Life Sciences Research for Lifelong Health


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Title / Authors / Details Open Access Download

The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation.
Tiedje C, Diaz-Muñoz MD, Trulley P, Ahlfors H, Laaß K, Blackshear PJ, Turner M, Gaestel M

RNA-binding proteins (RBPs) facilitate post-transcriptional control of eukaryotic gene expression at multiple levels. The RBP tristetraprolin (TTP/Zfp36) is a signal-induced phosphorylated anti-inflammatory protein guiding unstable mRNAs of pro-inflammatory proteins for degradation and preventing translation. Using iCLIP, we have identified numerous mRNA targets bound by wild-type TTP and by a non-MK2-phosphorylatable TTP mutant (TTP-AA) in 1 h LPS-stimulated macrophages and correlated their interaction with TTP to changes at the level of mRNA abundance and translation in a transcriptome-wide manner. The close similarity of the transcriptomes of TTP-deficient and TTP-expressing macrophages upon short LPS stimulation suggested an effective inactivation of TTP by MK2, whereas retained RNA-binding capacity of TTP-AA to 3'UTRs caused profound changes in the transcriptome and translatome, altered NF-κB-activation and induced cell death. Increased TTP binding to the 3'UTR of feedback inhibitor mRNAs, such as Ier3, Dusp1 or Tnfaip3, in the absence of MK2-dependent TTP neutralization resulted in a strong reduction of their protein synthesis contributing to the deregulation of the NF-κB-signaling pathway. Taken together, our study uncovers a role of TTP as a suppressor of feedback inhibitors of inflammation and highlights the importance of fine-tuned TTP activity-regulation by MK2 in order to control the pro-inflammatory response.

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Nucleic acids research, , 1362-4962, , 2016

PMID: 27220464

Open Access

Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.
Brucklacher-Waldert V, Ferreira C, Innocentin S, Kamdar S, Withers DR, Kullberg MC, Veldhoen M

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2016

PMID: 27183623

Open Access

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.
Roychoudhuri R, Clever D, Li P, Wakabayashi Y, Quinn KM, Klebanoff CA, Ji Y, Sukumar M, Eil RL, Yu Z, Spolski R, Palmer DC, Pan JH, Patel SJ, Macallan DC, Fabozzi G, Shih HY, Kanno Y, Muto A, Zhu J, Gattinoni L, O'Shea JJ, Okkenhaug K, Igarashi K, Leonard WJ, Restifo NP

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

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Nature immunology, , 1529-2916, , 2016

PMID: 27158840

Application of virtual screening to the discovery of novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with potential for the treatment of cancer and axonopathies.
Clark DE, Waszkowycz B, Wong M, Lockey PM, Adalbert R, Gilley J, Clark J, Coleman MP

NAMPT may represent a novel target for drug discovery in various therapeutic areas, including oncology and inflammation. Additionally, recent work has suggested that targeting NAMPT has potential in treating axon degeneration. In this work, publicly available X-ray co-crystal structures of NAMPT and the structures of two known NAMPT inhibitors were used as the basis for a structure- and ligand-based virtual screening campaign. From this, two novel series of NAMPT inhibitors were identified, one of which showed a statistically significant protective effect when tested in a cellular model of axon degeneration.

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Bioorganic & medicinal chemistry letters, , 1464-3405, , 2016

PMID: 27158141

Open Access

CHiCP: a web-based tool for the integrative and interactive visualization of promoter capture Hi-C datasets.
Schofield EC, Carver T, Achuthan P, Freire-Pritchett P, Spivakov M, Todd JA, Burren OS

Promoter capture Hi-C (PCHi-C) allows the genome-wide interrogation of physical interactions between distal DNA regulatory elements and gene promoters in multiple tissue contexts. Visual integration of the resultant chromosome interaction maps with other sources of genomic annotations can provide insight into underlying regulatory mechanisms. We have developed Capture HiC Plotter (CHiCP), a web-based tool that allows interactive exploration of PCHi-C interaction maps and integration with both public and user-defined genomic datasets.

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Bioinformatics (Oxford, England), 32, 1367-4811, 2511-3, 2016

PMID: 27153610

Open Access

The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.
Novo CL, Tang C, Ahmed K, Djuric U, Fussner E, Mullin NP, Morgan NP, Hayre J, Sienerth AR, Elderkin S, Nishinakamura R, Chambers I, Ellis J, Bazett-Jones DP, Rugg-Gunn PJ

An open and decondensed chromatin organization is a defining property of pluripotency. Several epigenetic regulators have been implicated in maintaining an open chromatin organization, but how these processes are connected to the pluripotency network is unknown. Here, we identified a new role for the transcription factor NANOG as a key regulator connecting the pluripotency network with constitutive heterochromatin organization in mouse embryonic stem cells. Deletion of Nanog leads to chromatin compaction and the remodeling of heterochromatin domains. Forced expression of NANOG in epiblast stem cells is sufficient to decompact chromatin. NANOG associates with satellite repeats within heterochromatin domains, contributing to an architecture characterized by highly dispersed chromatin fibers, low levels of H3K9me3, and high major satellite transcription, and the strong transactivation domain of NANOG is required for this organization. The heterochromatin-associated protein SALL1 is a direct cofactor for NANOG, and loss of Sall1 recapitulates the Nanog-null phenotype, but the loss of Sall1 can be circumvented through direct recruitment of the NANOG transactivation domain to major satellites. These results establish a direct connection between the pluripotency network and chromatin organization and emphasize that maintaining an open heterochromatin architecture is a highly regulated process in embryonic stem cells.

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Genes & development, , 1549-5477, , 2016

PMID: 27125671

Open Access

Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation.
Murray A, Sienerth AR, Hemberger M

Gene loci that are hypermethylated and repressed in embryonic (ESCs) but hypomethylated and expressed in trophoblast (TSCs) stem cells are very rare and may have particularly important roles in early developmental cell fate decisions, as previously shown for Elf5. Here, we assessed another member of this small group of genes, Placenta Expressed Transcript 1 (Plet1), for its function in establishing trophoblast lineage identity and modulating trophoblast differentiation. We find that Plet1 is tightly repressed by DNA methylation in ESCs but expressed on the cell surface of TSCs and trophoblast giant cells. In hypomethylated ESCs that are prone to acquire some trophoblast characteristics, Plet1 is required to confer a trophoblast-specific gene expression pattern, including up-regulation of Elf5. Plet1 displays an unusual biphasic expression profile during TSC differentiation and thus may be pivotal in balancing trophoblast self-renewal and differentiation. Furthermore, overexpression and CRISPR/Cas9-mediated knockout in TSCs showed that high Plet1 levels favour differentiation towards the trophoblast giant cell lineage, whereas lack of Plet1 preferentially induces syncytiotrophoblast formation. Thus, the endogenous dynamics of Plet1 expression establish important patterning cues within the trophoblast compartment by promoting differentiation towards the syncytiotrophoblast or giant cell pathway in Plet1-low and Plet1-high cells, respectively.

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Scientific reports, 6, 2045-2322, 25112, 2016

PMID: 27121762

Open Access

RNA-binding proteins ZFP36L1 and ZFP36L2 promote cell quiescence.
Galloway A, Saveliev A, Łukasiak S, Hodson DJ, Bolland D, Balmanno K, Ahlfors H, Monzón-Casanova E, Mannurita SC, Bell LS, Andrews S, Díaz-Muñoz MD, Cook SJ, Corcoran A, Turner M

Progression through the stages of lymphocyte development requires coordination of the cell cycle. Such coordination ensures genomic integrity while cells somatically rearrange their antigen receptor genes [in a process called variable-diversity-joining (VDJ) recombination] and, upon successful rearrangement, expands the pools of progenitor lymphocytes. Here we show that in developing B lymphocytes, the RNA-binding proteins (RBPs) ZFP36L1 and ZFP36L2 are critical for maintaining quiescence before precursor B cell receptor (pre-BCR) expression and for reestablishing quiescence after pre-BCR-induced expansion. These RBPs suppress an evolutionarily conserved posttranscriptional regulon consisting of messenger RNAs whose protein products cooperatively promote transition into the S phase of the cell cycle. This mechanism promotes VDJ recombination and effective selection of cells expressing immunoglobulin-μ at the pre-BCR checkpoint.

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Science (New York, N.Y.), 352, 1095-9203, 453-9, 2016

PMID: 27102483

Single-cell epigenomics: powerful new methods for understanding gene regulation and cell identity.
Clark SJ, Lee HJ, Smallwood SA, Kelsey G, Reik W

Emerging single-cell epigenomic methods are being developed with the exciting potential to transform our knowledge of gene regulation. Here we review available techniques and future possibilities, arguing that the full potential of single-cell epigenetic studies will be realized through parallel profiling of genomic, transcriptional, and epigenetic information.

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Genome biology, 17, 1474-760X, 72, 2016

PMID: 27091476

Open Access

Digesting the Expanding Mechanisms of Autophagy.
Ktistakis NT, Tooze SA

Autophagy is a catabolic 'self-eating' pathway that is emerging as a crucial integration point in cell physiology. With its own set of genes, the autophagy pathway communicates with virtually all signalling networks and organelles. Recent advances have been made in understanding the origin of the autophagosomal membrane, novel regulators, and the mechanisms by which specific intracellular membranes become autophagy substrates. New studies on noncanonical autophagy, mediated by subsets of autophagy proteins, and the role of autophagy proteins in non-autophagy pathways are also emerging in many different biological contexts. Our understanding of canonical autophagy, including membrane origin and autophagy proteins, needs to be considered together with emerging noncanonical pathways.

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Trends in cell biology, , 1879-3088, , 2016

PMID: 27050762

The systems biology format converter.
Rodriguez N, Pettit JB, Dalle Pezze P, Li L, Henry A, van Iersel MP, Jalowicki G, Kutmon M, Natarajan KN, Tolnay D, Stefan MI, Evelo CT, Le Novère N

Interoperability between formats is a recurring problem in systems biology research. Many tools have been developed to convert computational models from one format to another. However, they have been developed independently, resulting in redundancy of efforts and lack of synergy.

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BMC bioinformatics, 17, 1471-2105, 154, 2016

PMID: 27044654

Open Access

Inhibition of fatty acid desaturation is detrimental to cancer cell survival in metabolically compromised environments.
Peck B, Schug ZT, Zhang Q, Dankworth B, Jones DT, Smethurst E, Patel R, Mason S, Jiang M, Saunders R, Howell M, Mitter R, Spencer-Dene B, Stamp G, McGarry L, James D, Shanks E, Aboagye EO, Critchlow SE, Leung HY, Harris AL, Wakelam MJ, Gottlieb E, Schulze A

Enhanced macromolecule biosynthesis is integral to growth and proliferation of cancer cells. Lipid biosynthesis has been predicted to be an essential process in cancer cells. However, it is unclear which enzymes within this pathway offer the best selectivity for cancer cells and could be suitable therapeutic targets.

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Cancer & metabolism, 4, 2049-3002, 6, 2016

PMID: 27042297

Open Access

Effector γδ T Cell Differentiation Relies on Master but Not Auxiliary Th Cell Transcription Factors.
Barros-Martins J, Schmolka N, Fontinha D, Pires de Miranda M, Simas JP, Brok I, Ferreira C, Veldhoen M, Silva-Santos B, Serre K

γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αβ Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αβ T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1β plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αβ T cell subsets.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2016

PMID: 26994218

Can follicular helper T cells be targeted to improve vaccine efficacy?
Linterman MA, Hill DL

The success of most vaccines relies on the generation of antibodies to provide protection against subsequent infection; this in turn depends on a robust germinal centre (GC) response that culminates in the production of long-lived antibody-secreting plasma cells. The size and quality of the GC response are directed by a specialised subset of CD4 (+) T cells: T follicular helper (Tfh) cells. Tfh cells provide growth and differentiation signals to GC B cells and mediate positive selection of high-affinity B cell clones in the GC, thereby determining which B cells exit the GC as plasma cells and memory B cells. Because of their central role in the production of long-lasting humoral immunity, Tfh cells represent an interesting target for rational vaccine design.

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F1000Research, 5, 2046-1402, , 2016

PMID: 26989476

Open Access

Tumor cells with KRAS or BRAF mutations or ERK5/MAPK7 amplification are not addicted to ERK5 activity for cell proliferation.
Lochhead PA, Clark J, Wang LZ, Gilmour L, Squires M, Gilley R, Foxton C, Newell DR, Wedge SR, Cook SJ

ERK5, encoded by MAPK7, has been proposed to play a role in cell proliferation, thus attracting interest as a cancer therapeutic target. While oncogenic RAS or BRAF cause sustained activation of the MEK1/2-ERK1/2 pathway, ERK5 is directly activated by MEK5. It has been proposed that RAS and RAF proteins can also promote ERK5 activation. Here we investigated the interplay between RAS-RAF-MEK-ERK and ERK5 signaling and studied the role of ERK5 in tumor cell proliferation in 2 disease-relevant cell models. We demonstrate that although an inducible form of CRAF (CRAF:ER*) can activate ERK5 in fibroblasts, the response is delayed and reflects feed-forward signaling. Additionally, oncogenic KRAS and BRAF do not activate ERK5 in epithelial cells. Although KRAS and BRAF do not couple directly to MEK5-ERK5, ERK5 signaling might still be permissive for proliferation. However, neither the selective MEK5 inhibitor BIX02189 or ERK5 siRNA inhibited proliferation of colorectal cancer cells harbouring KRAS(G12C/G13D) or BRAF(V600E). Furthermore, there was no additive or synergistic effect observed when BIX02189 was combined with the MEK1/2 inhibitor Selumetinib (AZD6244), suggesting that ERK5 was neither required for proliferation nor a driver of innate resistance to MEK1/2 inhibitors. Finally, even cancer cells with MAPK7 amplification were resistant to BIX02189 and ERK5 siRNA, showing that ERK5 amplification does not confer addiction to ERK5 for cell proliferation. Thus ERK5 signaling is unlikely to play a role in tumor cell proliferation downstream of KRAS or BRAF or in tumor cells with ERK5 amplification. These results have important implications for the role of ERK5 as an anti-cancer drug target.

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Cell cycle (Georgetown, Tex.), 15, 1551-4005, 506-18, 2016

PMID: 26959608

Open Access

The inositol-3-phosphate synthase biosynthetic enzyme has distinct catalytic and metabolic roles.
Frej AD, Clark J, Roy CL, Lilla S, Thomason P, Otto GP, Churchill G, Insall R, Claus SP, Hawkins P, Stephens L, Williams RS

Inositol levels, maintained by the biosynthetic enzyme inositol-3-phosphate synthase (Ino1), are altered in a range of disorders including bipolar disorder and Alzheimer's disease. To date, most inositol studies have focused on the molecular and cellular effects of inositol depletion without considering Ino1 levels. Here we employ a simple eukaryote, Dictyostelium, to demonstrate distinct effects of loss of Ino1 and inositol depletion. We show that loss of Ino1 results in inositol auxotrophy that can only be partially rescued by exogenous inositol. Removal of inositol supplementation from the ino1(-) mutant results in a rapid 56% reduction in inositol levels, triggering the induction of autophagy, reduced cytokinesis and substrate adhesion. Inositol depletion also caused a dramatic generalised decrease in phosphoinositide levels that was rescued by inositol supplementation. However, loss of Ino1 triggered broad metabolic changes consistent with the induction of a catabolic state that was not rescued by inositol supplementation. These data suggest a metabolic role for Ino1 independent of inositol biosynthesis. To characterise this role, an Ino1 binding partner containing SEL1L1 domains (Q54IX5) was identified with homology to mammalian macromolecular complex adaptor proteins. Our findings therefore identify a new role for Ino1, independent of inositol biosynthesis, with broad effects on cell metabolism.

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Molecular and cellular biology, , 1098-5549, , 2016

PMID: 26951199

Open Access

Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.
Guo G, von Meyenn F, Santos F, Chen Y, Reik W, Bertone P, Smith A, Nichols J

Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals.

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Stem cell reports, , 2213-6711, , 2016

PMID: 26947977

Mislocalization of neuronal tau in the absence of tangle pathology in phosphomutant tau knockin mice.
Gilley J, Ando K, Seereeram A, Rodríguez-Martín T, Pooler AM, Sturdee L, Anderton BH, Brion JP, Hanger DP, Coleman MP

Hyperphosphorylation and fibrillar aggregation of the microtubule-associated protein tau are key features of Alzheimer's disease and other tauopathies. To investigate the involvement of tau phosphorylation in the pathological process, we generated a pair of complementary phosphomutant tau knockin mouse lines. One exclusively expresses phosphomimetic tau with 18 glutamate substitutions at serine and/or threonine residues in the proline-rich and first microtubule-binding domains to model hyperphosphorylation, whereas its phosphodefective counterpart has matched alanine substitutions. Consistent with expected effects of genuine phosphorylation, association of the phosphomimetic tau with microtubules and neuronal membranes is severely disrupted in vivo, whereas the phosphodefective mutations have more limited or no effect. Surprisingly, however, age-related mislocalization of tau is evident in both lines, although redistribution appears more widespread and more pronounced in the phosphomimetic tau knockin. Despite these changes, we found no biochemical or immunohistological evidence of pathological tau aggregation in mice of either line up to at least 2 years of age. These findings raise important questions about the role of tau phosphorylation in driving pathology in human tauopathies.

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Neurobiology of aging, 39, 1558-1497, 1-18, 2016

PMID: 26923397

Open Access

Follicular Helper T Cells.
Vinuesa CG, Linterman MA, Yu D, MacLennan IC

Although T cell help for B cells was described several decades ago, it was the identification of CXCR5 expression by B follicular helper T (Tfh) cells and the subsequent discovery of their dependence on BCL6, that led to the recognition of Tfh cells as an independent helper subset and accelerated the pace of discovery. More than 20 transcription factors, together with RNAbinding proteins and microRNAs, control the expression of chemotactic receptors and molecules important for the function and homeostasis of Tfh cells. Tfh cells prime B cells to initiate extrafollicular and germinal center antibody responses and are crucial for affinity maturation and maintenance of humoral memory. In addition to the roles that Tfh cells have in antimicrobial defense, cancer, and as HIV reservoirs, regulation of these cells is critical to prevent autoimmunity. The realization that follicular T cells are heterogeneous, comprising helper and regulatory subsets, has raised questions regarding a possible division of labor in germinal center B cell selection and elimination. Expected final online publication date for the Annual Review of Immunology Volume 34 is May 20, 2016. Please see for revised estimates.

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Annual review of immunology, , 1545-3278, , 2016

PMID: 26907215

Truncating PREX2 mutations activate its GEF activity and alter gene expression regulation in NRAS-mutant melanoma.
Lissanu Deribe Y, Shi Y, Rai K, Nezi L, Amin SB, Wu CC, Akdemir KC, Mahdavi M, Peng Q, Chang QE, Hornigold K, Arold ST, Welch HC, Garraway LA, Chin L

PREX2 (phosphatidylinositol-3,4,5-triphosphate-dependent Rac-exchange factor 2) is a PTEN (phosphatase and tensin homolog deleted on chromosome 10) binding protein that is significantly mutated in cutaneous melanoma and pancreatic ductal adenocarcinoma. Here, genetic and biochemical analyses were conducted to elucidate the nature and mechanistic basis of PREX2 mutation in melanoma development. By generating an inducible transgenic mouse model we showed an oncogenic role for a truncating PREX2 mutation (PREX2(E824)*) in vivo in the context of mutant NRAS. Using integrative cross-species gene expression analysis, we identified deregulated cell cycle and cytoskeleton organization as significantly perturbed biological pathways in PREX2 mutant tumors. Mechanistically, truncation of PREX2 activated its Rac1 guanine nucleotide exchange factor activity, abolished binding to PTEN and activated the PI3K (phosphatidyl inositol 3 kinase)/Akt signaling pathway. We further showed that PREX2 truncating mutations or PTEN deletion induces down-regulation of the tumor suppressor and cell cycle regulator CDKN1C (also known as p57(KIP2)). This down-regulation occurs, at least partially, through DNA hypomethylation of a differentially methylated region in chromosome 11 that is a known regulatory region for expression of the CDKN1C gene. Together, these findings identify PREX2 as a mediator of NRAS-mutant melanoma development that acts through the PI3K/PTEN/Akt pathway to regulate gene expression of a cell cycle regulator.

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Proceedings of the National Academy of Sciences of the United States of America, , 1091-6490, , 2016

PMID: 26884185

Open Access

Transient inhibition of ROR-γt therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells.
Withers DR, Hepworth MR, Wang X, Mackley EC, Halford EE, Dutton EE, Marriott CL, Brucklacher-Waldert V, Veldhoen M, Kelsen J, Baldassano RN, Sonnenberg GF

RAR-related orphan receptor-γt (ROR-γt) directs differentiation of proinflammatory T helper 17 (TH17) cells and is a potential therapeutic target in chronic autoimmune and inflammatory diseases. However, ROR-γt-dependent group 3 innate lymphoid cells ILC3s provide essential immunity and tissue protection in the intestine, suggesting that targeting ROR-γt could also result in impaired host defense after infection or enhanced tissue damage. Here, we demonstrate that transient chemical inhibition of ROR-γt in mice selectively reduces cytokine production from TH17 but not ILCs in the context of intestinal infection with Citrobacter rodentium, resulting in preserved innate immunity. Temporal deletion of Rorc (encoding ROR-γt) in mature ILCs also did not impair cytokine response in the steady state or during infection. Finally, pharmacologic inhibition of ROR-γt provided therapeutic benefit in mouse models of intestinal inflammation and reduced the frequency of TH17 cells but not ILCs isolated from primary intestinal samples of individuals with inflammatory bowel disease (IBD). Collectively, these results reveal differential requirements for ROR-γt in the maintenance of TH17 cell and ILC3 responses and suggest that transient inhibition of ROR-γt is a safe and effective therapeutic approach during intestinal inflammation.

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Nature medicine, , 1546-170X, , 2016

PMID: 26878233

The cellular composition of the human immune system is shaped by age and cohabitation.
Carr EJ, Dooley J, Garcia-Perez JE, Lagou V, Lee JC, Wouters C, Meyts I, Goris A, Boeckxstaens G, Linterman MA, Liston A

Detailed population-level description of the human immune system has recently become achievable. We used a 'systems-level' approach to establish a resource of cellular immune profiles of 670 healthy individuals. We report a high level of interindividual variation, with low longitudinal variation, at the level of cellular subset composition of the immune system. Despite the profound effects of antigen exposure on individual antigen-specific clones, the cellular subset structure proved highly elastic, with transient vaccination-induced changes followed by a return to the individual's unique baseline. Notably, the largest influence on immunological variation identified was cohabitation, with 50% less immunological variation between individuals who share an environment (as parents) than between people in the wider population. These results identify local environmental conditions as a key factor in shaping the human immune system.

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Nature immunology, , 1529-2916, , 2016

PMID: 26878114

What Is Trophoblast? A Combination of Criteria Define Human First-Trimester Trophoblast.
Lee CQ, Gardner L, Turco M, Zhao N, Murray MJ, Coleman N, Rossant J, Hemberger M, Moffett A

Controversy surrounds reports describing the derivation of human trophoblast cells from placentas and embryonic stem cells (ESC), partly due to the difficulty in identifying markers that define cells as belonging to the trophoblast lineage. We have selected criteria that are characteristic of primary first-trimester trophoblast: a set of protein markers, HLA class I profile, methylation of ELF5, and expression of microRNAs (miRNAs) from the chromosome 19 miRNA cluster (C19MC). We tested these criteria on cells previously reported to show some phenotypic characteristics of trophoblast: bone morphogenetic protein (BMP)-treated human ESC and 2102Ep, an embryonal carcinoma cell line. Both cell types only show some, but not all, of the four trophoblast criteria. Thus, BMP-treated human ESC have not fully differentiated to trophoblast. Our study identifies a robust panel, including both protein and non-protein-coding markers that, in combination, can be used to reliably define cells as characteristic of early trophoblast.

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Stem cell reports, 6, 2213-6711, 257-72, 2016

PMID: 26862703

Open Access

Emerging evidence of signalling roles for PI(3,4)P2 in Class I and II PI3K-regulated pathways.
Hawkins PT, Stephens LR

There are eight members of the phosphoinositide family of phospholipids in eukaryotes; PI, PI3P, PI4P, PI5P, PI(4,5)P2, PI(3,4)P2, PI(3,5)P2 and PI(3,4,5)P3. Receptor activation of Class I PI3Ks stimulates the phosphorylation of PI(4,5)P2 to form PI(3,4,5)P3. PI(3,4,5)P3 is an important messenger molecule that is part of a complex signalling network controlling cell growth and division. PI(3,4,5)P3 can be dephosphorylated by both 3- and 5-phosphatases, producing PI(4,5)P2 and PI(3,4)P2, respectively. There is now strong evidence that PI(3,4)P2 generated by this route does not merely represent another pathway for removal of PI(3,4,5)P3, but can act as a signalling molecule in its own right, regulating macropinocytosis, fast endophilin-mediated endocytosis (FEME), membrane ruffling, lamellipodia and invadopodia. PI(3,4)P2 can also be synthesized directly from PI4P by Class II PI3Ks and this is important for the maturation of clathrin-coated pits [clathrin-mediated endocytosis (CME)] and signalling in early endosomes. Thus PI(3,4)P2 is emerging as an important signalling molecule involved in the coordination of several specific membrane and cytoskeletal responses. Further, its inappropriate accumulation contributes to pathology caused by mutations in genes encoding enzymes responsible for its degradation, e.g. Inpp4B.

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Biochemical Society transactions, 44, 1470-8752, 307-14, 2016

PMID: 26862220

Disallowance of Acot7 in β-Cells Is Required for Normal Glucose Tolerance and Insulin Secretion.
Martinez-Sanchez A, Pullen TJ, Chabosseau P, Zhang Q, Haythorne E, Cane MC, Nguyen-Tu MS, Sayers SR, Rutter GA

Encoding acyl-CoA thioesterase-7 (Acot7) is one of ∼60 genes expressed ubiquitously across tissues but relatively silenced, or disallowed, in pancreatic β-cells. The capacity of ACOT7 to hydrolyze long-chain acyl-CoA esters suggests potential roles in β-oxidation, lipid biosynthesis, signal transduction, or insulin exocytosis. We explored the physiological relevance of β-cell-specific Acot7 silencing by re-expressing ACOT7 in these cells. ACOT7 overexpression in clonal MIN6 and INS1(832/13) β-cells impaired insulin secretion in response to glucose plus fatty acids. Furthermore, in a panel of transgenic mouse lines, we demonstrate that overexpression of mitochondrial ACOT7 selectively in the adult β-cell reduces glucose tolerance dose dependently and impairs glucose-stimulated insulin secretion. By contrast, depolarization-induced secretion was unaffected, arguing against a direct action on the exocytotic machinery. Acyl-CoA levels, ATP/ADP increases, membrane depolarization, and Ca(2+) fluxes were all markedly reduced in transgenic mouse islets, whereas glucose-induced oxygen consumption was unchanged. Although glucose-induced increases in ATP/ADP ratio were similarly lowered after ACOT7 overexpression in INS1(832/13) cells, changes in mitochondrial membrane potential were unaffected, consistent with an action of Acot7 to increase cellular ATP consumption. Because Acot7 mRNA levels are increased in human islets in type 2 diabetes, inhibition of the enzyme might provide a novel therapeutic strategy.

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Diabetes, 65, 1939-327X, 1268-82, 2016

PMID: 26861785