The rockefeller university

Activation-induced cytidine deaminase (AID) initiates both somatic hypermutation (SHM) for antibody affinity maturation and DNA breakage for antibody class switch recombination (CSR) via transcription-dependent cytidine deamination of single-stranded DNA targets. Though largely specific for immunoglobulin genes, AID also acts on a limited set of off-targets, generating oncogenic translocations and mutations that contribute to B cell lymphoma. How AID is recruited to off-targets has been a long-standing mystery. Based on deep GRO-seq studies of mouse and human B lineage cells activated for CSR or SHM, we report that most robust AID off-target translocations occur within highly focal regions of target genes in which sense and antisense transcription converge. Moreover, we found that such AID-targeting "convergent'' transcription arises from antisense transcription that emanates from super-enhancers within sense transcribed gene bodies. Our findings provide an explanation for AID off-targeting to a small subset of mostly lineage-specific genes in activated B cells.

Heterotrimers composed of B cell CLL/Iymphoma 10 (BCL10), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and caspase recruitment domain-containing (CARD) family adaptors play a role in NF-kappa B activation and have been shown to be involved in both the innate and the adaptive arms of immunity in murine models. Moreover, individuals with inherited defects of MALT1, CARD9, and CARD11 present with immunological and clinical phenotypes. Here, we characterized a case of autosomal-recessive, complete BCL10 deficiency in a child with a broad immunodeficiency, including defects of both hematopoietic and nonhematopoietic immunity. The patient died at 3 years of age and was homozygous for a loss-of-expression, loss-of-function BCL10 mutation. The effect of BCL10 deficiency was dependent on the signaling pathway, and, for some pathways, the cell type affected. Despite the noted similarities to BCL10 deficiency in mice, including a deficient adaptive immune response, human BCL10 deficiency in this patient resulted in a number of specific features within cell populations. Treatment of the patient's myeloid cells with a variety of pathogen-associated molecular pattern molecules (PAMPs) elicited a normal response; however, NF-kappa B-mediated fibroblast functions were dramatically impaired. The results of this study indicate that inherited BCL10 deficiency should be considered in patients with combined immunodeficiency with B cell, T cell, and fibroblast defects.

There is an increasing worldwide epidemic of type 2 diabetes that poses major health problems. We have identified a novel physiological system that increases glucose uptake in skeletal muscle but not in white adipocytes. Activation of this system improves glucose tolerance in Goto-Kakizaki rats or mice fed a high-fat diet, which are established models for type 2 diabetes. The pathway involves activation of beta(2)-adrenoceptors that increase cAMP levels and activate cAMP-dependent protein kinase, which phosphorylates mammalian target of rapamycin complex 2 (mTORC2) at S2481. The active mTORC2 causes translocation of GLUT4 to the plasma membrane and glucose uptake without the involvement of Akt or AS160. Stimulation of glucose uptake into skeletal muscle after activation of the sympathetic nervous system is likely to be of high physiological relevance because mTORC2 activation was observed at the cellular, tissue, and whole-animal level in rodent and human systems. This signaling pathway provides new opportunities for the treatment of type 2 diabetes.

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

The visualization of molecularly labeled structures within large intact tissues in three dimensions is an area of intense focus. We describe a simple, rapid, and inexpensive method, iDISCO, that permits whole-mount immunolabeling with volume imaging of large cleared samples ranging from perinatal mouse embryos to adult organs, such as brains or kidneys. iDISCO is modeled on classical histology techniques, facilitating translation of section staining assays to intact tissues, as evidenced by compatibility with 28 antibodies to both endogenous antigens and transgenic reporters like GFP. When applied to degenerating neurons, iDISCO revealed unexpected variability in number of apoptotic neurons within individual sensory ganglia despite tight control of total number in all ganglia. It also permitted imaging of single degenerating axons in adult brain and the first visualization of cleaved Caspase-3 in degenerating embryonic sensory axons in vivo, even single axons. iDISCO enables facile volume imaging of immunolabeled structures in complex tissues.

A search for excited quarks decaying into the gamma + jet final state is presented. The analysis is based on data corresponding to an integrated luminosity of 19.7 fb(-1) collected by the CMS experiment in proton-proton collisions at root s = 8 TeV at the LHC. Events with photons and jets with high transverse momenta are selected and the gamma + jet invariant mass distribution is studied to search for a resonance peak. The 95% confidence level upper limits on the product of cross section and branching fraction are evaluated as a function of the excited quark mass. Limits on excited quarks are presented as a function of their mass and coupling strength; masses below 3.5 TeV are excluded at 95% confidence level for unit couplings to their standard model partners. (C) 2014 The Authors. Published by Elsevier B.V.

AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.

Neuroinflammation is a feedback mechanism against infection, with recent studies suggesting a neuromodulatory role. The chemokine, (C-C motif) ligand 2 (CCL2), and its receptor, (C-C motif) receptor type 2 (CCR2), affect neuromodulation and migration in response to damage. Although CCL2 co-localizes with neuropeptides in the hypothalamus that control voluntary behavior, the function of CCL2/CCR2 is unknown. This led us to consider the possibility that CCL2 acting through CCR2, under natural conditions, may affect the migration and peptide levels of hypothalamic neurons that control voluntary behavior. This study used primary embryonic hypothalamic neurons to examine the effect of CCL2 on migratory behavior and on levels of the peptides, enkephalin (ENK) and galanin. Treatment with CCL2 led to a significant, dose-dependent increase in the number of migrated neurons and an increase in the velocity and distance traveled. CCL2 also significantly increased the number of ENK-expressing and CCR2/ENK co-expressing neurons and the percentage of neurons that contain higher levels of ENK. Lastly, CCL2 produced a dose-dependent increase in expression of ENK and galanin. These results provide evidence for a stimulatory effect of CCL2 on embryonic hypothalamic neurons involving changes in migratory behavior, expression, and synthesis of neuropeptides that function in controlling behavior.

High plasma levels of estradiol (E2) are associated with use of a place memory system over a response memory system. We examined whether infusing estradiol into the medial prefrontal cortex (mPFC) or anterior cingulate cortex (AC) could affect memory system bias in female rats. We also examined the ultrastructural distribution of estrogen receptor (ER)-alpha, ER beta,and G protein-coupled estrogen receptor 1 (GPER1) in the mPFC of female rats as a mechanism for the behavioral effects of E2 in the mPFC. Each rat was infused bilaterally with either E2 (0.13 mu g) or vehicle into the mPFC or AC. The majority of E2 mPFC rats used place memory. In contrast, the majority of mPFC vehicle rats and AC E2 or vehicle rats used response memory. These data show that mPFC E2 rapidly biases females to use place memory. Electron microscopic analysis demonstrated that ER alpha, ER beta, and GPER1 are localized in the mPFC, almost exclusively at extranuclear sites. This is the first time that GPER1 has been localized to the mPFC of rats and the first time that ER alpha and ER beta have been described at extranuclear sites in the rat mPFC. The majority of receptors were observed on axons and axon terminals, suggesting that estrogens alter presynaptic transmission in the mPFC. This provides a mechanism via which ERs could rapidly alter transmission in the mPFC to alter PFC-dependent behaviors, such as memory system bias. The discrete nature of immunolabeling for these membrane-associated ERs may explain the discrepancy in previous light microscopy studies.