- mice on a C57BL/6 background prone to develop airway fibrosis,and tracheal basal stem cells derived from WT mice,global Glrx-/- mice,or bi-transgenic mice allowing conditional ablation of the Glrx gene. Herein we show that absence of Glrx led to enhanced HDM-induced collagen deposition,elevated levels of transforming growth factor beta 1 (TGFB1) in the bronchoalveolar lavage,and resulted in increases in airway hyperresponsiveness. Airway epithelial cells isolated from Glrx-/- mice or following conditional ablation of Glrx showed spontaneous increases in secretion of TGFB1. Glrx-/- basal cells also showed spontaneous TGFB pathway activation,in association with increased expression of mesenchymal genes,including collagen 1a1 and fibronectin. Overall,these findings suggest that GLRX regulates airway fibrosis via a mechanism(s) that involve the plasticity of basal cells,the stem cells of the airways. View Publication

Murine models of myeloid neoplasia show how leukemia infiltration alters the hematopoietic stem cell (HSC) niche to reinforce malignancy at the expense of healthy hematopoiesis. However,little is known about the bone marrow architecture in humans and its impact on clinical outcome. Here,we dissect the bone marrow niche in patients with acute myeloid leukemia (AML) at first diagnosis. We combined immunohistochemical stainings with global gene expression analyses from these AML patients and correlated them with clinical features. Mesenchymal stem and progenitor cells (MSPCs) lost quiescence and significantly expanded in the bone marrow of AML patients. Strikingly,their HSC- and niche-regulating capacities were impaired with significant inhibition of osteogenesis and bone formation in a cell contact-dependent manner through inhibition of cytoplasmic $\beta$-catenin. Assessment of bone metabolism by quantifying peripheral blood osteocalcin levels revealed 30{\%} lower expression in AML patients at first diagnosis than in non-leukemic donors. Furthermore,patients with osteocalcin levels ≤11 ng/mL showed inferior overall survival with a 1-year survival rate of 38.7{\%} whereas patients with higher osteocalcin levels reached a survival rate of 66.8{\%}. These novel insights into the human AML bone marrow microenvironment help translate findings from preclinical models and detect new targets which might pave the way for niche-targeted therapies in AML patients. View Publication

Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes,but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study,we demonstrated that stabilizing actin filaments by inhibiting gene expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs,enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro,as well as heterotopic bone formation in vivo. Similarly,treating hMSC with Phalloidin,which is known to stabilize polymerized actin filaments,increased hMSCs viability and OB differentiation. Conversely,Cytocholasin D,an inhibitor of actin polymerization,reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level,preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1 (LIMK1) decreased cell viability and impaired OB differentiation of hMSCs. Moreover,depolymerizing actin reduced FAK,p38 and JNK activation during OB differentiation of hMSCs,while polymerizing actin enhanced these signaling pathways. Our results demonstrate that the actin dynamic reassembly and Cofilin phosphorylation loop is involved in the control of hMSC proliferation and osteoblasts differentiation. View Publication

The adoption of Warburg metabolism is critical for the activation of macrophages in response to lipopolysaccharide. Macrophages stimulated with lipopolysaccharide increase their expression of nicotinamide phosphoribosyltransferase (NAMPT),a key enzyme in NAD+ salvage,and loss of NAMPT activity alters their inflammatory potential. However,the events that lead to the cells' becoming dependent on NAD+ salvage remain poorly defined. We found that depletion of NAD+ and increased expression of NAMPT occurred rapidly after inflammatory activation and coincided with DNA damage caused by reactive oxygen species (ROS). ROS produced by complex III of the mitochondrial electron-transport chain were required for macrophage activation. DNA damage was associated with activation of poly(ADP-ribose) polymerase,which led to consumption of NAD+. In this setting,increased NAMPT expression allowed the maintenance of NAD+ pools sufficient for glyceraldehyde-3-phosphate dehydrogenase activity and Warburg metabolism. Our findings provide an integrated explanation for the dependence of inflammatory macrophages on the NAD+ salvage pathway. View Publication

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2,increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl,however,accelerated the rate of actin polymerization,although still lowering the final steady state viscosity. Cytochalasin B,at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl,accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2,cytochalasin D uncoupled the actin ATPase activity from actin polymerization,increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and,to a much greater extent,stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D,the initial rate of actin ATPase activity,when little or no polymerization had occurred,was directly proportional to the actin concentration and,therefore,apparently was independent of actin-actin interactions. To rationalize all these data,a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP,A . ATP,to monomeric actin-bound ADP and Pi,A* . ADP . Pi,which,like the preferred growing end of an actin filament,can bind cytochalasins. View Publication

INTRODUCTION One method for assessing the in vitro response to CFTR-modulating compounds is by analysis of epithelial monolayers in an Ussing chamber,where the apical and basolateral surfaces are isolated and the potential difference,short-circuit current,and transepithelial resistance can be monitored. The effect of a chloride ion gradient across airway epithelia on transepithelial chloride transport and the magnitude of CFTR modulator efficacy were examined. METHODS CFTR-mediated changes in the potential difference and transepithelial currents of primary human nasal epithelial cell cultures were quantified in Ussing chambers with either symmetrical solutions or reduced chloride solutions in the apical chamber. CFTR activity in homozygous F508del CFTR epithelia was rescued by treatment with VX-661,C4/C18,4-phenylbutyrate (4-PBA) for 24 hr at 37°C or by incubation at 29°C for 48 hr. RESULTS Imposing a chloride gradient increased CFTR-mediated and CaCC-mediated ion transport. Treatment of F508del CFTR homozygous cells with CFTR modulating compounds increased CFTR activity,which was significantly more evident in the presence of a chloride gradient. This observation was recapitulated with temperature-mediated F508del CFTR correction. CONCLUSIONS Imposing a chloride gradient during Ussing chamber measurements resulted in increased CFTR-mediated ion transport in expanded non-CF and F508del CFTR homozygous epithelia. In F508del CFTR homozygous epithelia,the magnitude of response to CFTR modulating compounds or low temperature was greater when assayed with a chloride gradient compared to symmetrical chloride,resulting in an apparent increase in measured efficacy. Future work may direct which methodologies utilized to quantify CFTR modulator response in vitro are most appropriate for the estimation of in vivo efficacy. View Publication

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo,miniaturization of respiratory drug discovery assays is of pivotal importance. Thus,a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air-liquid interface (ALI). After four weeks of ALI culture,a pseudostratified epithelium containing basal,club,goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition,ciliary beating frequency,and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements,together with dextran permeability measurements,confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor $\beta$1 (TGF-$\beta$1) and tumor necrosis factor $\alpha$ (TNF-$\alpha$) in a concentration dependent manner. Thus,the miniaturized cellular model system enables the recapitulation of a physiologically responsive,differentiated small airway epithelium,and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery,for instance,in respect of fibrotic distal airway/lung diseases. View Publication

NAD is a multifunctional molecule involved in both metabolic processes and signaling pathways. Such signalling pathways consume NAD which is replenished via one of several biosynthesis pathways. We show that influx of NAD across the plasma membrane may be able to contribute to the homeostasis of intracellular NAD levels. Indeed,extracellular application of NAD was able to replete NAD levels that had been lowered pharmacologically using the novel drug FK866 and was also able to rescue cells from FK866-induced cell death. A marked lag between the drop in NAD levels and cell death prompted us to investigate the mechanism of cell death. We were unable to find evidence of apoptosis as assessed by immunoblotting for the Caspase 3 activation fragment and immunostaining for cytochrome C and AIF translocation. We,therefore,investigated whether autophagy was initiated by FK866. Indeed,we were able to observe the formation of LC3-positive vesicles that had fused with lysosomes in FK866-treated but not control cells. Furthermore,this autophagic phenotype could be reverted by the addition of NAD to the extracellular medium. View Publication

Cellular immunotherapeutics targeting the human papillomavirus (HPV)-16 E6 and E7 proteins have achieved limited success in HPV-positive oropharyngeal cancer (OPC). Here we have conducted proteome-wide profiling of HPV-16-specific T cell responses in a cohort of 66 patients with HPV-associated OPC and 22 healthy individuals. Unexpectedly,HPV-specific T cell responses from OPC patients were not constrained to the E6 and E7 antigens; they also recognized E1,E2,E4,E5,and L1 proteins as dominant targets for virus-specific CD8+ and CD4+ T cells. Multivariate analysis incorporating tumor staging,treatment status,and smoking history revealed that treatment status had the most significant impact on HPV-specific CD8+ and CD4+ T cell immunity. Specifically,the breadth and overall strength of HPV-specific T cell responses were significantly higher before the commencement of curative therapy than after therapy. These data provide the first glimpse of the overall human T cell response to HPV in a clinical setting and offer groundbreaking insight into future development of cellular immunotherapies for HPV-associated OPC patients. View Publication