Differential Coupling of Self-Renewal Signaling Pathways in Murine Induced Pluripotent Stem Cells

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors

Garden and landscape-scale correlates of moths of differing conservation status: significant effects of urbanization and habitat diversity

Moths are abundant and ubiquitous in vegetated terrestrial environments and are pollinators, important herbivores of wild plants, and food for birds, bats and rodents. In recent years, many once abundant and widespread species have shown sharp declines that have been cited by some as indicative of a widespread insect biodiversity crisis. Likely causes of these declines include agricultural intensification, light pollution, climate change, and urbanization; however, the real underlying cause(s) is still open to conjecture. We used data collected from the citizen science Garden Moth Scheme (GMS) to explore the spatial association between the abundance of 195 widespread British species of moth, and garden habitat and landscape features, to see if spatial habitat and landscape associations varied for species of differing conservation status. We found that associations with habitat and landscape composition were species-specific, but that there were consistent trends in species richness and total moth abundance. Gardens with more diverse and extensive microhabitats were associated with higher species richness and moth abundance; gardens near to the coast were associated with higher richness and moth abundance; and gardens in more urbanized locations were associated with lower species richness and moth abundance. The same trends were also found for species classified as increasing, declining and vulnerable under IUCN (World Conservation Union) criteria

Short and medium-term effects of an education self-management program for individuals with osteoarthritis of the knee, designed and delivered by health professionals: A quality assurance study

<p>Abstract</p> <p>Background</p> <p>Self-management (SM) programs are effective for some chronic conditions, however the evidence for arthritis SM is inconclusive. The aim of this case series project was to determine whether a newly developed specific self-management program for people with osteoarthritis of the knee (OAK), implemented by health professionals could achieve and maintain clinically meaningful improvements.</p> <p>Methods</p> <p><it>Participants: </it>79 participants enrolled; mean age 66, with established osteoarthritis of the knee. People with coexisting inflammatory joint disease or serious co-morbidities were excluded.</p> <p><it>Intervention: </it>6-week disease (OA) and site (knee) specific self-management education program that included disease education, exercise advice, information on healthy lifestyle and relevant information within the constructs of self-management. This program was conducted in a community health care setting and was delivered by health professionals thereby utilising their knowledge and expertise.</p> <p><it>Measurements: </it>Pain, physical function and mental health scales were assessed at baseline, 8 weeks, 6 and 12 months using WOMAC and SF-36 questionnaires. Changes in pain during the 8-week intervention phase were monitored with VAS.</p> <p>Results</p> <p>Pain improved during the intervention phase: mean (95% CI) change 15 (8 to 22) mm. Improvements (0.3 to 0.5 standard deviation units) in indices of pain, mental health and physical functioning, assessed by SF-36 and WOMAC questionnaires were demonstrated from baseline to 12 months.</p> <p>Conclusion</p> <p>This disease and site-specific self-management education program improved health status of people with osteoarthritis of the knee in the short and medium term.</p

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Zscan4 is regulated by PI3-kinase and DNA-damaging agents and directly interacts with the transcriptional repressors LSD1 and CtBP2 in mouse embryonic stem cells.

The Zscan4 family of genes, encoding SCAN-domain and zinc finger-containing proteins, has been implicated in the control of early mammalian embryogenesis as well as the regulation of pluripotency and maintenance of genome integrity in mouse embryonic stem cells. However, many features of this enigmatic family of genes are poorly understood. Here we show that undifferentiated mouse embryonic stem cell (ESC) lines simultaneously express multiple members of the Zscan4 gene family, with Zscan4c, Zscan4f and Zscan4-ps2 consistently being the most abundant. Despite this, between only 0.1 and 0.7% of undifferentiated mouse pluripotent stem cells express Zscan4 protein at a given time, consistent with a very restricted pattern of Zscan4 transcripts reported previously. Herein we demonstrate that Zscan4 expression is regulated by the p110α catalytic isoform of phosphoinositide 3-kinases and is induced following exposure to a sub-class of DNA-damage-inducing agents, including Zeocin and Cisplatin. Furthermore, we observe that Zscan4 protein expression peaks during the G2 phase of the cell cycle, suggesting that it may play a critical role at this checkpoint. Studies with GAL4-fusion proteins suggest a role for Zscan4 in transcriptional regulation, further supported by the fact that protein interaction analyses demonstrate that Zscan4 interacts with both LSD1 and CtBP2 in ESC nuclei. This study advances and extends our understanding of Zscan4 expression, regulation and mechanism of action. Based on our data we propose that Zscan4 may regulate gene transcription in mouse ES cells through interaction with LSD1 and CtBP2

Mutant Escherichia coli Heat-Labile Toxin B Subunit That Separates Toxoid-Mediated Signaling and Immunomodulatory Action from Trafficking and Delivery Functions

The homopentameric B-subunit components of Escherichia coli heat-labile enterotoxin (EtxB) and cholera toxin (CtxB) possess the capacity to enter mammalian cells and to activate cell-signaling events in leukocytes that modulate immune cell function. Both properties have been attributed to the ability of the B subunits to bind to GM1-ganglioside receptors, a ubiquitous glycosphingolipid found in the plasma membrane. Here we describe the properties of EtxB(H57S), a mutant B subunit with a His→Ser substitution at position 57. The mutant was found to be severely defective in inducing leukocyte signaling, as shown by failure to (i) trigger caspase 3-mediated CD8(+)-T-cell apoptosis, (ii) activate nuclear translocation of NF-κB in Jurkat T cells, (iii) induce a potent anti-B-subunit response in mice, or (iv) serve as a mucosal adjuvant. However, its GM1 binding, cellular uptake, and delivery functions remained intact. This was further validated by the finding that EtxB(H57S) was as effective as EtxB in delivering a conjugated model class I epitope into the major histocompatibility complex class I pathway of a dendritic cell line. These observations imply that GM1 binding alone is not sufficient to trigger the signaling events responsible for the potent immunomodulatory properties of EtxB. Moreover, they demonstrate that its signaling properties play no role in EtxB uptake and trafficking. Thus, EtxB(H57S) represents a novel tool for evaluating the complex cellular interactions and signaling events occurring after receptor interaction, as well as offering an alternative means of delivering attached peptides in the absence of the potent immunomodulatory signals induced by wild-type B subunits

Glycogen Synthase Kinase-3 Inhibition Enhances Translation of Pluripotency-Associated Transcription Factors to Contribute to Maintenance of Mouse Embryonic Stem Cell Self-Renewal

Maintenance of embryonic stem cell (ESC) self-renewal and pluripotency are controlled by extrinsic factors, molecular signaling pathways and transcriptional regulators. While many of the key players have been studied in depth, how the molecular signals interact with transcription factors of the pluripotency network to regulate their action remains less well understood. Inhibition of glycogen synthase kinase 3 (Gsk-3) has been implicated in the maintenance of mouse ESC pluripotency, although there is contradictory data on its role, with enhancement of cell survival and metabolism, stabilisation of c-Myc and activation of Wnt signalling proposed as potential mechanisms. We have discovered that suppression of Gsk-3 activity leads to enhanced protein levels of key transcriptional regulators of the pluripotency network, notably Nanog, Tbx3 and c-Myc. Protein stability was unchanged following Gsk-3 inhibition, although interestingly, Nanog and Tbx3 proteins were found to have half-lives of 1–3 h, while that of Oct4 protein was longer, at 6 h. We demonstrate that the effects on protein levels seen following inhibition of Gsk-3 are due to both enhanced de novo synthesis of Nanog protein and increases in the proportion of Nanog and Tbx3 RNAs bound to polysomes, findings consistent with Gsk-3 regulating translation of these factors. These effects were not due to changes in regulators of general translation initiation machinery nor mediated via the 5′ or 3′ UTR sequences of Nanog alone. The data we present provide both new conceptual insight into the mechanisms regulated by Gsk-3 that may contribute to ESC self-renewal and, importantly, establish control of protein translation as an additional mechanism involved in modulation of ESC pluripotency

Zscan4 gene expression is regulated by the p110α isoform of PI3Ks.

<p><b>A.</b> (<b>i</b>) and (<b>ii</b>) E14 ESCs were cultured in the presence of LIF, with the addition of DMSO (control) or the inhibitors indicated: 5 µM LY294002 (broad spectrum PI3K inhibitor), 10 µM TGX121 (p110β), 5 µM IC87114 (p110δ), 25 nM PIK75 (p110α), 100 nM TGX221 (p110β), for 48 h. RNA was extracted and levels of Zscan4 expression analysed by qRT-PCR and normalised to levels of β-actin. Mean values are shown with standard deviations (n = 4). **, p<0.005, ***, p<0.0005 in a Student's t-test. <b>B.</b> ESCs were cultured in LIF and treated with either DMSO as a control or with 5 µM LY294002 (LY), 10 nM PIK75, 50 nM TGX221 (TGX), or 5 µM IC87114 for 48 h prior to immunostaining for Zscan4. The mean percentage of Zscan4 positive cells with SEM are shown. *, p<0.05, **, p<0.005 following an ANOVA and Tukey's post-hoc test. <b>C.</b> ESC clones over-expressing myristoylated p110α catalytic subunit of PI3Ks were cultured in the presence or absence of LIF. As a control parental OCRG9 ESCs were grown in presence and absence of LIF for 4 days. Expression of Zscan4 was analysed by qRT-PCR and Zscan4 expression normalised to levels of GAPDH. The averages and SEM of triplicate samples from each of three independent biological replicates are shown: ***, p<0.0005, in a Student's t-test.</p

Zscan4 protein expression is highly restricted in mouse ESC and iPSC lines.

<p><b>A.</b> The ZE3-MC1 Zscan4 reporter line <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089821#pone.0089821-Zalzman1" target="_blank">[25]</a> was cultured in serum replacement plus LIF, cells were fixed and immunostaining was carried out with pan-Zscan4 antibodies (Red). Expression of the Emerald reporter is shown (Green) and cells were counterstained with DAPI (Blue). (<b>i</b>) Scale bars = 100 µm. (<b>ii</b>) Scale bars = 5 µm. <b>B.</b> The mouse ESC and iPSC lines indicated were cultured in the presence of serum replacement plus LIF and subsequently fixed and immunostained with the pan-Zscan4 antibody, while nuclei were counter-stained with DAPI. The proportion of Zscan4 positive cells present were determined by counting several thousand cells for each cell line sample. Data represent average of three independent replicates ± SEM, unless otherwise indicated. <b>C.</b> A colony of Nanog-GFP reporter cells stained with the anti-Zscan4 antibody. (<b>i</b>) The GFP reporter (Green), Zscan4 (Red) and nuclei (DAPI, Blue) are shown, along with a merged image. (<b>ii</b>) Nanog-GFP cells were stained for Zscan4 protein. The levels of Nanog-GFP and Zscan4 protein expression were calculated using Cell-P (Olympus) using fluorescent images captured using an Olympus IX51 epifluorescence microscope. Mean fluorescence intensity for GFP (Nanog) was compared between all cells, Zscan4 expressing (+ve) and non-expressing (−ve) cells.</p