����������̳

Axonal and synaptic degeneration is a hallmark of peripheral neuropathy, brain injury, and neurodegenerative disease. Axonal degeneration has been proposed to be mediated by an active autodestruction program, akin to apoptotic cell death; however, loss-of-function mutations capable of potently blocking axon self-destruction have not been described. Here, we show that loss of the Drosophila Toll receptor adaptor dSarm (sterile α/Armadillo/Toll-Interleukin receptor homology domain protein) cell-autonomously suppresses Wallerian degeneration for weeks after axotomy. Severed mouse Sarm1 null axons exhibit remarkable long-term survival both in vivo and in vitro, indicating that Sarm1 prodegenerative signaling is conserved in mammals. Our results provide direct evidence that axons actively promote their own destruction after injury and identify dSarm/Sarm1 as a member of an ancient axon death signaling pathway.

The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ∼150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.

Grb10 is an intracellular adaptor protein that acts as a negative regulator of insulin and insulin-like growth factor 1 (IGF1) receptors. Since global deletion of Grb10 in mice causes hypermuscularity, we have characterized the skeletal muscle physiology underlying this phenotype. Compared to wild-type (WT) controls, adult mice deficient in Grb10 have elevated body mass and muscle mass throughout adulthood, up to 12 mo of age. The muscle enlargement is not due to increased myofiber size, but rather an increase in myofiber number (142% of WT, P<0.01). There is no change in myofiber type proportions between WT and Grb10-deficient muscles, nor are the metabolic properties of the muscles altered on Grb10 deletion. Notably, the weight and cross-sectional area of hindlimbs from neonatal mice are increased in Grb10-deficient animals (198 and 137% of WT, respectively, both P<0.001). Functional gene signatures for myogenic signaling and proliferation are up-regulated in Grb10-deficient neonatal muscle. Our findings indicate that Grb10 plays a previously unrecognized role in regulating the development of fiber number during murine embryonic growth. In addition, Grb10-ablated muscle from adult mice shows coordinate gene changes that oppose those of muscle wasting pathologies, highlighting Grb10 as a potential therapeutic target for these conditions.

PTEN, one of the most commonly mutated or lost tumor suppressors in human cancers, antagonizes signaling by the PI3K pathway. Mice with thymocyte-specific deletion of Pten rapidly develop peripheral lymphomas and autoimmunity, which may be caused by failed negative selection of thymocytes or from dysregulation of postthymic T cells. We induced conditional deletion of Pten from CD4 Th cells using a Cre knocked into the Tnfrsf4 (OX40) locus to generate OX40(Cre)Pten(f) mice. Pten-deficient Th cells proliferated more and produced greater concentrations of cytokines. The OX40(Cre)Pten(f) mice had a general increase in the number of lymphocytes in the lymph nodes, but not in the spleen. When transferred into wild-type (WT) mice, Pten-deficient Th cells enhanced anti-Listeria responses and the clearance of tumors under conditions in which WT T cells had no effect. Moreover, inflammatory responses were exaggerated and resolved later in OX40(Cre)Pten(f) mice than in WT mice. However, in contrast with models of thymocyte-specific Pten deletion, lymphomas and autoimmunity were not observed, even in older OX40(Cre)Pten(f) mice. Hence loss of Pten enhances Th cell function without obvious deleterious effects.

Eukaryotic cells must constantly degrade both intracellular and extracellular material to maintain cellular and organismal homeostasis. Two engulfment pathways, autophagy and phagocytosis, contribute to the turnover of intracellular and extracellular substrates by delivering material to the lysosome. Historically these are thought to be separate pathways, but recent studies have revealed the direct participation of autophagy proteins in phagocytosis. Autophagy proteins lipidate LC3 onto phagosomes and other macroendocytic vacuole membranes, and are required for lysosomal degradation of engulfed cargo, demonstrating an autophagosome-independent role for autophagy proteins in mediating the turnover of extracellular substrates. This review discusses the biological systems in which autophagy proteins have been found to regulate lysosome fusion to non-autophagic membranes.

Sequence-specific RNA-binding proteins (RBP) and the regulation of RNA decay have long been recognized as important regulators of the inflammatory response. RBP influence gene expression throughout the lifespan of the mRNA by regulating splicing, polyadenylation, cellular localization, translation, and decay. Increasing evidence now indicates that these proteins, together with the RNA decay machinery that they recruit, also regulate the development and activation of lymphocytes. The activity of RBP is regulated by the same signal transduction pathways that govern lymphocyte development and differentiation in response to antigen and cytokine receptor engagement. Roles for these proteins in regulating the diverse functions of lymphocytes are becoming increasingly apparent.

The RV144 trial demonstrated that an experimental AIDS vaccine can prevent human immunodeficiency virus type 1 (HIV-1) infection in humans. Because of its limited efficacy, further understanding of the mechanisms of preventive AIDS vaccines remains a priority, and nonhuman primate (NHP) models of lentiviral infection provide an opportunity to define immunogens, vectors, and correlates of immunity. In this study, we show that prime-boost vaccination with a mismatched SIV envelope (Env) gene, derived from simian immunodeficiency virus SIVmac239, prevents infection by SIVsmE660 intrarectally. Analysis of different gene-based prime-boost immunization regimens revealed that recombinant adenovirus type 5 (rAd5) prime followed by replication-defective lymphocytic choriomeningitis virus (rLCMV) boost elicited robust CD4 and CD8 T-cell and humoral immune responses. This vaccine protected against infection after repetitive mucosal challenge with efficacies of 82% per exposure and 62% cumulatively. No effect was seen on viremia in infected vaccinated monkeys compared to controls. Protection correlated with the presence of neutralizing antibodies to the challenge viruses tested in peripheral blood mononuclear cells. These data indicate that a vaccine expressing a mismatched Env gene alone can prevent SIV infection in NHPs and identifies an immune correlate that may guide immunogen selection and immune monitoring for clinical efficacy trials.

Computing accurate nucleic acid melting temperatures has become a crucial step for the efficiency and the optimisation of numerous molecular biology techniques such as in situ hybridization, PCR, antigene targeting, and microarrays. MELTING is a free open source software which computes the enthalpy, entropy and melting temperature of nucleic acids. MELTING 4.2 was able to handle several types of hybridization such as DNA/DNA, RNA/RNA, DNA/RNA and provided corrections to melting temperatures due to the presence of sodium. The program can use either an approximative approach or a more accurate Nearest-Neighbor approach.

LibSBGN is a software library for reading, writing and manipulating Systems Biology Graphical Notation (SBGN) maps stored using the recently developed SBGN-ML file format. The library (available in C++ and Java) makes it easy for developers to add SBGN support to their tools, whereas the file format facilitates the exchange of maps between compatible software applications. The library also supports validation of maps, which simplifies the task of ensuring compliance with the detailed SBGN specifications. With this effort we hope to increase the adoption of SBGN in bioinformatics tools, ultimately enabling more researchers to visualize biological knowledge in a precise and unambiguous manner.

Axonal degeneration is a major contributor to neuronal dysfunction in many neurological conditions and has additional roles in development. It can be triggered by divergent stimuli including mechanical, metabolic, infectious, toxic, hereditary and inflammatory stresses. Axonal mitochondria are an important convergence point as regulators of bioenergetic metabolism, reactive oxygen species (ROS), Ca²⁺ homeostasis and protease activation. The challenges likely to render axonal mitochondria more vulnerable than their cellular counterparts are reviewed, including axonal transport, replenishing nuclear-encoded proteins and maintenance of quality control, fusion and fission in locations remote from the cell body. The potential for mitochondria to act as a decision node in axon loss is considered, highlighting the need to understand the biology of axonal mitochondria and their contributions to degenerative mechanisms for novel therapeutic strategies.

Trophoblast cells are required for the growth and survival of the fetus during pregnancy, and failure to maintain appropriate trophoblast regulation is associated with placental insufficiencies and intrauterine growth restriction. Development of the trophoblast lineage is mediated by interactions between genetic and epigenetic factors. This review will focus on new insights that have been gained from analysis of mouse models into the epigenetic mechanisms that are required for the early establishment of the trophoblast lineage and for the development of specialized cell types of the fetal placenta. In particular, the importance of DNA methylation, 5-hydroxymethylcytosine and histone modifications in orchestrating trophoblast gene expression and functional outcome will be discussed. These insights are beginning to be extended towards human studies and initial results suggest that the causes and consequences of a variety of placental pathologies are related to epigenetic processes. Furthermore, the epigenetic landscape that regulates trophoblast cells seems to be particularly vulnerable to perturbation during development. This has major implications for diet and other environmental factors during pregnancy.

The chorioallantoic placenta is the defining organ of eutherians that has enabled prolonged intrauterine gestation. As such, normal placental development and function are essential for mammalian reproductive success. Reflecting the key role of this organ in providing nutrients to the embryo, the characteristic cell type that forms substantial parts of the placenta is called 'trophoblast' (from Greek trephein 'to feed' and blastos 'germinator'). However, in addition to regulating nutrient supply, the placenta also exerts a number of other pivotal functions that highlight the importance of normal trophoblast differentiation for a successful pregnancy. In this guest symposium, 'Trophoblast Development', several contributors summarize insights gained from recent studies in the mouse that have advanced our understanding of trophoblast biology. This includes how the earliest trophoblast cells are set aside to expand in a stem- or progenitor-cell compartment under tight genetic and epigenetic control and how subsequent differentiation into the various placental cell types is controlled to ensure normal placentation. The relevance of these contributions range from early developmental cell fate decisions, stem cell biology and placental development for healthy pregnancy to the impact of placental failures on long-term health, with important clinical implications for assisted reproductive technology procedures and pregnancy-associated complications.

The host response following malaria infection depends on a fine balance between levels of pro-inflammatory and anti-inflammatory mediators resulting in the resolution of the infection or immune-mediated pathology. Whilst other components of the innate immune system contribute to the pro-inflammatory milieu, T cells play a major role. For blood-stage malaria, CD4(+) and γδ T cells are major producers of the IFN-γ that controls parasitemia, however, a role for TH17 cells secreting IL-17A and other cytokines, including IL-17F and IL-22 has not yet been investigated in malaria. TH17 cells have been shown to play a role in some protozoan infections, but they also are a source of pro-inflammatory cytokines known to be involved in protection or pathogenicity of infections. In the present study, we have investigated whether IL-17A and IL-22 are induced during a Plasmodium chabaudi infection in mice, and whether these cytokines contribute to either protection or to pathology induced during the infection. Although small numbers of IL-17- and IL-22-producing CD4 T cells are induced in the spleens of infected mice, a more pronounced induction is observed in the liver, where increases in mRNA for IL-17A and, to a lesser extent, IL-22 were observed and CD8(+) T cells, rather than CD4 T cells, are a major source of these cytokines in this organ. Although the lack of IL-17 did not affect the outcome of infection or pathology, lack of IL-22 resulted in 50% mortality within 12 days after infection with significantly greater weight loss at the peak of infection and significant increase in alanine transaminase in the plasma in the acute infection. As parasitemias and temperature were similar in IL-22 KO and wild-type control mice, our observations support the idea that IL-22 but not IL-17 provides protection from the potentially lethal effects of liver damage during a primary P. chabaudi infection.

Mutations in the autoimmune regulator (AIRE) gene lead to autoimmune polyendocrinopathy syndrome type 1 (APS1), characterized by the development of multi-organ autoimmune damage. The mechanism by which defects in AIRE result in autoimmunity has been the subject of intense scrutiny. At the cellular level, the working model explains most of the clinical and immunological characteristics of APS1, with AIRE driving the expression of tissue-restricted antigens (TRAs) in the epithelial cells of the thymic medulla. This TRA expression results in effective negative selection of TRA-reactive thymocytes, preventing autoimmune disease. At the molecular level, the mechanism by which AIRE initiates TRA expression in the thymic medulla remains unclear. Multiple different models for the molecular mechanism have been proposed, ranging from classical transcriptional activity, to random induction of gene expression, to epigenetic tag recognition effect, to altered cell biology. In this review, we evaluate each of these models and discuss their relative strengths and weaknesses.

The ERK1/2 (extracellular signal-regulated kinase 1 and 2) pathway, comprising the protein kinases RAF (v-raf-1 murine leukemia viral oncogene homolog 1), MEK1/2 (mitogen-activated protein kinase or ERK kinase 1 and 2) and ERK1/2 is frequently de-regulated in human cancers, due to mutations in RAS or BRAF (v-raf-1 murine leukemia viral oncogene homolog B1). New, highly selective inhibitors of BRAF and MEK1/2 have shown promise in clinical trials, including in previously intractable diseases such as melanoma. However, drug-resistant tumour cells invariably emerge leading to disease progression. It is important to understand the mechanisms underlying such acquired resistance since this may lead to the development of rational strategies either to delay its onset or to overcome it once established. It also offers unique insights into the plasticity of signalling pathways, which may in turn inform our understanding of the basic biology of these pathways and lead to the validation of new drug targets. Several recent reports have identified diverse mechanisms of acquired resistance to MEK1/2 or BRAF inhibitors. In this article, we review these studies, discuss the different mechanisms, identify common themes and consider their therapeutic implications.

An improved system for cell-free expression of protein arrays based on DNA arrays is presented. Our technology uses an array of DNA constructs for cell-free expression, which acts as a template instructing the generation of the corresponding protein array. Proteins are expressed locally from these templates by a cell-free transcription and translation system, and are immobilised on a separate capture surface overlayed on the DNA array. By simplifying the setup to allow protein diffusion between the slides across a gap filled with the cell-free system, we have markedly improved the evenness of the resulting protein microarrays.

5-Methylcytosine can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. We introduce oxidative bisulfite sequencing (oxBS-Seq), the first method for quantitative mapping of 5hmC in genomic DNA at single-nucleotide resolution. Selective chemical oxidation of 5hmC to 5-formylcytosine (5fC) enables bisulfite conversion of 5fC to uracil. We demonstrate the utility of oxBS-Seq to map and quantify 5hmC at CpG islands (CGIs) in mouse embryonic stem (ES) cells and identify 800 5hmC-containing CGIs that have on average 3.3% hydroxymethylation. High levels of 5hmC were found in CGIs associated with transcriptional regulators and in long interspersed nuclear elements, suggesting that these regions might undergo epigenetic reprogramming in ES cells. Our results open new questions on 5hmC dynamics and sequence-specific targeting by TETs.

The thymus is the primary organ for T-cell differentiation and maturation. Unlike other major organs, the thymus is highly dynamic, capable of undergoing multiple rounds of almost complete atrophy followed by rapid restoration. The process of thymic atrophy, or involution, results in decreased thymopoiesis and emigration of naïve T cells to the periphery. Multiple processes can trigger transient thymic involution, including bacterial and viral infection(s), aging, pregnancy and stress. Intense investigations into the mechanisms that underlie thymic involution have revealed diverse cellular and molecular mediators, with elaborate control mechanisms. This review outlines the disparate pathways through which involution can be mediated, from the transient infection-mediated pathway, tightly controlled by microRNA, to the chronic changes that occur through aging.

Regulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as a crucial regulator of long-range chromatin interactions. CTCF often controls specific interactions by preventing inappropriate communication between neighboring regulatory elements or independent chromatin domains. Although recent gene targeting experiments demonstrated that the presence of the CTCF protein is not required for the process of V(D)J recombination per se, CTCF turned out to be essential to control order, lineage specificity and to balance the Ig V gene repertoire. Moreover, CTCF was shown to restrict activity of κ enhancer elements to the Ig κ locus. In this review, we discuss CTCF function in the regulation of V(D)J recombination on the basis of established knowledge on CTCF-mediated chromatin loop domains in various other loci, including the imprinted H19-Igf2 locus as well as the complex β-globin, MHC class II and IFN-γ loci. Moreover, we discuss that loss of CTCF-mediated restriction of enhancer activity may well contribute to oncogenic activation, when in chromosomal translocations Ig enhancer elements and oncogenes appear in a novel genomic context.

Environmental factors during early life are critical for the later metabolic health of the individual and of future progeny. In our obesogenic environment, it is of great socioeconomic importance to investigate the mechanisms that contribute to the risk of metabolic ill health. Imprinted genes, a class of functionally mono-allelic genes critical for early growth and metabolic axis development, have been proposed to be uniquely susceptible to environmental change. Furthermore, it has also been suggested that perturbation of the epigenetic reprogramming of imprinting control regions (ICRs) may play a role in phenotypic heritability following early life insults. Alternatively, the presence of multiple layers of epigenetic regulation may in fact protect imprinted genes from such perturbation. Unbiased investigation of these alternative hypotheses requires assessment of imprinted gene expression in the context of the response of the whole transcriptome to environmental assault. We therefore analyse the role of imprinted genes in multiple tissues in two affected generations of an established murine model of the developmental origins of health and disease using microarrays and quantitative RT-PCR. We demonstrate that, despite the functional mono-allelicism of imprinted genes and their unique mechanisms of epigenetic dosage control, imprinted genes as a class are neither more susceptible nor protected from expression perturbation induced by maternal undernutrition in either the F1 or the F2 generation compared to other genes. Nor do we find any evidence that the epigenetic reprogramming of ICRs in the germline is susceptible to nutritional restriction. However, we propose that those imprinted genes that are affected may play important roles in the foetal response to undernutrition and potentially its long-term sequelae. We suggest that recently described instances of dosage regulation by relaxation of imprinting are rare and likely to be highly regulated.

Human and mouse studies performed over the last decade have established that follicular helper T (Tfh) cells are a CD4(+) helper subset specialized in the provision of help to B cells. Tfh differentiation is driven by expression of the transcriptional repressor B-cell lymphoma-6 (Bcl-6), which turns on a program that guides T cells close to B-cell areas where Tfh cells first provide help to B cells. Sustained Bcl-6 expression promotes the entry of Tfh cells into follicles and modulates their cytokine expression profile so they can support and select germinal center B cells that have acquired affinity-enhancing mutations in their immunoglobulin genes. Forkhead box 3 protein (Foxp3)(+) regulatory T cells and invariant natural killer T (NKT) cells can also co-opt the Bcl-6-dependent follicular differentiation pathway to migrate into B-cell follicles and regulate antibody responses. The resulting NKT follicular helper cells drive a distinctive type of T-dependent B-cell response to lipid-containing antigens, whereas FoxP3(+) follicular regulatory (Tfr) cells exert a suppressive function on germinal centers. Elucidating how Tfr cells are functionally and numerically regulated and the factors that control the balance between Tfh and Tfr cells is likely to be critical for improved understanding of the pathogenesis and progression of autoimmunity and lymphomas of germinal center origin, and generation of effective vaccines.

Maternal deletion of the NESP55 differentially methylated region (DMR) (delNESP55/ASdel3-4(m), delNAS(m)) from the GNAS locus in humans causes autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-Ib(delNASm)), a disorder of proximal tubular parathyroid hormone (PTH) resistance associated with loss of maternal GNAS methylation imprints. Mice carrying a similar, maternally inherited deletion of the Nesp55 DMR (ΔNesp55(m)) replicate these Gnas epigenetic abnormalities and show evidence for PTH resistance, yet these mice demonstrate 100% mortality during the early postnatal period. We investigated whether the loss of extralarge αs (XLαs) imprinting and the resultant biallelic expression of XLαs are responsible for the early postnatal lethality in ΔNesp55(m) mice. First, we found that ΔNesp55(m) mice are hypoglycemic and have reduced stomach-to-body weight ratio. We then generated mice having the same epigenetic abnormalities as the ΔNesp55(m) mice but with normalized XLαs expression due to the paternal disruption of the exon giving rise to this Gnas product. These mice (ΔNesp55(m)/Gnasxl(m+/p-)) showed nearly 100% survival up to postnatal day 10, and a substantial number of them lived to adulthood. The hypoglycemia and reduced stomach-to-body weight ratio observed in 2-d-old ΔNesp55(m) mice were rescued in the ΔNesp55(m)/Gnasxl(m+/p-) mice. Surviving double-mutant animals had significantly reduced Gαs mRNA levels and showed hypocalcemia, hyperphosphatemia, and elevated PTH levels, thus providing a viable model of human AD-PHP-Ib. Our findings show that the hypoglycemia and early postnatal lethality caused by the maternal deletion of the Nesp55 DMR result from biallelic XLαs expression. The double-mutant mice will help elucidate the pathophysiological mechanisms underlying AD-PHP-Ib.

Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.