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Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.

The specifications of the Systems Biology Markup Language (SBML) define standards for storing and exchanging computer models of biological processes in text files. In order to perform model simulations, graphical visualizations and other software manipulations, an in-memory representation of SBML is required. We developed JSBML for this purpose. In contrast to prior implementations of SBML APIs, JSBML has been designed from the ground up for the Java programming language, and can therefore be used on all platforms supported by a Java Runtime Environment. This offers important benefits for Java users, including the ability to distribute software as Java Web Start applications. JSBML supports all SBML Levels and Versions through Level 3 Version 1, and we have strived to maintain the highest possible degree of compatibility with the popular library libSBML. JSBML also supports modules that can facilitate the development of plugins for end user applications, as well as ease migration from a libSBML-based backend.

Despite using the same Rag recombinase machinery expressed in both lymphocyte lineages, V(D)J recombination of immunoglobulins only occurs in B cells and T cell receptor recombination is confined to T cells. This vital segregation of recombination targets is governed by the coordinated efforts of several epigenetic mechanisms that control both the general chromatin accessibility of these loci to the Rag recombinase, and the movement and synapsis of distal gene segments in these enormous multigene AgR loci, in a lineage and developmental stage-specific manner. These mechanisms operate both locally at individual gene segments and AgR domains, and globally over large distances in the nucleus. Here we will discuss the roles of several epigenetic components that regulate V(D)J recombination of the immunoglobulin heavy chain locus in B cells, both in the context of the locus itself, and of its 3D nuclear organization, focusing in particular on non-coding RNA transcription. We will also speculate about how several newly described epigenetic mechanisms might impact on AgR regulation.

How oscillatory brain rhythms alone, or in combination, influence cortical information processing to support learning has yet to be fully established. Local field potential and multi-unit neuronal activity recordings were made from 64-electrode arrays in the inferotemporal cortex of conscious sheep during and after visual discrimination learning of face or object pairs. A neural network model has been developed to simulate and aid functional interpretation of learning-evoked changes.

In pancreatic β cells, elevated glucose concentrations stimulate mitochondrial oxidative metabolism to raise intracellular ATP/ADP levels, prompting insulin secretion. Unusually low levels of expression of genes encoding the plasma membrane monocarboxylate transporter, MCT1 (SLC16A1), as well as lactate dehydrogenase A (LDHA) ensure that glucose-derived pyruvate is efficiently metabolized by mitochondria, while exogenous lactate or pyruvate is unable to stimulate metabolism and hence insulin secretion inappropriately. We show here that whereas DNA methylation at the Mct1 promoter is unlikely to be involved in cell-type-specific transcriptional repression, three microRNAs (miRNAs), miR-29a, miR-29b, and miR-124, selectively target both human and mouse MCT1 3' untranslated regions. Mutation of the cognate miR-29 or miR-124 binding sites abolishes the effects of the corresponding miRNAs, demonstrating a direct action of these miRNAs on the MCT1 message. However, despite reports of its expression in the mouse β-cell line MIN6, miR-124 was not detectably expressed in mature mouse islets. In contrast, the three isoforms of miR-29 are highly expressed and enriched in mouse islets. We show that inhibition of miR-29a in primary mouse islets increases Mct1 mRNA levels, demonstrating that miR-29 isoforms contribute to the β-cell-specific silencing of the MCT1 transporter and may thus affect insulin release.

The t(12;21) translocation that generates the ETV6-RUNX1 (TEL-AML1) fusion gene, is the most common chromosomal rearrangement in childhood cancer and is exclusively associated with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The translocation arises in utero and is necessary but insufficient for the development of leukemia. Single-nucleotide polymorphism array analysis of ETV6-RUNX1 patient samples has identified multiple additional genetic alterations; however, the role of these lesions in leukemogenesis remains undetermined. Moreover, murine models of ETV6-RUNX1 ALL that faithfully recapitulate the human disease are lacking. To identify novel genes that cooperate with ETV6-RUNX1 in leukemogenesis, we generated a mouse model that uses the endogenous Etv6 locus to coexpress the Etv6-RUNX1 fusion and Sleeping Beauty transposase. An insertional mutagenesis screen was performed by intercrossing these mice with those carrying a Sleeping Beauty transposon array. In contrast to previous models, a substantial proportion (20%) of the offspring developed BCP-ALL. Isolation of the transposon insertion sites identified genes known to be associated with BCP-ALL, including Ebf1 and Epor, in addition to other novel candidates. This is the first mouse model of ETV6-RUNX1 to develop BCP-ALL and provides important insight into the cooperating genetic alterations in ETV6-RUNX1 leukemia.

The Bcl-2 protein, best known for its ability to inhibit apoptosis, interacts with the inositol 1,4,5-trisphosphate receptor (IP(3)R) Ca(2+) channel to regulate IP(3)-mediated Ca(2+) release from the endoplasmic reticulum. This review summarizes the current state of knowledge regarding the interaction of Bcl-2, and also its homologue Bcl-xl, with the IP(3)R and how these interactions regulate Ca(2+) signaling. The dual role of these interactions in promoting prosurvival Ca(2+) signals, while at the same time inhibiting proapoptotic Ca(2+) signals, is discussed. Moreover, this review will elucidate the recently recognized importance of the Bcl-2-IP(3)R interaction in human disease.

NAD(+) synthesizing enzyme NMNAT1 constitutes most of the sequence of neuroprotective protein Wld(S), which delays axon degeneration by 10-fold. NMNAT1 activity is necessary but not sufficient for Wld(S) neuroprotection in mice and 70 amino acids at the N-terminus of Wld(S), derived from polyubiquitination factor Ube4b, enhance axon protection by NMNAT1. NMNAT1 activity can confer neuroprotection when redistributed outside the nucleus or when highly overexpressed in vitro and partially in Drosophila. However, the role of endogenous NMNAT1 in normal axon maintenance and in Wallerian degeneration has not been elucidated yet. To address this question we disrupted the Nmnat1 locus by gene targeting. Homozygous Nmnat1 knockout mice do not survive to birth, indicating that extranuclear NMNAT isoforms cannot compensate for its loss. Heterozygous Nmnat1 knockout mice develop normally and do not show spontaneous neurodegeneration or axon pathology. Wallerian degeneration after sciatic nerve lesion is neither accelerated nor delayed in these mice, consistent with the proposal that other endogenous NMNAT isoforms play a principal role in Wallerian degeneration.

In mammals, silencing of one of the two X chromosomes in female cells compensates for the different number of X chromosomes between the sexes. The noncoding Xist RNA initiates X chromosome inactivation. Xist spreads from its transcription site over the X chromosome territory and triggers the formation of a repressive chromatin domain. To understand localization of Xist over one X chromosome we aimed to develop a system for investigating Xist in living cells. Here we report successful visualization of transgenically expressed MS2-tagged Xist in mouse embryonic stem cells. Imaging of Xist during an entire cell cycle shows that Xist spreads from a single point to a steady state when the chromosome is covered with a constant amount of Xist. Photobleaching experiments of the established Xist cluster indicate that chromosome-bound Xist is dynamic and turns over on the fully Xist covered chromosome. It appears that in interphase the loss of bound Xist and newly produced Xist are in equilibrium. We also show that the turnover of bound Xist requires transcription, and Xist binding becomes stable when transcription is inhibited. Our data reveal a strategy for visualizing Xist and indicate that spreading over the chromosome might involve dynamic binding and displacement.

The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fcγ-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.

DNA methylation is one of the most extensively studied, and one of the most stable, of all epigenetic modifications. Two drugs that target DNA methyltransferase enzymes are licensed for clinical use in oncology but relatively little attention has focused on the enzymatic pathways by which DNA methylation can be reversed. Recent breakthroughs have identified at least two classes of enzymes that can achieve functional reversal. This review discusses the significance of DNA demethylation in a range of human diseases, the candidate proteins that mediate the demethylation and the opportunities and challenges in targeting these candidates to develop new therapeutics.

Defective V(D)J rearrangement of immunoglobulin heavy or light chain (IgH or IgL) or class switch recombination (CSR) can initiate chromosomal translocations. The DNA-damage kinase ATM is required for the suppression of chromosomal translocations but ATM regulation is incompletely understood. Here, we show that mice lacking the ATM cofactor ATMIN in B cells (ATMIN(ΔB/ΔB)) have impaired ATM signaling and develop B cell lymphomas. Notably, ATMIN(ΔB/ΔB) cells exhibited defective peripheral V(D)J rearrangement and CSR, resulting in translocations involving the Igh and Igl loci, indicating that ATMIN is required for efficient repair of DNA breaks generated during somatic recombination. Thus, our results identify a role for ATMIN in regulating the maintenance of genomic stability and tumor suppression in B cells.

CD4(+) T helper (T(H)) cells play a critical role in orchestrating a pleiotropy of immune activities against a large variety of pathogens. It is generally thought that this is achieved through the acquisition of highly specialized functions after activation followed by the differentiation into various functional subsets. The differentiation process of naive precursor T(H) cells into defined effector subsets is controlled by cells of the innate immune system and their complex array of effector molecules such as secreted cytokines and membrane bound costimulatory molecules. These provide a unique quantitative or qualitative signal initiating T(H) development, which is subsequently reinforced via T cell-mediated feedback signals and selective survival and proliferative cues, ultimately resulting in the predominance of a particular T cell subset. In recent years, the number of defined T(H)cell subsets has expanded and the once rigid division of labor among them has been blurred with reports of plasticity among the subsets. In this chapter, we summarize and speculate on the current knowledge of the differentiation requirements of T(H) cell lineages, with particular focus on the T(H)17 subset.

Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.

The Rap family of small GTPases regulate the adhesion of cells to extracellular matrices. Several Rap-binding proteins have been shown to function as effectors that mediate Rap-induced adhesion. However, little is known regarding the relationships between these effectors, or about other proteins that are downstream of or act in parallel to the effectors. To establish whether an array of effectors was required for Rap-induced cell adhesion and spreading, and to find new components involved in Rap-signal transduction, we performed a small-scale siRNA screen in A549 lung epithelial cells. Of the Rap effectors tested, only Radil blocked Rap-induced spreading. Additionally, we identified a novel role for Ezrin downstream of Rap1. Ezrin was necessary for Rap-induced cell spreading, but not Rap-induced cell adhesion or basal adhesion processes. Furthermore, Ezrin depletion inhibited Rap-induced cell spreading in several cell lines, including primary human umbilical vein endothelial cells. Interestingly, Radixin and Moesin, two proteins with high homology to Ezrin, are not required for Rap-induced cell spreading and cannot compensate for loss of Ezrin to rescue Rap-induced cell spreading. Here, we present a novel function for Ezrin in Rap1-induced cell spreading and evidence of a non-redundant role of an ERM family member.

Heavy chain diseases (HCDs) are B-cell proliferative disorders characterized by the production of monoclonal, incomplete, immunoglobulin (Ig) heavy chains (HCs) without associated light chains (LCs). These abnormal HCs are produced as a consequence of HC gene alterations in the neoplastic B cells. HC gene alterations will also impact on surface HC, which is part of the B-cell receptor (BCR), a crucial player in lymphocyte activation by antigen. The selective advantage conferred to mutant cells by abnormal BCR without an antigen-binding domain may be explained by activation of ligand-independent signaling, in analogy to what has been shown for mutated oncogenic growth factor receptors. Here we review data obtained from mouse models showing abnormal, constitutive activity of HCD-BCR, and we discuss the possible mechanism involved, namely, aberrant spontaneous self-aggregation. This self-aggregation might occur as a consequence of escape from the chaperone immunoglobulin binding protein (BiP) and from the anti-aggregation effect of LC association. The concept of misfolding-induced signaling elaborated here may extend to other pathologies termed conformational diseases.

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

DNA methylation is involved in a number of important processes such as maintaining genome stability, silencing of retrotransposons, co-ordinating mono-alleleic expression of parentally imprinted genes and ensuring transcriptional repression of genes on the inactive X chromosome. Further, correct DNA methylation patterns are necessary for normal development and lineage commitment. DNA methylation provides a stable and heritable epigenetic mark. However, it can be removed, either actively or passively, during periods of reprogramming in primordial germ cells and preimplantation embryos. By combining immunofluorescence data with recent insights from genome-wide studies utilizing techniques such as Bisulphite-seq and MeDIP-ChIP, a clearer picture of the dynamic patterns of DNA methylation throughout gametogenesis, preimplantation development and early lineage commitment is beginning to emerge. The continuing use of these next-generation technologies to elucidate genome-wide methylation patterns in a variety of cellular contexts will further understanding of how this epigenetic mark contributes to lineage commitment, differentiation and pluripotency and, ultimately, to human health and disease.

The mysterious secrets of long noncoding RNAs, often referred to as the Dark Matter of the genome, are gradually coming to light. Several recent papers dig deep to reveal surprisingly complex and diverse functions of these enigmatic molecules.

A combination of bisulfite treatment of DNA and high-throughput sequencing (BS-Seq) can capture a snapshot of a cell's epigenomic state by revealing its genome-wide cytosine methylation at single base resolution. Bismark is a flexible tool for the time-efficient analysis of BS-Seq data which performs both read mapping and methylation calling in a single convenient step. Its output discriminates between cytosines in CpG, CHG and CHH context and enables bench scientists to visualize and interpret their methylation data soon after the sequencing run is completed. Availability and implementation: Bismark is released under the GNU GPLv3+ licence. The source code is freely available from www.bioinformatics.bbsrc.ac.uk/projects/bismark/.

Tauopathies are characterized by hyperphosphorylation of the microtubule-associated protein tau and its accumulation into fibrillar aggregates. Toxic effects of aggregated tau and/or dysfunction of soluble tau could both contribute to neural defects in these neurodegenerative diseases. We have generated a novel knockin mouse model of an inherited tauopathy, frontotemporal dementia with parkinsonism linked to tau mutations on chromosome 17 (FTDP-17T). We incorporated a single mutation, homologous to the common FTDP-17T P301L mutation, directly into the endogenous mouse gene, mimicking the human disease situation. These mice express P301L-equivalent mutant tau at normal physiological levels from the knockin allele. Importantly, in contrast to existing transgenic mouse models that overexpress human P301L mutant tau, no overt tau pathology developed during the normal lifespan of the knockin mice. In fact, overall phosphorylation of tau was reduced, perhaps due to reduced microtubule binding. However, homozygous knockin mice did display intriguing age-dependent changes in axonal transport of mitochondria, and increased spontaneous locomotor activity in old age. These could represent early consequences of the tau dysfunction that eventually precipitates pathogenesis in humans.

Neutrophils form a vital part of the innate immune response, but at the same time their inappropriate activation contributes to autoimmune diseases. Many molecular components are involved in fine-tuning neutrophil function. We report here the first characterization of the role of ARAP3, a PI3K and Rap-regulated GTPase-activating protein for RhoA and Arf6 in murine neutrophils. We show that neutrophils lacking ARAP3 are preactivated in vitro and in vivo, exhibiting increased β2 integrin affinity and avidity. ARAP3-deficient neutrophils are hyperresponsive in several adhesion-dependent situations in vitro, including the formation of reactive oxygen species, adhesion, spreading, and granule release. ARAP3-deficient cells adhere more firmly under flow conditions in vitro and to the vessel wall in vivo. Finally, loss of ARAP3 interferes with integrin-dependent neutrophil chemotaxis. The results of the present study suggest an important function of ARAP3 downstream of Rap. By modulating β2 integrin activity, ARAP3 guards neutrophils in their quiescent state unless activated.