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The 海角社区论坛 Publications database contains details of all publications resulting from our research groups and  Pre-prints by Institute authors can be viewed on the Institute's . We believe that free and open access to the outputs of publicly鈥恌unded research offers significant social and economic benefits, as well as aiding the development of new research. We are working to provide Open Access to as many publications as possible and these can be identified below by the padlock icon. Where this hasn't been possible, subscriptions may be required to view the full text.
 

AG Torres, MM Fabani, E Vigorito, MJ Gait

MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.

+view abstract RNA (New York, N.Y.), PMID: 21441346 2011

Open Access
A Collins, SL Hewitt, J Chaumeil, M Sellars, M Micsinai, J Allinne, F Parisi, EP Nora, DJ Bolland, AE Corcoran, Y Kluger, R Bosselut, W Ellmeier, MM Chong, DR Littman, JA Skok

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T脗聽cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells.

+view abstract Immunity, PMID: 21435585 2011

G Kelsey

Transcriptome sequencing has identified more than a thousand potentially imprinted genes in the mouse brain. This comes as a revelation to someone who cut his teeth on the identification of imprinted genes when only a handful was known. Genomic imprinting, an epigenetic mechanism that determines expression of alleles according to sex of transmitting parent, was discovered over 25芒鈧 years ago in mice but remains an enigmatic phenomenon. Why do these genes disobey the normal Mendelian logic of inheritance, do they function in specific processes, and how is their imprinting conferred? Next generation sequencing technologies are providing an unprecedented opportunity to survey the whole genome for imprinted genes and are beginning to reveal that imprinting may be more pervasive than we had come to believe. Such advances should lay the foundation for a definitive account of imprinting, but may also challenge accepted views on what it means to be imprinted. Editor's suggested further reading in BioEssays RNA as the substrate for epigenome-environment interactions Abstract.

+view abstract BioEssays : news and reviews in molecular, cellular and developmental biology, PMID: 21425303 2011

SJ Gaunt, YL Paul

Cdx1, an upstream regulator of Hox genes, is best characterized for its homeotic effects upon the developing axial skeleton, particularly in the neck. It responds to retinoic acid (RA) in both mouse embryos and embryonal carcinoma (EC) cells. By use of beta-galactosidase chemiluminescence, we show that a mouse Cdx1/lacZ reporter expressed in P19 EC cells responds to RA by the combined activities of an intron retinoic acid response element (RARE) and an upstream RARE. In contrast, a chicken Cdx1/lacZ reporter responds only by activity of the intron RARE. Database analyses upon Cdx1 from twenty three vertebrate species reveal that the intron RARE is structurally conserved in amniotes (eutherian mammals, marsupials, birds and Anole lizard), but not in Xenopus or fish. The upstream RARE is structurally conserved only in eutherian mammals. We conclude that the intron RARE originated at around the amphibian/amniote division, and the upstream RARE appeared around the marsupial/eutherian mammal division. In view of the site of action of Cdx1, we propose that acquisition of the intron RARE may have facilitated the substantial changes that occurred in the neck and anterior thorax at the advent of the amniotes. We present evidence that Cdx1 is also a developmental regulator of the female urogenital system, and we suggest that acquisition of the upstream RARE may have contributed to morphological divergence of marsupial and eutherian mammals.

+view abstract The International journal of developmental biology, PMID: 21425084 2011

Open Access
Flatz L,Roychoudhuri R,Honda M,Filali-Mouhim A,Goulet JP,Kettaf N,Lin M,Roederer M,Haddad EK,S脙漏kaly RP,Nabel GJ Immunology

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

+view abstract Proceedings of the National Academy of Sciences of the United States of America, PMID: 21422297 2011

G Str眉bbe, C Popp, A Schmidt, A Pauli, L Ringrose, C Beisel, R Paro

The maintenance of specific gene expression patterns during cellular proliferation is crucial for the identity of every cell type and the development of tissues in multicellular organisms. Such a cellular memory function is conveyed by the complex interplay of the Polycomb and Trithorax groups of proteins (PcG/TrxG). These proteins exert their function at the level of chromatin by establishing and maintaining repressed (PcG) and active (TrxG) chromatin domains. Past studies indicated that a core PcG protein complex is potentially associated with cell type or even cell stage-specific sets of accessory proteins. In order to better understand the dynamic aspects underlying PcG composition and function we have established an inducible version of the biotinylation tagging approach to purify Polycomb and associated factors from Drosophila embryos. This system enabled fast and efficient isolation of Polycomb containing complexes under near physiological conditions, thereby preserving substoichiometric interactions. Novel interacting proteins were identified by highly sensitive mass spectrometric analysis. We found many TrxG related proteins, suggesting a previously unrecognized extent of molecular interaction of the two counteracting chromatin regulatory protein groups. Furthermore, our analysis revealed an association of PcG protein complexes with the cohesin complex and showed that Polycomb-dependent silencing of a transgenic reporter depends on cohesin function.

+view abstract Proceedings of the National Academy of Sciences of the United States of America, PMID: 21415365 2011

M Wossidlo, T Nakamura, K Lepikhov, CJ Marques, V Zakhartchenko, M Boiani, J Arand, T Nakano, W Reik, J Walter

The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular mechanisms. We detect 5-hydroxymethylcytosine (5hmC) as a novel modification in mouse, bovine and rabbit zygotes. On zygotic development 5hmC accumulates in the paternal pronucleus along with a reduction of 5mC. A knockdown of the 5hmC generating dioxygenase Tet3 simultaneously affects the patterns of 5hmC and 5mC in the paternal pronucleus. This finding links the loss of 5mC to its conversion into 5hmC. The maternal pronucleus seems to be largely protected against this mechanism by PGC7/Dppa3/Stella, as in PGC7 knockout zygotes 5mC also becomes accessible to oxidation into 5hmC. In summary, our data suggest an important role of 5hmC and Tet3 for DNA methylation reprogramming processes in the mammalian zygote.

+view abstract Nature communications, PMID: 21407207 2011

Open Access
DT Patton, MD Wilson, WC Rowan, DR Soond, K Okkenhaug Immunology

The PI3K pathway has emerged as a key regulator of regulatory T cell (Treg) development and homeostasis and is required for full Treg-mediated suppression. To identify new genes involved in PI3K-dependent suppression, we compared the transcriptome of WT and p110脦麓(D910A) Tregs. Among the genes that were differentially expressed was the gene for the transmembrane cyclic ADP ribose hydrolase CD38. Here we show that CD38 is expressed mainly by a subset of Foxp3(+)CD25(+)CD4(+) T cells originating in the thymus and on Tregs in the spleen. CD38(high) WT Tregs showed superior suppressive activity to CD38(low) Tregs, which failed to upregulate CD73, a surface protein which is important for suppression. However, Tregs from heterozygous CD38(+/-) mice were unimpaired despite lower levels of CD38 expression. Therefore, CD38 can be used as a marker for Tregs with high suppressive activity and the impaired Treg function in p110脦麓(D910A) mice can in part be explained by the failure of CD38(high) cells to develop.

+view abstract PloS one, PMID: 21390257 2011

Open Access
CM Wiggins, P Tsvetkov, M Johnson, CL Joyce, CA Lamb, NJ Bryant, D Komander, Y Shaul, SJ Cook

BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)脦鈥滽K, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase 脦虏-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

+view abstract Journal of cell science, PMID: 21378313 2011

CW Paxton, RA Cosgrove, AC Drozd, EL Wiggins, S Woodhouse, RA Watson, HJ Spence, BW Ozanne, JM Pell Signalling

The BTB-Kelch protein Krp1 is highly and specifically expressed in skeletal muscle, where it is proposed to have a role in myofibril formation. We observed significant upregulation of Krp1 in C2 cells early in myoblast differentiation, well before myofibrillogenesis. Krp1 has a role in cytoskeletal organization and cell motility; since myoblast migration and elongation/alignment are important events in early myogenesis, we hypothesized that Krp1 is involved with earlier regulation of differentiation. Krp1 protein levels were detectable by 24 h after induction of differentiation in C2 cells and were significantly upregulated by 48 h, i.e., following the onset myogenin expression and preceding myosin heavy chain (MHC) upregulation. Upregulation of Krp1 required a myogenic stimulus as signaling derived from increased myoblast cell density was insufficient to activate Krp1 expression. Examination of putative Krp1 proximal promoter regions revealed consensus E box elements associated with myogenic basic helix-loop-helix binding. The activity of a luciferase promoter-reporter construct encompassing this 2,000-bp region increased in differentiating C2 myoblasts and in C2 cells transfected with myogenin and/or MyoD. Knockdown of Krp1 via short hairpin RNA resulted in increased C2 cell number and proliferation rate as assessed by bromodeoxyuridine incorporation, whereas overexpression of Krp1-myc had the opposite effect; apoptosis was unchanged. No effects of changed Krp1 protein levels on cell migration were observed, either by scratch wound assay or live cell imaging. Paradoxically, both knockdown and overexpression of Krp1 inhibited myoblast differentiation assessed by expression of myogenin, MEF2C, MHC, and cell fusion.

+view abstract American journal of physiology. Cell physiology, PMID: 21368295 2011

Open Access
X Zhang, O Vadas, O Perisic, KE Anderson, J Clark, PT Hawkins, LR Stephens, RL Williams Signalling,Biological Chemistry

Phosphoinositide 3-kinases (PI3Ks) are essential for cell growth, migration, and survival. The structure of a p110脦虏/p85脦虏 complex identifies an inhibitory function for the C-terminal SH2 domain (cSH2) of the p85 regulatory subunit. Mutagenesis of a cSH2 contact residue activates downstream signaling in cells. This inhibitory contact ties up the C-terminal region of the p110脦虏 catalytic subunit, which is essential for lipid kinase activity. In脗聽vitro, p110脦虏 basal activity is tightly restrained by contacts with three p85 domains: the cSH2, nSH2, and iSH2. RTK phosphopeptides relieve inhibition by nSH2 and cSH2 using completely different mechanisms. The binding site for the RTK's pYXXM motif is exposed on the cSH2, requiring an extended RTK motif to reach and disrupt the inhibitory contact with p110脦虏. This contrasts with the nSH2 where the pY-binding site itself forms the inhibitory contact. This establishes an unusual mechanism by which p85 SH2 domains contribute to RTK signaling specificities.

+view abstract Molecular cell, PMID: 21362552 2011

Open Access
CJ Petry, ML Evans, DL Wingate, KK Ong, W Reik, M Const芒ncia, DB Dunger Epigenetics

At the beginning of the third week of pregnancy, mouse fetuses with targeted disruption of their paternally-transmitted insulin-like growth factor 2 gene placental-specific transcripts have growth-restricted placentas but normal body weights due to upregulated placental nutrient transport. We assessed whether increased placental glucose transport rates were associated with raised maternal glucose concentrations by performing intraperitoneal glucose tolerance tests (ipGTT) in pregnant mice carrying knockout pups and comparing them with mice carrying genotype-matched phenotypically wild type pups. Mean 脗卤 SD body weights of affected pups were 95 脗卤 8% of control values at e16 and 73 脗卤 7% at e18. There were no differences in areas under the maternal ipGTT curves at either e16 (mean 脗卤 SD being 99.0 脗卤 9.1% of control values; P = .9) or e18 (91.4 脗卤 13.4%; P = .3), suggesting that effects on transplacental glucose transport in these mice are not mediated through changes in maternal glucose concentrations.

+view abstract Experimental diabetes research, PMID: 21331382 2011

Open Access
Altin JA, Tian L, Liston A, Bertram EM, Goodnow CC, Cook MC Immunology

Allergy, the most common disease of immune dysregulation, has a substantial genetic component that is poorly understood. Although complete disruption of T-cell receptor (TCR) signaling causes profound immunodeficiency, little is known about the consequences of inherited genetic variants that cause partial quantitative decreases in particular TCR-signaling pathways, despite their potential to dysregulate immune responses and cause immunopathology.

+view abstract The Journal of allergy and clinical immunology, PMID: 21320717 2011

M Coleman Signalling

Axons depend critically on axonal transport both for supplying materials and for communicating with cell bodies. This chapter looks at each activity, asking what aspects are essential for axon survival. Axonal transport declines in neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis, and multiple sclerosis, and in normal ageing, but whether all cargoes are equally affected and what limits axon survival remains unclear. Cargoes can be differentially blocked in some disorders, either individually or in groups. Each missing protein cargo results in localized loss-of-function that can be partially modeled by disrupting the corresponding gene, sometimes with surprising results. The axonal response to losing specific proteins also depends on the rates of protein turnover and on whether the protein can be locally synthesized. Among cargoes with important axonal roles are components of the PI3 kinase, Mek/Erk, and Jnk signaling pathways, which help to communicate with cell bodies and to regulate axonal transport itself. Bidirectional trafficking of Bdnf, NT-3, and other neurotrophic factors contribute to intra- and intercellular signaling, affecting the axon's cellular environment and survival. Finally, several adhesion molecules and gangliosides are key determinants of axon survival, probably by mediating axon-glia interactions. Thus, failure of long-distance intracellular transport can deprive axons of one, few, or many cargoes. This can lead to axon degeneration either directly, through the absence of essential axonal proteins, or indirectly, through failures in communication with cell bodies and nonneuronal cells.

+view abstract Advances in genetics, PMID: 21310297 2011

F Krueger, SR Andrews, CS Osborne Bioinformatics

Massively parallel DNA sequencing is capable of sequencing tens of millions of DNA fragments at the same time. However, sequence bias in the initial cycles, which are used to determine the coordinates of individual clusters, causes a loss of fidelity in cluster identification on Illumina Genome Analysers. This can result in a significant reduction in the numbers of clusters that can be analysed. Such low sample diversity is an intrinsic problem of sequencing libraries that are generated by restriction enzyme digestion, such as e4C-seq or reduced-representation libraries. Similarly, this problem can also arise through the combined sequencing of barcoded, multiplexed libraries. We describe a procedure to defer the mapping of cluster coordinates until low-diversity sequences have been passed. This simple procedure can recover substantial amounts of next generation sequencing data that would otherwise be lost.

+view abstract PloS one, PMID: 21305042 2011

Open Access
Z Madeja, H Yadi, R Apps, S Boulenouar, SJ Roper, L Gardner, A Moffett, F Colucci, M Hemberger Epigenetics

The mammalian fetus represents a semiallograft within the maternal uterus yet is not rejected. This situation is particularly pronounced in species with a hemochorial type of placentation, such as humans and rodents, where maternal tissues and blood are in direct contact with fetal trophoblast and thus potentially with paternal antigens. The main polymorphic antigens responsible for graft rejection are MHC antigens. In humans the trophoblast cells invading into the decidua have a unique pattern of MHC class I expression characterized by both classical (HLA-C) and nonclassical (HLA-G and HLA-E) molecules. Whether such an unusual MHC repertoire on the surface of trophoblast is a conserved feature between species with hemochorial placentation has not been resolved. Here we demonstrate, using a range of methods, that C57BL/6 mouse trophoblast predominantly expresses only one MHC class I antigen, H2-K, at the cell surface of giant cells but lacks expression of nonclassical MHC molecules. Antigenic disparity between parental MHCs affects trophoblast-induced transformation of the uterine vasculature and, consequently, placental and fetal gowth. Maternal uterine blood vessels were more dilated, allowing for increased blood supply, in certain combinations of maternal and paternal MHC haplotypes, and these allogeneic fetuses and placentas were heavier at term compared with syngeneic controls. Thus, maternal-fetal immune interactions are instrumental to optimize reproductive success. This cross-talk has important implications for human disorders of pregnancy, such as preeclampsia and fetal growth restriction.

+view abstract Proceedings of the National Academy of Sciences of the United States of America, PMID: 21300875 2011

MD Bootman, I Smyrnias, R Thul, S Coombes, HL Roderick

Whereas Ca(2+) signalling in ventricular cardiomyocytes is well described, much less is known regarding the Ca(2+) signals within atrial cells. This is surprising given that atrial cardiomyocytes make an important contribution to the refilling of ventricles with blood, which enhances the subsequent ejection of blood from the heart. The dependence of cardiac function on the contribution of atria becomes increasingly important with age and exercise. Disruption of the rhythmic beating of atrial cardiomyocytes can lead to life-threatening conditions such as atrial fibrillation. Atrial and ventricular myocytes have many structural and functional similarities. However, one key structural difference, the lack of transverse tubules ("T-tubules") in atrial myocytes, make these two cell types display vastly different calcium patterns in response to electrical excitation. The lack of T-tubules in atrial myocytes means that depolarisation provokes calcium signals that originate around the periphery of the cells. Under resting conditions, such Ca(2+) signals do not propagate towards the centre of the atrial cells and so do not fully engage the contractile machinery. Consequently, contraction of atrial myocytes under resting conditions is modest. However, when atrial myocytes are stimulated with a positive inotropic agonist, such as isoproterenol, the peripheral Ca(2+) signals trigger a global wave of Ca(2+) that propagates in a centripetal manner into the cells. Enhanced centripetal movement of Ca(2+) in atrial myocytes leads to increased contraction and a more substantial contribution to blood pumping. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

+view abstract Biochimica et biophysica acta, PMID: 21295621 2011

Open Access
AN Macintyre, D Finlay, G Preston, LV Sinclair, CM Waugh, P Tamas, C Feijoo, K Okkenhaug, DA Cantrell Immunology

In cytotoxic T cells (CTL), Akt, also known as protein kinase B, is activated by the T cell antigen receptor (TCR) and the cytokine interleukin 2 (IL-2). Akt can control cell metabolism in many cell types but whether this role is important for CTL function has not been determined. Here we have shown that Akt does not mediate IL-2- or TCR-induced cell metabolic responses; rather, this role is assumed by other Akt-related kinases. There is, however, a nonredundant role for sustained and strong activation of Akt in CTL to coordinate the TCR- and IL-2-induced transcriptional programs that control expression of key cytolytic effector molecules, adhesion molecules, and cytokine and chemokine receptors that distinguish effector versus memory and naive T cells. Akt is thus dispensable for metabolism, but the strength and duration of Akt activity dictates the CTL transcriptional program and determines CTL fate.

+view abstract Immunity, PMID: 21295499 2011

B Stockinger, K Hirota, J Duarte, M Veldhoen Immunology

The aryl hydrocarbon receptor (AhR), subject of intensive research over three decades by the pharmacology/toxicology field has recently made its entry into mainstream immunology research and is set to continue to intrigue with ever more complex modes of modulating immune responses. The discovery of high and selective AhR expression on Th17 cells and its role in induction of the cytokine IL-22 attributed new immunological functions to this transcription factor and stimulated further research into physiological functions of the AhR in the immune system. A number of recent reviews have highlighted potential new avenues of research. This review addresses recent new insight into physiological roles of AhR in the immune system.

+view abstract Seminars in immunology, PMID: 21288737 2011

Jeremiah NM, Liston A Immunology

The discovery of Foxp3 as a reliable marker for murine regulatory T cells has led to an explosion in the development of genetic tools for investigating the biology of regulatory T cells. More than 25 Foxp3-based mouse strains have been published with a variety of characteristics. The effects of Foxp3 expression can be analyzed using null, hypomorphic, conditional, altered control, and over-expression strains. Reporter strains are available to efficiently isolate Foxp3+ cells, with various reporter designs in terms of construct (fusion, replacement, and bicistronic positioning), and reporter system (GFP, YFP, RFP, Luciferase, Thy1.1). Multifunction strain fusion, replacement, and bicistronic positionings add functional proteins under the control of the Foxp3 promoter allowing induced apoptosis or lineage-specific Cre recombinase activity. In this chapter, we discuss the uses of the cornucopia of genetic tools, in isolation and in combination, for research on Foxp3(+) regulatory T cells.

+view abstract Methods in molecular biology (Clifton, N.J.), PMID: 21287332 2011

Open Access
E Angiolini, PM Coan, I Sandovici, OH Iwajomo, G Peck, GJ Burton, CP Sibley, W Reik, AL Fowden, M Const芒ncia Epigenetics

The healthy development of the fetus depends on an optimal balance between fetal genetic drive for growth and the maternal ability to provide nutrients through the placenta. Nothing is known about fetal-placental signaling in response to increased fetal demand in the situation of overgrowth. Here, we examined this question using the H19(脦鈥13) mouse model, shown previously to result in elevated levels of Igf2. Fetal and placental weights in H19(脦鈥13) were increased by 23% and 45%, respectively, at E19, when compared with wild-type mice. Unexpectedly, we found that disproportionately large H19(脦鈥13) placentas transport 20-35% less (per gram placenta) glucose and system A amino acids and have similar reductions in passive permeability, despite a significantly greater surface area for nutrient exchange and theoretical diffusion capacity compared with wild-type mice. Expression of key transporter genes Slc2a3 and Slc38a4 was reduced by 芒藛录20%. Decreasing the overgrowth of the H19(脦鈥13) placenta by genetically reducing levels of Igf2P0 resulted in up-regulation of system A activity and maintenance of fetal overgrowth. Our results provide direct evidence that large placentas can modify their nutrient transfer capacity to regulate fetal nutrient acquisition. Our findings are indicative of fetal-placental signaling mechanisms that limit total demand for maternal nutrients.

+view abstract FASEB journal : official publication of the Federation of American Societies for Experimental Biology, PMID: 21282203 2011

Open Access
J Clark, KE Anderson, V Juvin, TS Smith, F Karpe, MJ Wakelam, LR Stephens, PT Hawkins Signalling,Biological Chemistry

Class I phosphoinositide-3-kinase (PI3K) isoforms generate the intracellular signaling lipid, phosphatidylinositol(3,4,5)trisphosphate (PtdIns(3,4,5)P(3)). PtdIns(3,4,5)P(3) regulates major aspects of cellular behavior, and the use of both genetic and pharmacological intervention has revealed important isoform-specific roles for PI3Ks in health and disease. Despite this interest, current methods for measuring PtdIns(3,4,5)P(3) have major limitations, including insensitivity, reliance on radiolabeling, low throughput and an inability to resolve different fatty-acyl species. We introduce a methodology based on phosphate methylation coupled to high-performance liquid chromatography-mass spectrometry (HPLC-MS) to solve many of these problems and describe an integrated approach to quantify PtdIns(3,4,5)P(3) and related phosphoinositides (regio-isomers of PtdInsP and PtdInsP(2) are not resolved). This methodology can be used to quantify multiple fatty-acyl species of PtdIns(3,4,5)P(3) in unstimulated mouse and human cells (芒鈥奥10(5)) or tissues (芒鈥奥0.1 mg) and their increase upon appropriate stimulation.

+view abstract Nature methods, PMID: 21278744 2011

Open Access
K Hirota, JH Duarte, M Veldhoen, E Hornsby, Y Li, DJ Cua, H Ahlfors, C Wilhelm, M Tolaini, U Menzel, A Garefalaki, AJ Potocnik, B Stockinger Immunology

Here we describe a reporter mouse strain designed to map the fate of cells that have activated interleukin 17A (IL-17A). We found that IL-17-producing helper T cells (T(H)17 cells) had distinct plasticity in different inflammatory settings. Chronic inflammatory conditions in experimental autoimmune encephalomyelitis (EAE) caused a switch to alternative cytokines in T(H)17 cells, whereas acute cutaneous infection with Candida albicans did not result in the deviation of T(H)17 cells to the production of alternative cytokines, although IL-17A production was shut off in the course of the infection. During the development of EAE, interferon-脦鲁 (IFN-脦鲁) and other proinflammatory cytokines in the spinal cord were produced almost exclusively by cells that had produced IL-17 before their conversion by IL-23 ('ex-T(H)17 cells'). Thus, this model allows the actual functional fate of effector T cells to be related to T(H)17 developmental origin regardless of IL-17 expression.

+view abstract Nature immunology, PMID: 21278737 2011

NJ Shimwell, W Wei, S Wilson, MJ Wakelam, T Ismail, T Iqbal, PJ Johnson, A Martin, DG Ward Signalling

Patients with colorectal cancer often present with advanced disease and concomitant poor prognosis. The best known serum biomarker, carcinoembryonic antigen (CEA) is not recommended for screening because of its limited specificity and sensitivity. A number of other circulating proteins have been suggested to be diagnostically useful but individually none of these has proved to be of sufficient sensitivity or specificity to establish a role in routine clinical practice. Here, we test the hypothesis that combining several of these biomarkers will improve diagnostic efficacy.

+view abstract Cancer biomarkers : section A of Disease markers, PMID: 21263188 2010