The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte-like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca(2+) signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca(2+)-sensitive fluorescent indicator (Ca(2+) measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca(2+) signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca(2+) influx and Ca(2+) release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca(2+) signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca(2+) signals and contractile responses was dramatically influenced by inherent spontaneous Ca(2+) activity. In PVCs with relatively low spontaneous Ca(2+) activity (<1 Hz), entrainment with electrical pacing was good. However, in PVCs with higher frequencies of spontaneous Ca(2+) activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca(2+) activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca(2+) cycling properties. Heterogeneous spontaneous Ca(2+) activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity.

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

Gene therapy of the lung has the potential to treat life-threatening diseases such as cystic fibrosis and α(1)-antitrypsin or surfactant deficiencies. A major hurdle for successful gene therapy is the development of an immune response against the transgene and/or viral vector. We hypothesized that by targeting the airways in the perinatal period, induction of an immune response against the vector particle could be prevented because of immaturity of the immune system, in turn allowing repeated gene transfer later in adult life to ensure long-term gene expression. Therefore, we readministered recombinant adeno-associated viral vector serotype 5 (rAAV2/5) to mouse airways 3 and 6 months after initial perinatal gene transfer. Our findings demonstrate that perinatal rAAV2/5-mediated gene transfer to the airways avoids a strong immune response. This immunological ignorance allows the readministration of an autologous vector later in adult life, resulting in efficient and stable gene transfer up to 7 months, without evidence of a decrease in transgene expression. Together, these data provide a basis to further explore perinatal gene therapy for pulmonary conditions with adequate gene expression up to 7 months.

BIK (BCL2-interacting killer) is a pro-apoptotic BH3 (BCL2 homology domain 3)-only protein and a member of the BCL2 protein family. It was proposed recently that BIK abundance is controlled by ERK1/2 (extracellular-signal-regulated kinase 1/2)-catalysed phosphorylation, which targets the protein for proteasome-dependent destruction. In the present study, we examined ERK1/2-dependent regulation of BIK, drawing comparisons with BIM(EL) (BCL2-interacting mediator of cell death; extra long), a well-known target of ERK1/2. In many ERK1/2-dependent tumour cell lines, inhibition of BRAF(V600E) (v-raf murine sarcoma viral oncogene homologue B1, V600E mutation) or MEK1/2 (mitogen-activated protein kinase/ERK kinase 1/2) had very little effect on BIK expression, whereas BIM(EL) was strongly up-regulated. In some cell lines we observed a modest increase in BIK expression; however, this was not apparent until ~16 h or later, whereas BIM(EL) expression increased rapidly within a few hours. Although BIK was degraded by the proteasome, we found no evidence that this was regulated by ERK1/2 signalling. Rather, the delayed increase in BIK expression was prevented by actinomycin D, and was accompanied by increases in BIK mRNA. Finally, the delayed increase in BIK expression following ERK1/2 inhibition was phenocopied by a highly selective CDK4/6 (cyclin-dependent kinases 4 and 6) inhibitor, which caused a strong G₁ cell-cycle arrest without inhibiting ERK1/2 signalling. In contrast, BIM(EL) expression was induced by ERK1/2 inhibition, but not by CDK4/6 inhibition. We conclude that BIK expression is not subject to direct regulation by the ERK1/2 pathway; rather, we propose that BIK expression is cell-cycle-dependent and increases as a consequence of the G₁ cell-cycle arrest which results from inhibition of ERK1/2 signalling.

Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage.

The GTPase Ras is a molecular switch engaged downstream of G-protein coupled receptors and receptor tyrosine kinases that controls multiple cell fate-determining signalling pathways. Ras signalling is frequently deregulated in cancer underlying associated changes in cell phenotype. Although Ca(2+) signalling pathways control some overlapping functions with Ras, and altered Ca(2+) signalling pathways are emerging as important players in oncogenic transformation, how Ca(2+) signalling is remodelled during transformation and whether it has a causal role remains unclear. We have investigated Ca(2+) signalling in two human colorectal cancer cell lines and their isogenic derivatives in which the mutated K-Ras allele (G13D) has been deleted by homologous recombination. We show that agonist-induced Ca(2+) release from intracellular stores is enhanced by loss of K-Ras(G13D) through an increase in the ER store content and a modification of IP3R subtype abundance. Consistently, uptake of Ca(2+) into mitochondria and sensitivity to apoptosis was enhanced as a result of K-Ras(G13D) loss. These results suggest that suppression of Ca(2+) signalling is a common response to naturally occurring levels of K-Ras(G13D) that contributes to a survival advantage during oncogenic transformation.

A plethora of non-protein coding RNAs are produced throughout eukaryotic genomes, many of which are transcribed antisense to protein-coding genes and could potentially instigate RNA interference (RNAi) responses. Here we have used a synthetic RNAi system to show that gene copy number is a key factor controlling RNAi for transcripts from endogenous loci, since transcripts from multi-copy loci form double stranded RNA more efficiently than transcripts from equivalently expressed single-copy loci. Selectivity towards transcripts from high-copy DNA is therefore an emergent property of a minimal RNAi system. The ability of RNAi to selectively degrade transcripts from high-copy loci would allow suppression of newly emerging transposable elements, but such a surveillance system requires transcription. We show that low-level genome-wide pervasive transcription is sufficient to instigate RNAi, and propose that pervasive transcription is part of a defense mechanism capable of directing a sequence-independent RNAi response against transposable elements amplifying within the genome. DOI: http://dx.doi.org/10.7554/eLife.01581.001.

Gimap3 (IAN4) and Gimap5 (IAN5) are highly homologous GTP-binding proteins of the Gimap family. Gimap3 and Gimap5, whose transcripts are abundant in mature lymphocytes, can associate with antiapoptotic Bcl-2 family proteins. While it is established that Gimap5 regulates T-cell survival, the in vivo role of Gimap3 is unclear. Here we report the preparation and characteristics of mouse strains lacking Gimap3 and/or Gimap5. We found that the number of T cells was markedly reduced in mice deficient in both Gimap3 and Gimap5. The defects in T-cell cellularity were more severe in mice lacking both Gimap3 and Gimap5 than in mice lacking only Gimap5. No defects in the cellularity of T cells were detected in mice lacking only Gimap3, whereas bone marrow cells from Gimap3-deficient mice showed reduced T-cell production in a competitive hematopoietic environment. Moreover, retroviral overexpression and short hairpin RNAs-mediated silencing of Gimap3 in bone marrow cells elevated and reduced, respectively, the number of T cells produced in irradiated mice. These results suggest that Gimap3 is a regulator of T-cell numbers in the mouse and that multiple Gimap family proteins cooperate to maintain T-cell survival.

The binding sites of two ruthenium(II) organometallic complexes of the form [(η(6)-arene)Ru(N,N)Cl](+), where arene/N,N = biphenyl (bip)/bipyridine (bipy) for complex AH076, and biphenyl (bip)/o-phenylenediamine (o-pda) for complex AH078, on the peptides angiotensin and bombesin have been investigated using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Fragmentation was performed using collisionally activated dissociation (CAD), with, in some cases, additional data being provided by electron capture dissociation (ECD). The primary binding sites were identified as methionine and histidine, with further coordination to phenylalanine, potentially through a π-stacking interaction, which has been observed here for the first time. This initial peptide study was expanded to investigate protein binding through reaction with insulin, on which the binding sites proposed are histidine, glutamic acid, and tyrosine. Further reaction of the ruthenium complexes with the oxidized B chain of insulin, in which two cysteine residues are oxidized to cysteine sulfonic acid (Cys-SO3H), and glutathione, which had been oxidized with hydrogen peroxide to convert the cysteine to cysteine sulfonic acid, provided further support for histidine and glutamic acid binding, respectively.

Regulatory T (TReg) cells constitute an essential counterbalance to adaptive immune responses. Failure to maintain appropriate TReg cell numbers or function leads to autoimmune, malignant and immunodeficient conditions. Dynamic homeostatic processes preserve the number of forkhead box P3-expressing (FOXP3(+)) TReg cells within a healthy range, with high rates of cell division being offset by apoptosis under steady-state conditions. Recent studies have shown that TReg cells become specialized for different environmental contexts, tailoring their functions and homeostatic properties to a wide range of tissues and immune conditions. In this Review, we describe new insights into the molecular controls that maintain the steady-state homeostasis of TReg cells and the cues that drive TReg cell adaptation to inflammation and/or different locations. We highlight how differing local milieu might drive context-specific TReg cell function and restoration of immune homeostasis, and how dysregulation of these processes can precipitate disease.

The cytokine IL-21 is a potent immune modulator with diverse mechanisms of action on multiple cell types. IL-21 is in clinical use to promote tumor rejection and is an emerging target for neutralization in the setting of autoimmunity. Despite its clinical potential, the biological actions of IL-21 are not yet fully understood and the full range of effects of this pleiotropic cytokine are still being uncovered. In this study, we identify a novel role for IL-21 as an inducer of the costimulatory ligand CD86 on B lymphocytes. CD86 provides critical signals through T cell-expressed CD28 that promote T cell activation in response to Ag engagement. Expression levels of CD86 are tightly regulated in vivo, being actively decreased by regulatory T cells and increased in response to pathogen-derived signals. In this study, we demonstrate that IL-21 can trigger potent and sustained CD86 upregulation through a STAT3 and PI3K-dependent mechanism. We show that elevated CD86 expression has functional consequences for the magnitude of CD4 T cell responses both in vitro and in vivo. These data pinpoint CD86 upregulation as an additional mechanism by which IL-21 can elicit immunomodulatory effects.

Systemic juvenile idiopathic arthritis (JIA) is unique among the rheumatic diseases of childhood, given its distinctive systemic inflammatory character. Inappropriate control of innate immune responses following an initially harmless trigger is thought to account for the excessive inflammatory reaction. The aim of this study was to generate a similar systemic inflammatory syndrome in mice by injecting a relatively innocuous, yet persistent, immune system trigger: Freund's complete adjuvant (CFA), containing heat-killed mycobacteria.

Glycation is a post-translational modification (PTM) that affects the physiological properties of peptides and proteins. In particular, during hyperglycaemia, glycation by α-dicarbonyl compounds generate α-dicarbonyl-derived glycation products also called α-dicarbonyl-derived advanced glycation end products. Glycation by the α-dicarbonyl compound known as glyoxal was studied in model peptides by MS/MS using a Fourier transform ion cyclotron resonance mass spectrometer. An unusual type of glyoxal-derived AGE with a mass addition of 21.98436 Da is reported in peptides containing combinations of two arginine-two lysine, and one arginine-three lysine amino acid residues. Electron capture dissociation and collisionally activated dissociation results supported that the unusual glyoxal-derived AGE is formed at the guanidino group of arginine, and a possible structure is proposed to illustrate the 21.9843 Da mass addition.

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

Perturbations in endoplasmic reticulum (ER) homeostasis, including depletion of Ca(2+) or altered redox status, induce ER stress due to protein accumulation, misfolding and oxidation. This activates the unfolded protein response (UPR) to re-establish the balance between ER protein folding capacity and protein load, resulting in cell survival or, following chronic ER stress, promotes cell death. The mechanisms for the transition between adaptation to ER stress and ER stress-induced cell death are still being understood. However, the identification of numerous points of cross-talk between the UPR and mitogen-activated protein kinase (MAPK) signalling pathways may contribute to our understanding of the consequences of ER stress. Indeed, the MAPK signalling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses. In this article, we review UPR signalling and the activation of MAPK signalling pathways in response to ER stress. In addition, we highlight components of the UPR that are modulated in response to MAPK signalling and the consequences of this cross-talk. We also describe several diseases, including cancer, type II diabetes and retinal degeneration, where activation of the UPR and MAPK signalling contribute to disease progression and highlight potential avenues for therapeutic intervention. This article is part of a Special Issue entitled: Calcium Signaling In Health and Disease.

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3(Cre/+) , devoid of mast cells but with intact Kit signaling.

Resource description framework (RDF) is an emerging technology for describing, publishing and linking life science data. As a major provider of bioinformatics data and services, the European Bioinformatics Institute (EBI) is committed to making data readily accessible to the community in ways that meet existing demand. The EBI RDF platform has been developed to meet an increasing demand to coordinate RDF activities across the institute and provides a new entry point to querying and exploring integrated resources available at the EBI.

Beta-2 microglobulin (β2m) is an essential component of the major histocompatibility complex (MHC) class I proteins and in the nervous system β2m is predominantly expressed in motor neurons. As β2m can promote nerve regeneration, we investigated its potential role in amyotrophic lateral sclerosis (ALS) by investigating its expression level as well as the effect of genetically removing β2m on the disease process in mutant superoxide dismutase 1 (SOD1 (G93A) ) mice, a model of ALS. We observed a strong upregulation of β2m in motor neurons during the disease process and ubiquitous removal of β2m dramatically shortens the disease duration indicating that β2m plays an essential and positive role during the disease process. We hypothesize that β2m contributes to plasticity that is essential for muscle reinnervation. Absence of this plasticity will lead to faster muscle denervation and counteracting this process could be a relevant therapeutic target.

Insulin activation of phosphoinositide 3-kinase (PI3K) signaling regulates glucose homeostasis through the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3). The dual-specificity phosphatase and tensin homolog deleted on chromosome 10 (PTEN) blocks PI3K signaling by dephosphorylating PIP3, and is inhibited through its interaction with phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 2 (P-REX2). The mechanism of inhibition and its physiological significance are not known. Here, we report that P-REX2 interacts with PTEN via two interfaces. The pleckstrin homology (PH) domain of P-REX2 inhibits PTEN by interacting with the catalytic region of PTEN, and the inositol polyphosphate 4-phosphatase domain of P-REX2 provides high-affinity binding to the postsynaptic density-95/Discs large/zona occludens-1-binding domain of PTEN. P-REX2 inhibition of PTEN requires C-terminal phosphorylation of PTEN to release the P-REX2 PH domain from its neighboring diffuse B-cell lymphoma homology domain. Consistent with its function as a PTEN inhibitor, deletion of Prex2 in fibroblasts and mice results in increased Pten activity and decreased insulin signaling in liver and adipose tissue. Prex2 deletion also leads to reduced glucose uptake and insulin resistance. In human adipose tissue, P-REX2 protein expression is decreased and PTEN activity is increased in insulin-resistant human subjects. Taken together, these results indicate a functional role for P-REX2 PH-domain-mediated inhibition of PTEN in regulating insulin sensitivity and glucose homeostasis and suggest that loss of P-REX2 expression may cause insulin resistance.

The mechanistic target of rapamycin (mTOR) protein kinase coordinates responses to nutrients and growth factors and is an anti-cancer drug target. To anticipate how cells will respond and adapt to chronic mTOR complex (mTORC)1 and mTORC2 inhibition, we have generated SW620 colon cancer cells with acquired resistance to the ATP-competitive mTOR kinase inhibitor AZD8055 (SW620:8055R). AZD8055 inhibited mTORC1 and mTORC2 signalling and caused a switch from cap-dependent to internal ribosome entry site (IRES)-dependent translation in parental SW620 cells. In contrast, SW620:8055R cells exhibited a loss of S6K signalling, an increase in expression of the eukaryotic translation initiation factor eIF4E and increased cap-dependent mRNA translation. As a result, the expression of CCND1 and MCL1, proteins encoded by eIF4E-sensitive and cap-dependent transcripts, was refractory to AZD8055 in SW620:8055R cells. RNAi-mediated knockdown of eIF4E reversed acquired resistance to AZD8055 in SW620:8055R cells; furthermore, increased expression of eIF4E was sufficient to reduce sensitivity to AZD8055 in a heterologous cell system. Finally, although the combination of MEK1/2 inhibitors with mTOR inhibitors is an attractive rational drug combination, SW620:8055R cells were actually cross-resistant to the MEK1/2 inhibitor selumetinib (AZD6244). These results exemplify the convergence of ERK1/2 and mTOR signalling at eIF4E, and the key role of eIF4E downstream of mTOR in maintaining cell proliferation. They also have important implications for therapeutic strategies based around mTOR and the MEK1/2-ERK1/2 pathway.