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.

Lipidomic methodologies have developed such that determination in lipid species content of cells and tissues is increasingly achievable. Adoption of these methods is highlighting the physiological importance of individual lipid molecular species rather than changes in an overall lipid class. In this article the use of such methodologies is considered and the potential for understanding the importance of lipid changes in malignancy assessed.

During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epigenetic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however re-localisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with γH2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming.

Qualitative frameworks, especially those based on the logical discrete formalism, are increasingly used to model regulatory and signalling networks. A major advantage of these frameworks is that they do not require precise quantitative data, and that they are well-suited for studies of large networks. While numerous groups have developed specific computational tools that provide original methods to analyse qualitative models, a standard format to exchange qualitative models has been missing.

The post-translational modification known as glycation affects the physiological properties of peptides and proteins. Glycation is particularly important during hyperglycaemia where α-dicarbonyl compounds are generated. These compounds react with proteins to generate α-dicarbonyl-derived glycation products, which are correlated with diabetic complications such as nephropathy, retinopathy, and neuropathy, among others. One of these α-dicarbonyl compounds is ethanedial, also known as glyoxal. Thereby, glyoxal binding to protein/peptides is studied by electron capture dissociation (ECD) and collisionally activated dissociation (CAD).

The NAD-synthesizing enzyme NMNAT2 is critical for axon survival in primary culture and its depletion may contribute to axon degeneration in a variety of neurodegenerative disorders. Here we discuss several recent reports from our laboratory that establish a critical role for NMNAT2 in axon growth in vivo in mice and shed light on the delivery and turnover of this survival factor in axons. In the absence of NMNAT2, axons fail to extend more than a short distance beyond the cell body during embryonic development, implying a requirement for NMNAT2 in axon maintenance even during development. Furthermore, we highlight findings regarding the bidirectional trafficking of NMNAT2 in axons on a vesicle population that undergoes fast axonal transport in primary culture neurites and in mouse sciatic nerve axons in vivo. Surprisingly, loss of vesicle association boosts the axon protective capacity of NMNAT2, an effect that is at least partially mediated by a longer protein half-life of cytosolic NMNAT2 variants. Analysis of wild-type and variant NMNAT2 in mouse sciatic nerves and Drosophila olfactory receptor neuron axons supports the existence of a similar mechanism in vivo, highlighting the potential for regulation of NMNAT2 stability and turnover as a mechanism to modulate axon degeneration in vivo.

Regulation of lipid metabolism via activation of sterol regulatory element binding proteins (SREBPs) has emerged as an important function of the Akt/mTORC1 signaling axis. Although the contribution of dysregulated Akt/mTORC1 signaling to cancer has been investigated extensively and altered lipid metabolism is observed in many tumors, the exact role of SREBPs in the control of biosynthetic processes required for Akt-dependent cell growth and their contribution to tumorigenesis remains unclear.

DNA methylation in mammals is an epigenetic mark necessary for normal embryogenesis. During development active loss of methylation occurs in the male pronucleus during the first cell cycle after fertilisation. This is accompanied by major chromatin remodelling and generates a marked asymmetry between the paternal and maternal genomes. The mechanism(s) by which this is achieved implicate, among others, base excision repair (BER) components and more recently a major role for TET3 hydroxylase. To investigate these methylation dynamics further we have analysed DNA methylation and hydroxymethylation in fertilised mouse oocytes by indirect immunofluorescence (IF) and evaluated the relative contribution of different candidate factors for active demethylation in knock-out zygotes by three-dimensional imaging and IF semi-quantification.

In this issue of Chemistry & Biology, Winkler and colleagues describe the discovery and preclinical development of IPI-145, a new inhibitor of the phosphoinositide 3-kinase (PI3K) isoforms p110δ and p110γ that have entered clinical trials.

Quantitative reductions in T-cell receptor (TCR) signalling are associated with severe immunodeficiency, yet in certain cases can lead to autoimmunity. Mutation of the tyrosine kinase ZAP-70 can cause either of these outcomes, yet the limits of its signal transducing capacity are not well defined. To investigate these limits we have made use of mrtless: a chemically induced mutation of Zap70 associated with T-cell deficiency. Unlike cells devoid of ZAP-70, mrtless thymocytes showed partial induction of CD5 and CD69, and were sensitive to TCR stimulation with a dose-response shifted approximately 10-fold. However, essentially no T cells were able to compensate for the mrtless mutation and mature beyond the CD4⁺ CD8⁺ stage. This outcome contrasts with a ZAP-70 Src Homology 2 domain mutant strain, where high-affinity self-reactive TCR are positively selected rather than deleted. We discuss these data with respect to current models of TCR signalling in thymocyte selection.

The evolution of the biogenic amine signalling system in vertebrates is unclear. However, insights can be obtained from studying the structures and signalling properties of biogenic amine receptors from the protochordate, amphioxus, which is an invertebrate species that exists at the base of the chordate lineage. Here we describe the signalling properties of AmphiAmR11, an amphioxus (Branchiostoma floridae) G protein-coupled receptor which has structural similarities to vertebrate α2-adrenergic receptors but which functionally acts as a D2 dopamine-like receptor when expressed in Chinese hamster ovary -K1 cells. AmphiAmR11 inhibits forskolin-stimulated cyclic AMP levels with tyramine, phenylethylamine and dopamine being the most potent agonists. AmphiAmR11 also increases mitogen-activated protein kinase activity and calcium mobilisation, and in both pathways, dopamine was found to be more potent than tyramine. Thus, differences in the relative effectiveness of various agonists in the different second messenger assay systems suggest that the receptor displays agonist-specific coupling (biased agonism) whereby different agonists stabilize different conformations of the receptor which lead to the enhancement of one signalling pathway over another. The present study provides insights into the evolution of α2-adrenergic receptor signalling and support the hypothesis that α2-adrenergic receptors evolved from D2-dopamine receptors. The AmphiAmR11 receptor may represent a transition state between D2-dopamine receptors and α2-adrenergic receptors.

Many species of bacteria use quorum sensing to sense the amount of secreted metabolites and to adapt their growth according to their population density. We asked whether similar mechanisms would operate in lymphocyte homeostasis. We investigated the regulation of the size of interleukin-2 (IL-2)-producing CD4(+) T cell (IL-2p) pool using different IL-2 reporter mice. We found that in the absence of either IL-2 or regulatory CD4(+) T (T reg) cells, the number of IL-2p cells increases. Administration of IL-2 decreases the number of cells of the IL-2p cell subset and, pertinently, abrogates their ability to produce IL-2 upon in vivo cognate stimulation, while increasing T reg cell numbers. We propose that control of the IL-2p cell numbers occurs via a quorum sensing-like feedback loop where the produced IL-2 is sensed by both the activated CD4(+) T cell pool and by T reg cells, which reciprocally regulate cells of the IL-2p cell subset. In conclusion, IL-2 acts as a self-regulatory circuit integrating the homeostasis of activated and T reg cells as CD4(+) T cells restrain their growth by monitoring IL-2 levels, thereby preventing uncontrolled responses and autoimmunity.

Trophoblast stem cells (TSCs) are a self-renewing stem cell population derived from the early trophoblast lineage, analogous to embryonic stem cells (ESCs) that can be generated from the inner cell mass (ICM) of the mouse blastocyst. In that sense TSCs and ESCs reflect the earliest lineage differentiation event after fertilization. TSCs are characterized by an indefinite proliferation potential and by multipotency, i.e. the ability to differentiate into all the various trophoblast cell types of the placenta. These properties are driven by specific signalling pathways orchestrating characteristic transcriptional outputs. Here we review the recent advances in studying the signalling cascades and the transcriptional regulatory networks that define specification and maintenance of TSCs, and provide a future outlook of TSC research.

Normal ageing is accompanied by a decline in the function of the immune system that causes an increased susceptibility to infections and an impaired response to vaccination in older individuals. This results in an increased disease burden in the aged population, even with good immunisation programmes in place. The decreased response to vaccination is partly due to the diminution of the germinal centre response with age, caused by impaired T-cell help to B cells. Within the germinal centre, T-cell help is provided by a specialised subset of CD4(+) T cells; T follicular helper (Tfh) cells. Tfh cells provide survival and selection signals to germinal centre B cells, allowing them to egress from the germinal centre and become long-live plasma cells or memory B cells, and provide life-long protection against subsequent infection. This review will discuss the cellular and molecular changes in both Tfh cells and germinal centre B cells that occur with advancing age, which result in a smaller germinal centre response and a less effective response to immunisation.

The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

Dysregulation of Ca(2+) homeostasis is considered to contribute to the toxic action of the Alzheimer's disease (AD)-associated amyloid-β-peptide (Aβ). Ca(2+) fluxes across the plasma membrane and release from intracellular stores have both been reported to underlie the Ca(2+) fluxes induced by Aβ42. Here, we investigated the contribution of Ca(2+) release from the endoplasmic reticulum (ER) to the effects of Aβ42 upon Ca(2+) homeostasis and the mechanism by which Aβ42 elicited these effects. Consistent with previous reports, application of soluble oligomeric forms of Aβ42 induced an elevation in intracellular Ca(2+). The Aβ42-stimulated Ca(2+) signals persisted in the absence of extracellular Ca(2+) indicating a significant contribution of Ca(2+) release from the ER Ca(2+) store to the generation of these signals. Moreover, inositol 1,4,5-trisphosphate (InsP3) signaling contributed to Aβ42-stimulated Ca(2+) release. The Ca(2+) mobilizing effect of Aβ42 was also observed when applied to permeabilized cells deficient in InsP3 receptors, revealing an additional direct effect of Aβ42 upon the ER, and a mechanism for induction of toxicity by intracellular Aβ42.