TY - JOUR AB - Large language models (LLMs) are being increasingly incorporated into scientific workflows. However, we have yet to fully grasp the implications of this integration. How should the advancement of large language models affect the practice of science? For this opinion piece, we have invited four diverse groups of scientists to reflect on this query, sharing their perspectives and engaging in debate. Schulz et al. make the argument that working with LLMs is not fundamentally different from working with human collaborators, while Bender et al. argue that LLMs are often misused and overhyped, and that their limitations warrant a focus on more specialized, easily interpretable tools. Marelli et al. emphasize the importance of transparent attribution and responsible use of LLMs. Finally, Botvinick and Gershman advocate that humans should retain responsibility for determining the scientific roadmap. To facilitate the discussion, the four perspectives are complemented with a response from each group. By putting these different perspectives in conversation, we aim to bring attention to important considerations within the academic community regarding the adoption of LLMs and their impact on both current and future scientific practices. AU - Binz, M. AU - Alaniz, S. AU - Roskies, A.* AU - Aczel, B.* AU - Bergstrom, C.T.* AU - Allen, C.E.* AU - Schad, D.* AU - Wulff, D.U.* AU - West, J.D.* AU - Zhang, Q.* AU - Shiffrin, R.M.* AU - Gershman, S.J.* AU - Popov, V.* AU - Bender, E.M.* AU - Marelli, M.* AU - Botvinick, M.M.* AU - Akata, Z. AU - Schulz, E. C1 - 73185 C2 - 56950 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - How should the advancement of large language models affect the practice of science? JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 5 PB - Natl Acad Sciences PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - The chloroplast genome encodes key components of the photosynthetic light reaction machinery as well as the large subunit of the enzyme central for carbon fixation, Ribulose-1,5-bisphosphat-carboxylase/-oxygenase (RuBisCo). Its expression is predominantly regulated posttranscriptionally, with nuclear-encoded RNA-binding proteins (RBPs) playing a key role. Mutants of chloroplast gene expression factors often exhibit impaired chloroplast biogenesis, especially in cold conditions. Low temperatures pose a challenge for plants as this leads to electron imbalances and oxidative damage. A well-known response of plants to this problem is to increase the production of RuBisCo and other Calvin Cycle enzymes in the cold, but how this is achieved is unclear. The chloroplast RBP CP29A has been shown to be essential for cold resistance in growing leaf tissue of Arabidopsis thaliana. Here, we examined CP29A-RNA interaction sites at nucleotide resolution. We found that CP29A preferentially binds to the 5'-untranslated region of rbcL, downstream of the binding site of the pentatricopeptide repeat protein MATURATION OF RBCL 1 (MRL1). MRL1 is an RBP known to be necessary for the accumulation of rbcL. In Arabidopsis mutants lacking CP29A, we were unable to observe significant effects on rbcL, possibly due to CP29A's restricted role in a limited number of cells at the base of leaves. In contrast, CRISPR/Cas9-induced mutants of tobacco NtCP29A exhibit cold-dependent photosynthetic deficiencies throughout the entire leaf blade. This is associated with a parallel reduction in rbcL mRNA and RbcL protein accumulation. Our work indicates that a chloroplast RNA-binding protein contributes to cold acclimation of RbcL production. AU - Lenzen, B.* AU - Rösch, F.* AU - Legen, J.* AU - Ruwe, H.* AU - Kachariya, N. AU - Sattler, M. AU - Small, I.* AU - Schmitz-Linneweber, C.* C1 - 73213 C2 - 56957 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - The chloroplast RNA-binding protein CP29A supports rbcL expression during cold acclimation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 5 PB - Natl Acad Sciences PY - 2025 SN - 0027-8424 ER - TY - JOUR AU - Mishima, E. AU - O'Neill, T.J. AU - Hoefig, K.P. AU - Chen, D. AU - Behrens, G. AU - Henkelmann, B. AU - Ito, J.* AU - Nakagawa, K.* AU - Heissmeyer, V. AU - Conrad, M. AU - Krappmann, D. C1 - 74362 C2 - 57482 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - MALT1 inhibitor MI-2 induces ferroptosis by direct targeting of GPX4. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 20 PB - Natl Acad Sciences PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - In order to find extrinsic rewards, humans explore their environment even if exploration requires several intermediate, reward-free decisions. It has been hypothesized that intrinsic rewards, such as novelty, surprise, or information gain, guide this reward-free exploration. However, in artificial agents, different intrinsic reward signals induce exploration strategies that respond differently to stochasticity. In particular, some strategies are vulnerable to the "noisy TV problem," i.e., an attraction to irrelevant stochastic stimuli. Here, we ask whether humans exhibit a similar attraction to reward-free stochasticity. We design a multistep decision-making paradigm in which participants search for rewarding states in a complex environment containing a highly stochastic but reward-free subregion. We show that i) participants persistently explore the stochastic subregion, and ii) their decisions are best explained by a novelty-driven exploration strategy, compared to alternatives driven by information gain or surprise. Our findings suggest that novelty and extrinsic rewards jointly control human exploration in complex environments. AU - Modirshanechi, A. AU - Lin, W.H.* AU - Xu, H.A.* AU - Herzog, M.H.* AU - Gerstner, W.* C1 - 75627 C2 - 58056 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Novelty as a drive of human exploration in complex stochastic environments. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 39 PB - Natl Acad Sciences PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - Hepatitis B virus (HBV) poses a significant global health challenge, necessitating the urgent development of curative therapeutics. However, this progress is impeded by the lack of robust, immunocompetent preclinical animal models due to HBV's strict species specificity. We previously showed that vector-mediated expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), renders macaques fully susceptible to HBV infection. In this study, we have generated transgenic macaques expressing hNTCP, marking the creation of the first transgenic nonhuman primate model for infectious disease research. We used PiggyBac (PB) transposon technology to insert a liver-specific hNTCP expression cassette into rhesus macaque zygotes and transferred the resulting embryos into surrogate females, resulting in two healthy transgenic offspring. In both animals, hNTCP is highly and selectively expressed in the liver. Most importantly, we show that isolated hepatocytes from these monkeys are susceptible to HBV infection. These findings lay the foundation for the development of a nonhuman primate HBV model, facilitating the advancement and validation of curative HBV therapies. AU - Rust, L.N.* AU - Wettengel, J.M. AU - Biswas, S.* AU - Ryu, J.* AU - Piekarski, N.* AU - Yusova, S.* AU - Lutz, S.S.* AU - Naldiga, S.* AU - Hinrichs, B.J.* AU - Sullivan, M.N.* AU - Lo, J.O.* AU - Protzer, U. AU - Smedley, J.V.* AU - Sacha, J.B.* AU - Hanna, C.B.* AU - Bimber, B.N.* AU - Hennebold, J.D.* AU - Burwitz, B.J.* C1 - 73366 C2 - 57017 TI - Liver-specific transgenic expression of human NTCP in rhesus macaques confers HBV susceptibility on primary hepatocytes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 7 PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - Protein therapeutics such as antibodies require in-depth in vivo characterization during development and consequently account for a large proportion of laboratory animal consumption in the pharmaceutical industry. Currently, antibody candidates are exhaustively tested one-by-one in animal models to determine pharmacokinetic and pharmacodynamic (PK/PD) profiles. The simultaneous analysis of antibody mixtures in single animals, called cassette-dosing, could in principle overcome this bottleneck, but is currently limited to small cassette sizes. Here, we demonstrate how the use of genetically encoded peptide tags (flycodes), designed for maximal detectability in liquid chromatography-mass spectrometry, can allow for the simultaneous characterization of large pools of drug candidates, from single cassette-dosed mice. We demonstrate the simultaneous assessment of PK parameters for a group of >20 marketed/development-stage antibodies. Biodistribution experiments in mice bearing EGFR-expressing tumors correctly identified the two pool members recognizing EGFR, while organ analysis registered liver accumulation of an antibody targeting glucagon receptor, a protein profoundly expressed in that organ. In analogy to an early-phase drug development campaign, we performed biophysical and PK analysis for a cassette of 80 unique bispecific DARPin-sybody molecules. The data shown in this study originate from only 18 cassette-dosed mice, thereby demonstrating how flycode technology efficiently advances preclinical discovery pipelines allowing a direct comparison of drug candidates under identical experimental conditions. AU - Walter, J.D.* AU - Beffinger, M.* AU - Egloff, P.* AU - Zimmermann, I.* AU - Hürlimann, L.M.* AU - Ackle, F.* AU - Seifert, M.* AU - Kobold, S. AU - vom Berg, J.* AU - Seeger, M.A.* C1 - 73675 C2 - 57165 TI - Flycodes enable simultaneous preclinical analysis for dozens of antibodies in single cassette-dosed mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 12 PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - Yellow fever vaccination provides long-lasting protection and is a unique model for studying the immune response to an acute RNA virus infection in humans. To elucidate the early innate immune events preceding the rapid generation of protective immunity, we performed transcriptome analysis of human blood dendritic cell (DC) and monocyte subpopulations before and 3, 7, 14, and 28 d after vaccination. We detected temporary upregulation of IFN-stimulated genes (ISG) in all DC and monocyte subsets on days 3 and 7 after vaccination as well as cell type-specific responses and response kinetics. Single-cell RNA sequencing revealed rapid appearance of activated DC and monocyte clusters dominated by ISGs, inflammatory chemokines, and genes involved in antigen processing and presentation. This was confirmed by flow cytometric analysis in a large cohort of vaccinees. We identified SIGLEC1/CD169 upregulation as a sensitive indicator of the transient IFN-induced activation state elicited in DCs and monocytes by YF17D vaccination correlating with early protective IgM antibody responses. AU - Winheim, E.* AU - Santos-Peral, A.* AU - Ehm, T.* AU - Rinke, L.* AU - Riemer, S.* AU - Zaucha, M. AU - Goresch, S.* AU - Lehmann, L.* AU - Eisenächer, K.* AU - Pritsch, M.* AU - Barba-Spaeth, G.* AU - Straub, T.* AU - Rothenfußer, S. AU - Krug, A.B.* C1 - 74315 C2 - 57432 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Interferon-induced activation of dendritic cells and monocytes by yellow fever vaccination correlates with early antibody responses. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 122 IS - 19 PB - Natl Acad Sciences PY - 2025 SN - 0027-8424 ER - TY - JOUR AB - The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management. AU - Huang, Z.* AU - Zhang, Y.* AU - Wang, S.* AU - Qi, R.* AU - Tao, Y.* AU - Sun, Y.* AU - Jiang, D. AU - Jiang, X.* AU - Tao, J.* C1 - 69907 C2 - 55318 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain via m6A-dependent stabilization of 5-HT3A mRNA in sensory neurons. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 6 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases. AU - Immler, R.* AU - Nussbaumer, K.* AU - Doerner, A.* AU - El Bounkari, O.* AU - Huber, S.* AU - Abisch, J.* AU - Napoli, M.* AU - Schmidt, S.* AU - Margraf, A.* AU - Pruenster, M.* AU - Rohwedder, I.* AU - Lange-Sperandio, B.* AU - Mall, M.A.* AU - de Jong, R.J. AU - Ohnmacht, C. AU - Bernhagen, J.* AU - Voehringer, D.* AU - Marth, J.D.* AU - Frommhold, D.* AU - Sperandio, M.* C1 - 70591 C2 - 55585 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 19 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Hematopoietic stem cells surrender organelles during differentiation, leaving mature red blood cells (RBC) devoid of transcriptional machinery and mitochondria. The resultant absence of cellular repair capacity limits RBC circulatory longevity, and old cells are removed from circulation. The specific age-dependent alterations required for this apparently targeted removal of RBC, however, remain elusive. Here, we assessed the function of Piezo1, a stretch-activated transmembrane cation channel, within subpopulations of RBC isolated based on physical properties associated with aging. We subsequently investigated the potential role of Piezo1 in RBC removal, using pharmacological and mechanobiological approaches. Dense (old) RBC were separated from whole blood using differential density centrifugation. Tolerance of RBC to mechanical forces within the physiological range was assessed on single-cell and cell population levels. Expression and function of Piezo1 were investigated in separated RBC populations by monitoring accumulation of cytosolic Ca2+ and changes in cell morphology in response to pharmacological Piezo1 stimulation and in response to physical forces. Despite decreased Piezo1 activity with increasing cell age, tolerance to prolonged Piezo1 stimulation declined sharply in older RBC, precipitating lysis. Cell lysis was immediately preceded by an acute reversal of density. We propose a Piezo1-dependent mechanism by which RBC may be removed from circulation: Upon adherence of these RBC to other tissues, they are uniquely exposed to prolonged mechanical forces. The resultant sustained activation of Piezo1 leads to a net influx of Ca2+, overpowering the Ca2+-removal capacity of specifically old RBC, which leads to reversal of ion gradients, dysregulated cell hydration, and ultimately osmotic lysis. AU - Kuck, L.* AU - McNamee, A.P.* AU - Bordukova, M. AU - Sadafi, A. AU - Marr, C. AU - Peart, J.N.* AU - Simmonds, M.J.* C1 - 71552 C2 - 56277 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Lysis of human erythrocytes due to Piezo1-dependent cytosolic calcium overload as a mechanism of circulatory removal. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 36 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - The apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide (APOBEC) family is composed of nucleic acid editors with roles ranging from antibody diversification to RNA editing. APOBEC2, a member of this family with an evolutionarily conserved nucleic acid-binding cytidine deaminase domain, has neither an established substrate nor function. Using a cellular model of muscle differentiation where APOBEC2 is inducibly expressed, we confirmed that APOBEC2 does not have the attributed molecular functions of the APOBEC family, such as RNA editing, DNA demethylation, and DNA mutation. Instead, we found that during muscle differentiation APOBEC2 occupied a specific motif within promoter regions; its removal from those regions resulted in transcriptional changes. Mechanistically, these changes reflect the direct interaction of APOBEC2 with histone deacetylase (HDAC) transcriptional corepressor complexes. We also found that APOBEC2 could bind DNA directly, in a sequence-specific fashion, suggesting that it functions as a recruiter of HDAC to specific genes whose promoters it occupies. These genes are normally suppressed during muscle cell differentiation, and their suppression may contribute to the safeguarding of muscle cell fate. Altogether, our results reveal a unique role for APOBEC2 within the APOBEC family. AU - Lorenzo, J.P.* AU - Molla, L.* AU - Amro, E.M.* AU - Ibarra Del Rio, I.A. AU - Ruf, S.* AU - Neber, C.* AU - Gkougkousis, C.* AU - Ridani, J.* AU - Subramani, P.G.* AU - Boulais, J.* AU - Harjanto, D.* AU - Vonica, A.* AU - di Noia, J.M.* AU - Dieterich, C.* AU - Zaugg, J.B.* AU - Papavasiliou, F.N.* C1 - 70545 C2 - 55664 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - APOBEC2 safeguards skeletal muscle cell fate through binding chromatin and regulating transcription of non-muscle genes during myoblast differentiation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 17 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Sleep supports the consolidation of episodic memory. It is, however, a matter of ongoing debate how this effect is established, because, so far, it has been demonstrated almost exclusively for simple associations, which lack the complex associative structure of real-life events, typically comprising multiple elements with different association strengths. Because of this associative structure interlinking the individual elements, a partial cue (e.g., a single element) can recover an entire multielement event. This process, referred to as pattern completion, is a fundamental property of episodic memory. Yet, it is currently unknown how sleep affects the associative structure within multielement events and subsequent processes of pattern completion. Here, we investigated the effects of post-encoding sleep, compared with a period of nocturnal wakefulness (followed by a recovery night), on multielement associative structures in healthy humans using a verbal associative learning task including strongly, weakly, and not directly encoded associations. We demonstrate that sleep selectively benefits memory for weakly associated elements as well as for associations that were not directly encoded but not for strongly associated elements within a multielement event structure. Crucially, these effects were accompanied by a beneficial effect of sleep on the ability to recall multiple elements of an event based on a single common cue. In addition, retrieval performance was predicted by sleep spindle activity during post-encoding sleep. Together, these results indicate that sleep plays a fundamental role in shaping associative structures, thereby supporting pattern completion in complex multielement events. AU - Lutz, N.D.* AU - Martínez-Albert, E.* AU - Friedrich, H.* AU - Born, J. AU - Besedovsky, L.* C1 - 70054 C2 - 55385 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 9 TI - Sleep shapes the associative structure underlying pattern completion in multielement event memory. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 9 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two β-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation. AU - Suladze, S. AU - Sarkar, R. AU - Rodina, N.* AU - Bokvist, K.* AU - Krewinkel, M.* AU - Scheps, D.* AU - Nagel, N.* AU - Bardiaux, B.* AU - Reif, B. C1 - 70771 C2 - 55741 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Atomic resolution structure of full-length human insulin fibrils. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 23 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of humans and animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked negative effect on the male reproductive system, and particularly the production of spermatozoa, less is known about the immediate impact on male reproductive function following subchronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, we exposed unrestrained male mice to heat stress conditions that emulate a heat wave (daily cycle of 8 h at 35 °C followed by 16 h at 25 °C) for a period of 7 d. Neither the testes or epididymides of heat-exposed male mice exhibited evidence of gross histological change, and similarly, spermatozoa of exposed males retained their functionality and ability to support embryonic development. However, the embryos generated from heat-exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching, and increased fetal:placental weight ratio. Such changes were causally associated with an altered sperm small noncoding RNA (sncRNA) profile, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat-exposed spermatozoa. Such data highlight that even relatively modest excursions in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress. AU - Trigg, N.* AU - Schjenken, J.E.* AU - Martin, J.H.* AU - Skerrett-Byrne, D.A. AU - Smyth, S.P.* AU - Bernstein, I.R.* AU - Anderson, A.L.* AU - Stanger, S.J.* AU - Simpson, E.N.A.* AU - Tomar, A. AU - Teperino, R. AU - Conine, C.C.* AU - De Iuliis, G.N.* AU - Roman, S.D.* AU - Bromfield, E.G.* AU - Dun, M.D.* AU - Eamens, A.L.* AU - Nixon, B.* C1 - 72303 C2 - 56557 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Subchronic elevation in ambient temperature drives alterations to the sperm epigenome and accelerates early embryonic development in mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 121 IS - 47 PB - Natl Acad Sciences PY - 2024 SN - 0027-8424 ER - TY - JOUR AB - Iridoviridae, such as the lymphocystis disease virus-1 (LCDV-1) and other viruses, encode viral insulin–like peptides (VILPs) which are capable of triggering insulin receptors (IRs) and insulin-like growth factor receptors. The homology of VILPs includes highly conserved disulfide bridges. However, the binding affinities to IRs were reported to be 200- to 500-fold less effective compared to the endogenous ligands. We therefore speculated that these peptides also have noninsulin functions. Here, we report that the LCDV-1 VILP can function as a potent and highly specific inhibitor of ferroptosis. Induction of cell death by the ferroptosis inducers erastin, RSL3, FIN56, and FINO2 and nonferroptotic necrosis produced by the thioredoxin-reductase inhibitor ferroptocide were potently prevented by LCDV-1, while human insulin had no effect. Fas-induced apoptosis, necroptosis, mitotane-induced cell death and growth hormone–releasing hormone antagonist–induced necrosis were unaffected, suggesting the specificity to ferroptosis inhibition by the LCDV-1 VILP. Mechanistically, we identified the viral C-peptide to be required for inhibition of lipid peroxidation and ferroptosis inhibition, while the human C-peptide exhibited no antiferroptotic properties. In addition, the deletion of the viral C-peptide abolishes radical trapping activity in cell-free systems. We conclude that iridoviridae, through the expression of insulin-like viral peptides, are capable of preventing ferroptosis. In analogy to the viral mitochondrial inhibitor of apoptosis and the viral inhibitor of RIP activation (vIRA) that prevents necroptosis, we rename the LCDV-1 VILP a viral peptide inhibitor of ferroptosis-1. Finally, our findings indicate that ferroptosis may function as a viral defense mechanism in lower organisms. AU - Belavgeni, A.* AU - Maremonti, F.* AU - Tonnus, W.* AU - Stadtmüller, M.* AU - Gavali, S.* AU - Mallais, M.* AU - Flade, K.* AU - Brucker, A.J.* AU - Becker, J.N.* AU - Beer, K.* AU - Tmava, M.* AU - Stumpf, J.* AU - Gembardt, F.* AU - Hugo, C.* AU - Giacca, M.* AU - Hale, B.G.* AU - Perakakis, N.* AU - Sha, W.* AU - Pratt, D.A.* AU - Schally, A.V.* AU - Bornstein, S.R. AU - Linkermann, A.* C1 - 68559 C2 - 53673 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - vPIF-1 is an insulin-like antiferroptotic viral peptide. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 21 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Antibody delivery to the CNS remains a huge hurdle for the clinical application of antibodies targeting a CNS antigen. The blood-brain barrier and blood-CSF barrier restrict access of therapeutic antibodies to their CNS targets in a major way. The very high amounts of therapeutic antibodies that are administered systemically in recent clinical trials to reach CNS targets are barely viable cost-wise for broad, routine applications. Though global CNS delivery of antibodies can be achieved by intrathecal application, these procedures are invasive. A non-invasive method to bring antibodies into the CNS reliably and reproducibly remains an important unmet need in neurology. In the present study, we show that intranasal application of a mouse monoclonal antibody against the neurite growth-inhibiting and plasticity-restricting membrane protein Nogo-A leads to a rapid transfer of significant amounts of antibody to the brain and spinal cord in intact adult rats. Daily intranasal application for 2 wk of anti-Nogo-A antibody enhanced growth and compensatory sprouting of corticofugal projections and functional recovery in rats after large unilateral cortical strokes. These findings are a starting point for clinical translation for a less invasive route of application of therapeutic antibodies to CNS targets for many neurological indications. AU - Correa, D.* AU - Scheuber, M.I.* AU - Shan, H.* AU - Weinmann, O.W.* AU - Baumgartner, Y.A.* AU - Harten, A. AU - Wahl, A.S.* AU - Skaar, K.L.* AU - Schwab, M.E.* C1 - 67276 C2 - 54210 TI - Intranasal delivery of full-length anti-Nogo-A antibody: A potential alternative route for therapeutic antibodies to central nervous system targets. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 4 PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release. AU - Pena-Francesch, M.* AU - Vanoaica, L.D.* AU - Zhu, G.F.* AU - Stumpe, M.* AU - Sankar, D.S.* AU - Nowag, H.* AU - Valencia-Camargo, A.D.* AU - Hammerschmidt, W. AU - Dengjel, J.* AU - Ligeon, L.A.* AU - Münz, C.* C1 - 68000 C2 - 54478 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - The autophagy machinery interacts with EBV capsids during viral envelope release. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 34 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Exposure to stressful life events increases the risk for psychiatric disorders. Mechanistic insight into the genetic factors moderating the impact of stress can increase our understanding of disease processes. Here, we test 3,662 single nucleotide polymorphisms (SNPs) from preselected expression quantitative trait loci in massively parallel reporter assays to identify genetic variants that modulate the activity of regulatory elements sensitive to glucocorticoids, important mediators of the stress response. Of the tested SNP sequences, 547 were located in glucocorticoid-responsive regulatory elements of which 233 showed allele-dependent activity. Transcripts regulated by these functional variants were enriched for those differentially expressed in psychiatric disorders in the postmortem brain. Phenome-wide Mendelian randomization analysis in 4,439 phenotypes revealed potentially causal associations specifically in neurobehavioral traits, including major depression and other psychiatric disorders. Finally, a functional gene score derived from these variants was significantly associated with differences in the physiological stress response, suggesting that these variants may alter disease risk by moderating the individual set point of the stress response. AU - Penner-Goeke, S.* AU - Bothe, M.* AU - Rek, N.* AU - Kreitmaier, P. AU - Pöhlchen, D.* AU - Kühnel, A.* AU - Glaser, L.* AU - Kaya, E.* AU - Krontira, A.C.* AU - Röh, S.* AU - Czamara, D.* AU - Ködel, M.* AU - Monteserin-Garcia, J.L.* AU - Diener, L.* AU - Wölfel, B.* AU - Sauer, S.* AU - Rummel, C.* AU - Riesenberg, S.* AU - Arloth-Knauer, J.* AU - Ziller, M.J.* AU - Labeur, M.* AU - Meijsing, S.* AU - Binder, E.B.* C1 - 68903 C2 - 53758 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - High-throughput screening of glucocorticoid-induced enhancer activity reveals mechanisms of stress-related psychiatric disorders. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 49 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AU - Peters, A. C1 - 67163 C2 - 53562 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Ambient air pollution and Alzheimer's disease: The role of the composition of fine particles. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 3 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Constitutive activation of the MALT1 paracaspase in conventional T cells of Malt1TBM/TBM (TRAF6 Binding Mutant = TBM) mice causes fatal inflammation and autoimmunity, but the involved targets and underlying molecular mechanisms are unknown. We genetically rendered a single MALT1 substrate, the RNA-binding protein (RBP) Roquin-1, insensitive to MALT1 cleavage. These Rc3h1Mins/Mins mice showed normal immune homeostasis. Combining Rc3h1Mins/Mins alleles with those encoding for constitutively active MALT1 (TBM) prevented spontaneous T cell activation and restored viability of Malt1TBM/TBM mice. Mechanistically, we show how antigen/MHC recognition is translated by MALT1 into Roquin cleavage and derepression of Roquin targets. Increasing T cell receptor (TCR) signals inactivated Roquin more effectively, and only high TCR strength enabled derepression of high-affinity targets to promote Th17 differentiation. Induction of experimental autoimmune encephalomyelitis (EAE) revealed increased cleavage of Roquin-1 in disease-associated Th17 compared to Th1 cells in the CNS. T cells from Rc3h1Mins/Mins mice did not efficiently induce the high-affinity Roquin-1 target IκBNS in response to TCR stimulation, showed reduced Th17 differentiation, and Rc3h1Mins/Mins mice were protected from EAE. These data demonstrate how TCR signaling and MALT1 activation utilize graded cleavage of Roquin to differentially regulate target mRNAs that control T cell activation and differentiation as well as the development of autoimmunity. AU - Schmidt, H.* AU - Raj, T.* AU - O´Neill, T.J. AU - Muschaweckh, A.* AU - Giesert, F. AU - Negraschus, A.* AU - Hoefig, K.P. AU - Behrens, G.* AU - Esser, L.* AU - Baumann, Christina AU - Feederle, R. AU - Plaza-Sirvent, C.* AU - Geerlof, A. AU - Gewies, A. AU - Isay, S.E.* AU - Ruland, J.* AU - Schmitz, I.* AU - Wurst, W. AU - Korn, T.* AU - Krappmann, D. AU - Heissmeyer, V. C1 - 68863 C2 - 53684 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 11 TI - Unrestrained cleavage of Roquin-1 by MALT1 induces spontaneous T cell activation and the development of autoimmunity. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 48 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Osteoarthritis (OA) is a joint disease featuring cartilage breakdown and chronic pain. Although age and joint trauma are prominently associated with OA occurrence, the trigger and signaling pathways propagating their pathogenic aspects are ill defined. Following long-term catabolic activity and traumatic cartilage breakdown, debris accumulates and can trigger Toll-like receptors (TLRs). Here we show that TLR2 stimulation suppressed the expression of matrix proteins and induced an inflammatory phenotype in human chondrocytes. Further, TLR2 stimulation impaired chondrocyte mitochondrial function, resulting in severely reduced adenosine triphosphate (ATP) production. RNA-sequencing analysis revealed that TLR2 stimulation upregulated nitric oxide synthase 2 (NOS2) expression and downregulated mitochondria function-associated genes. NOS inhibition partially restored the expression of these genes, and rescued mitochondrial function and ATP production. Correspondingly, Nos2-/- mice were protected from age-related OA development. Taken together, the TLR2-NOS axis promotes human chondrocyte dysfunction and murine OA development, and targeted interventions may provide therapeutic and preventive approaches in OA. AU - Shen, P.* AU - Serve, S.* AU - Wu, P.* AU - Liu, X.* AU - Dai, Y.* AU - Durán-Hernández, N.* AU - Nguyen, D.T.M.* AU - Fuchs, M.* AU - Maleitzke, T.* AU - Reisener, M.J.* AU - Dzamukova, M.* AU - Nussbaumer, K.* AU - Brunner, T.M.* AU - Li, Y.* AU - Holecska, V.* AU - Heinz, G.* AU - Heinrich, F.* AU - Durek, P.* AU - Katsoula, G. AU - Gwinner, C.* AU - Jung, T.* AU - Zeggini, E. AU - Winkler, T.* AU - Mashreghi, M.F.* AU - Pumberger, M.* AU - Perka, C.* AU - Löhning, M.* C1 - 68128 C2 - 54606 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - NOS inhibition reverses TLR2-induced chondrocyte dysfunction and attenuates age-related osteoarthritis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 29 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - The vascular endothelium from individual organs is functionally specialized, and it displays a unique set of accessible molecular targets. These serve as endothelial cell receptors to affinity ligands. To date, all identified vascular receptors have been proteins. Here, we show that an endothelial lung-homing peptide (CGSPGWVRC) interacts with C16-ceramide, a bioactive sphingolipid that mediates several biological functions. Upon binding to cell surfaces, CGSPGWVRC triggers ceramide-rich platform formation, activates acid sphingomyelinase and ceramide production, without the associated downstream apoptotic signaling. We also show that the lung selectivity of CGSPGWVRC homing peptide is dependent on ceramide production in vivo. Finally, we demonstrate two potential applications for this lipid vascular targeting system: i) as a bioinorganic hydrogel for pulmonary imaging and ii) as a ligand-directed lung immunization tool against COVID-19. Thus, C16-ceramide is a unique example of a lipid-based receptor system in the lung vascular endothelium targeted in vivo by circulating ligands such as CGSPGWVRC. AU - Staquicini, D.I.* AU - Cardó-Vila, M.* AU - Rotolo, J.A.* AU - Staquicini, F.I.* AU - Tang, F.H.F.* AU - Smith, T.L.* AU - Ganju, A.* AU - Schiavone, C.* AU - Dogra, P.* AU - Wang, Z.* AU - Cristini, V.* AU - Giordano, R.J.* AU - Ozawa, M.G.* AU - Driessen, W.* AU - Proneth, B. AU - Souza, G.R.* AU - Brinker, L.M.* AU - Noureddine, A.* AU - Snider, A.J.* AU - Canals, D.* AU - Gelovani, J.G.* AU - Petrache, I.* AU - Tuder, R.M.* AU - Obeid, L.M.* AU - Hannun, Y.A.* AU - Kolesnick, R.N.* AU - Brinker, C.J.* AU - Pasqualini, R.* AU - Arap, W.* C1 - 68001 C2 - 54479 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Ceramide as an endothelial cell surface receptor and a lung-specific lipid vascular target for circulating ligands. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 34 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - Duchenne muscular dystrophy (DMD) is a fatal X-linked disease caused by mutations in the DMD gene, leading to complete absence of dystrophin and progressive degeneration of skeletal musculature and myocardium. In DMD patients and in a corresponding pig model with a deletion of DMD exon 52 (DMDΔ52), expression of an internally shortened dystrophin can be achieved by skipping of DMD exon 51 to reframe the transcript. To predict the best possible outcome of this strategy, we generated DMDΔ51-52 pigs, additionally representing a model for Becker muscular dystrophy (BMD). DMDΔ51-52 skeletal muscle and myocardium samples stained positive for dystrophin and did not show the characteristic dystrophic alterations observed in DMDΔ52 pigs. Western blot analysis confirmed the presence of dystrophin in the skeletal muscle and myocardium of DMDΔ51-52 pigs and its absence in DMDΔ52 pigs. The proteome profile of skeletal muscle, which showed a large number of abundance alterations in DMDΔ52 vs. wild-type (WT) samples, was normalized in DMDΔ51-52 samples. Cardiac function at age 3.5 mo was significantly reduced in DMDΔ52 pigs (mean left ventricular ejection fraction 58.8% vs. 70.3% in WT) but completely rescued in DMDΔ51-52 pigs (72.3%), in line with normalization of the myocardial proteome profile. Our findings indicate that ubiquitous deletion of DMD exon 51 in DMDΔ52 pigs largely rescues the rapidly progressing, severe muscular dystrophy and the reduced cardiac function of this model. Long-term follow-up studies of DMDΔ51-52 pigs will show if they develop symptoms of the milder BMD. AU - Stirm, M.* AU - Shashikadze, B.* AU - Blutke, A.* AU - Kemter, E.* AU - Lange, A.* AU - Stöckl, J.B.* AU - Jaudas, F.* AU - Laane, L.* AU - Kurome, M.* AU - Keßler, B.* AU - Zakhartchenko, V.* AU - Bähr, A.* AU - Klymiuk, N.* AU - Nagashima, H.* AU - Walter, M.* AU - Wurst, W. AU - Kupatt, C.* AU - Fröhlich, T.* AU - Wolf, E.* C1 - 68129 C2 - 54607 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Systemic deletion of DMD exon 51 rescues clinically severe Duchenne muscular dystrophy in a pig model lacking DMD exon 52. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 120 IS - 29 PB - Natl Acad Sciences PY - 2023 SN - 0027-8424 ER - TY - JOUR AB - The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans. AU - Aznaourova, M.* AU - Schmerer, N.* AU - Janga, H.* AU - Zhang, Z.* AU - Pauck, K.* AU - Bushe, J.* AU - Volkers, S.M.* AU - Wendisch, D.* AU - Georg, P.* AU - Ntini, E.* AU - Aillaud, M.* AU - Gündisch, M.* AU - Mack, E.* AU - Skevaki, C.* AU - Keller, C.* AU - Bauer, C.* AU - Bertrams, W.* AU - Marsico, A. AU - Nist, A.* AU - Stiewe, T.* AU - Gruber, A.D.* AU - Ruppert, C.* AU - Li, Y.* AU - Garn, H.* AU - Sander, L.E.* AU - Schulte, L.N.* C1 - 65993 C2 - 53030 TI - Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 36 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a human tumor virus which preferentially infects resting human B cells. Upon infection in vitro, EBV activates and immortalizes these cells. The viral latent protein EBV nuclear antigen 2 (EBNA2) is essential for B cell activation and immortalization; it targets and binds the cellular and ubiquitously expressed DNA-binding protein CBF1, thereby transactivating a plethora of viral and cellular genes. In addition, EBNA2 uses its N-terminal dimerization (END) domain to bind early B cell factor 1 (EBF1), a pioneer transcription factor specifying the B cell lineage. We found that EBNA2 exploits EBF1 to support key metabolic processes and to foster cell cycle progression of infected B cells in their first cell cycles upon activation. The α1-helix within the END domain was found to promote EBF1 binding. EBV mutants lacking the α1-helix in EBNA2 can infect and activate B cells efficiently, but activated cells fail to complete the early S phase of their initial cell cycle. Expression of MYC, target genes of MYC and E2F, as well as multiple metabolic processes linked to cell cycle progression are impaired in EBVΔα1-infected B cells. Our findings indicate that EBF1 controls B cell activation via EBNA2 and, thus, has a critical role in regulating the cell cycle of EBV-infected B cells. This is a function of EBF1 going beyond its well-known contribution to B cell lineage specification. AU - Beer, S. AU - Wange, L.E.* AU - Zhang, X. AU - Kuklik-Roos, C. AU - Enard, W.* AU - Hammerschmidt, W. AU - Scialdone, A. AU - Kempkes, B. C1 - 65767 C2 - 52907 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - EBNA2-EBF1 complexes promote MYC expression and metabolic processes driving S-phase progression of Epstein-Barr virus-infected B cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 30 PB - Natl Acad Sciences PY - 2022 SN - 0027-8424 ER - TY - JOUR AU - Belavgeni, A.* AU - Maremonti, F.* AU - Stadtmüller, M.* AU - Bornstein, S.R. AU - Linkermann, A.* C1 - 64889 C2 - 51978 TI - Schwann cell necroptosis in diabetic neuropathy. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 17 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - The use of spoken and written language is a fundamental human capacity. Individual differences in reading- and language-related skills are influenced by genetic variation, with twin-based heritability estimates of 30 to 80% depending on the trait. The genetic architecture is complex, heterogeneous, and multifactorial, but investigations of contributions of single-nucleotide polymorphisms (SNPs) were thus far underpowered. We present a multicohort genome-wide association study (GWAS) of five traits assessed individually using psychometric measures (word reading, nonword reading, spelling, phoneme awareness, and nonword repetition) in samples of 13,633 to 33,959 participants aged 5 to 26 y. We identified genome-wide significant association with word reading (rs11208009, P = 1.098 × 10-8) at a locus that has not been associated with intelligence or educational attainment. All five reading-/language-related traits showed robust SNP heritability, accounting for 13 to 26% of trait variability. Genomic structural equation modeling revealed a shared genetic factor explaining most of the variation in word/nonword reading, spelling, and phoneme awareness, which only partially overlapped with genetic variation contributing to nonword repetition, intelligence, and educational attainment. A multivariate GWAS of word/nonword reading, spelling, and phoneme awareness maximized power for follow-up investigation. Genetic correlation analysis with neuroimaging traits identified an association with the surface area of the banks of the left superior temporal sulcus, a brain region linked to the processing of spoken and written language. Heritability was enriched for genomic elements regulating gene expression in the fetal brain and in chromosomal regions that are depleted of Neanderthal variants. Together, these results provide avenues for deciphering the biological underpinnings of uniquely human traits. AU - Eising, E.* AU - Mirza-Schreiber, N. AU - de Zeeuw, E.L.* AU - Wang, C.A.* AU - Truong, D.T.* AU - Allegrini, A.G.* AU - Shapland, C.Y.* AU - Zhu, G.* AU - Wigg, K.G.* AU - Gerritse, M.L.* AU - Molz, B.* AU - Alagöz, G.* AU - Gialluisi, A.* AU - Abbondanza, F.* AU - Rimfeld, K.* AU - van Donkelaar, M.M.J.* AU - Liao, Z.* AU - Jansen, P.R.* AU - Andlauer, T.F.M.* AU - Bates, T.C.* AU - Bernard, M.* AU - Blokland, K.* AU - Bonte, M.* AU - Børglum, A.D.* AU - Bourgeron, T.* AU - Brandeis, D.* AU - Ceroni, F.* AU - Csépe, V.* AU - Dale, P.S.* AU - de Jong, P.F.* AU - DeFries, J.C.* AU - Demonet, J.F.* AU - Demontis, D.* AU - Feng, Y.* AU - Gordon, S.D.* AU - Guger, S.L.* AU - Hayiou-Thomas, M.E.* AU - Hernández-Cabrera, J.A.* AU - Hottenga, J.J.* AU - Hulme, C.* AU - Kere, J.* AU - Kerr, E.N.* AU - Koomar, T.* AU - Landerl, K.* AU - Leonard, G.T.* AU - Lovett, M.W.* AU - Lyytinen, H.* AU - Martin, N.G.* AU - Martinelli, A.* AU - Maurer, U.* AU - Michaelson, J.J.* AU - Moll, K.* AU - Monaco, A.P.* AU - Morgan, A.T.* AU - Nöthen, M.M.* AU - Pausova, Z.* AU - Pennell, C.E.* AU - Pennington, B.F.* AU - Price, K.M.* AU - Rajagopal, V.M.* AU - Ramus, F.* AU - Richer, L.* AU - Simpson, N.H.* AU - Smith, S.D.* AU - Snowling, M.J.* AU - Stein, J.* AU - Strug, L.J.* AU - Talcott, J.B.* AU - Tiemeier, H.* AU - van der Schroeff, M.P.* AU - Verhoef, E.* AU - Watkins, K.E.* AU - Wilkinson, M.* AU - Wright, M.J.* AU - Barr, C.L.* AU - Boomsma, D.I.* AU - Carreiras, M.* AU - Franken, M.J.* AU - Gruen, J.R.* AU - Luciano, M.* AU - Müller-Myhsok, B.* AU - Newbury, D.F.* AU - Olson, R.K.* AU - Paracchini, S.* AU - Paus, T.* AU - Plomin, R.* AU - Reilly, S.* AU - Schulte-Körne, G.* AU - Tomblin, J.B.* AU - van Bergen, E.* AU - Whitehouse, A.J.O.* AU - Willcutt, E.G.* AU - St Pourcain, B.* AU - Francks, C.* AU - Fisher, S.E.* C1 - 67019 C2 - 53373 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 35 PB - Natl Acad Sciences PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IR-deficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1α and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling. AU - Fernandez, A.M.* AU - Martinez-Rachadell, L.* AU - Navarrete, M.* AU - Pose-Utrilla, J.* AU - Davila, J.C.* AU - Pignatelli, J.* AU - Diaz-Pacheco, S.* AU - Guerra-Cantera, S.* AU - Viedma-Moreno, E.* AU - Palenzuela, R.* AU - Ruiz de Martin Esteban, S.* AU - Mostany, R.* AU - García-Cáceres, C. AU - Tschöp, M.H. AU - Iglesias, T.* AU - de Ceballos, M.L.* AU - Gutierrez, A.* AU - Torres Aleman, I.* C1 - 67284 C2 - 53542 TI - Insulin regulates neurovascular coupling through astrocytes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 29 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Apoptosis is widely believed to be crucial for epithelial cell death and shedding in the intestine, thereby shaping the overall architecture of the gastrointestinal tract, but also regulating tolerance induction, pinpointing a role of apoptosis intestinal epithelial cell (IEC) turnover and maintenance of barrier function, and in maintaining immune homeostasis. To experimentally address this concept, we generated IEC-specific knockout mice that lack both executioner caspase-3 and caspase-7 (Casp3/7 ΔIEC), which are the converging point of the extrinsic and intrinsic apoptotic pathway. Surprisingly, the overall architecture, cellular landscape, and proliferation rate remained unchanged in these mice. However, nonapoptotic cell extrusion was increased in Casp3/7 ΔIEC mice, compensating apoptosis deficiency, maintaining the same physiological level of IEC shedding. Microbiome richness and composition stayed unaffected, bearing no sign of dysbiosis. Transcriptome and single-cell RNA sequencing analyses of IECs and immune cells revealed no differences in signaling pathways of differentiation and inflammation. These findings demonstrate that during homeostasis, apoptosis per se is dispensable for IEC turnover at the top of intestinal villi intestinal tissue dynamics, microbiome, and immune cell composition. AU - Ghazavi, F.* AU - Huysentruyt, J.* AU - De Coninck, J.* AU - Kourula, S.* AU - Martens, S.* AU - Hassannia, B.* AU - Wartewig, T.* AU - Divert, T.* AU - Roelandt, R.* AU - Popper, B.* AU - Hiergeist, A.* AU - Tougaard, P.* AU - Vanden Berghe, T.* AU - Joossens, M.* AU - Berx, G.* AU - Takahashi, N.* AU - Wahida, A. AU - Vandenabeele, P.* C1 - 64256 C2 - 51934 TI - Executioner caspases 3 and 7 are dispensable for intestinal epithelium turnover and homeostasis at steady state. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 6 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - SignificanceWhile increasing evidence associates the disruption of circadian rhythms with pathologic conditions, including obesity, type 2 diabetes, and nonalcoholic fatty liver diseases (NAFLD), the involved mechanisms are still poorly described. Here, we show that, in both humans and mice, the pathogenesis of NAFLD is associated with the disruption of the circadian clock combined with perturbations of the growth hormone and sex hormone pathways. However, while this condition protects mice from the development of fibrosis and insulin resistance, it correlates with increased fibrosis in humans. This suggests that the perturbation of the circadian clock and its associated disruption of the growth hormone and sex hormone pathways are critical for the pathogenesis of metabolic and liver diseases. AU - Jouffe, C. AU - Weger, B.D.* AU - Martin, E.* AU - Atger, F.* AU - Weger, M.* AU - Gobet, C.* AU - Ramnath, D.* AU - Charpagne, A.* AU - Morin-Rivron, D.* AU - Powell, E.E.* AU - Sweet, M.J.* AU - Masoodi, M.* AU - Uhlenhaut, N.H. AU - Gachon, F.* C1 - 64523 C2 - 51951 TI - Disruption of the circadian clock component BMAL1 elicits an endocrine adaption impacting on insulin sensitivity and liver disease. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 10 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Plants are resistant to most microbial species due to nonhost resistance (NHR), providing broad-spectrum and durable immunity. However, the molecular components contributing to NHR are poorly characterised. We address the question of whether failure of pathogen effectors to manipulate nonhost plants plays a critical role in NHR. RxLR (Arg-any amino acid-Leu-Arg) effectors from two oomycete pathogens, Phytophthora infestans and Hyaloperonospora arabidopsidis, enhanced pathogen infection when expressed in host plants (Nicotiana benthamiana and Arabidopsis, respectively) but the same effectors performed poorly in distantly related nonhost pathosystems. Putative target proteins in the host plant potato were identified for 64 P. infestans RxLR effectors using yeast 2-hybrid (Y2H) screens. Candidate orthologues of these target proteins in the distantly related non-host plant Arabidopsis were identified and screened using matrix Y2H for interaction with RxLR effectors from both P. infestans and H. arabidopsidis. Few P. infestans effector-target protein interactions were conserved from potato to candidate Arabidopsis target orthologues (cAtOrths). However, there was an enrichment of H. arabidopsidis RxLR effectors interacting with cAtOrths. We expressed the cAtOrth AtPUB33, which unlike its potato orthologue did not interact with P. infestans effector PiSFI3, in potato and Nicotiana benthamiana. Expression of AtPUB33 significantly reduced P. infestans colonization in both host plants. Our results provide evidence that failure of pathogen effectors to interact with and/or correctly manipulate target proteins in distantly related non-host plants contributes to NHR. Moreover, exploiting this breakdown in effector-nonhost target interaction, transferring effector target orthologues from non-host to host plants is a strategy to reduce disease. AU - McLellan, H.* AU - Harvey, S.E.* AU - Steinbrenner, J.* AU - Armstrong, M.R.* AU - He, Q.* AU - Clewes, R.* AU - Pritchard, L.* AU - Wang, W.* AU - Wang, S.* AU - Nussbaumer, T. AU - Dohai, B.S.M. AU - Luo, Q.* AU - Kumari, P.* AU - Duan, H.* AU - Roberts, A.* AU - Boevink, P.C.* AU - Neumann, C.* AU - Champouret, N.* AU - Hein, I.* AU - Falter-Braun, P. AU - Beynon, J.* AU - Denby, K.* AU - Birch, P.R.J.* C1 - 65946 C2 - 52995 TI - Exploiting breakdown in nonhost effector-target interactions to boost host disease resistance. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 35 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - SignificanceAirborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or other pathogens is probably increased during indoor exercise, but data on the emission of aerosol particles by an exercising individual are lacking. Here, we report that aerosol particle emission increases on average 132-fold from 580 ± 489 particles/min at rest to 76,200 ± 48,000 particles/min during maximal exercise. Aerosol particle emission increases moderately up to an exercise intensity of ≈2 W/kg and exponentially at higher exercise intensities. These data not only explain SARS-CoV-2 transmissions during indoor group exercise but also can be used to design better targeted mitigation measures for physical activity indoors such as physical education in school, dance events during weddings, or high-intensity gym classes such as spinning. AU - Mutsch, B.* AU - Heiber, M.* AU - Grätz, F.* AU - Hain, R.* AU - Schönfelder, M.* AU - Kaps, S.* AU - Schranner, D. AU - Kähler, C.J.* AU - Wackerhage, H.* C1 - 65068 C2 - 52142 TI - Aerosol particle emission increases exponentially above moderate exercise intensity resulting in superemission during maximal exercise. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 22 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - SignificanceIn this study, we identify microRNA-32-5p (miR-32-5p) as a key functional noncoding RNA in trigeminal-mediated neuropathic pain. We report that injury-induced histone methylation attenuates the binding of glucocorticoid receptor to the promoter region of the miR-32-5p gene and decreases the expression of miR-32-5p, in turn promoting the development of neuropathic pain through regulation of Cav3.2 channels. miRNA-mediated gene regulation has been proposed as a therapeutic approach in neuropathic pain. Our findings identify miR-32-5p replenishment as a therapeutic strategy for treating chronic neuropathic pain. AU - Qi, R.* AU - Cao, J.* AU - Sun, Y.* AU - Li, Y.* AU - Huang, Z.* AU - Jiang, D. AU - Jiang, X.H.* AU - Snutch, T.P.* AU - Zhang, Y.* AU - Tao, J.* C1 - 64719 C2 - 51950 TI - Histone methylation-mediated microRNA-32-5p down-regulation in sensory neurons regulates pain behaviors via targeting Cav3.2 channels. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 14 PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Memory consolidation is promoted by sleep. However, there is also evidence for consolidation into long-term memory during wakefulness via processes that preferentially affect nonhippocampal representations. We compared, in rats, the effects of 2-h postencoding periods of sleep and wakefulness on the formation of long-term memory for objects and their associated environmental contexts. We employed a novel-object recognition (NOR) task, using object exploration and exploratory rearing as behavioral indicators of these memories. Remote recall testing (after 1 wk) confirmed significant long-term NOR memory under both conditions, with NOR memory after sleep predicted by the occurrence of EEG spindle-slow oscillation coupling. Rats in the sleep group decreased their exploratory rearing at recall testing, revealing successful recall of the environmental context. By contrast, rats that stayed awake after encoding showed equally high levels of rearing upon remote testing as during encoding, indicating that context memory was lost. Disruption of hippocampal function during the postencoding interval (by muscimol administration) suppressed long-term NOR memory together with context memory formation when animals slept, but enhanced NOR memory when they were awake during this interval. Testing remote recall in a context different from that during encoding impaired NOR memory in the sleep condition, while exploratory rearing was increased. By contrast, NOR memory in the wake rats was preserved and actually superior to that after sleep. Our findings indicate two distinct modes of long-term memory formation: Sleep consolidation is hippocampus dependent and implicates event-context binding, whereas wake consolidation is impaired by hippocampal activation and strengthens context-independent representations. AU - Sawangjit, A.* AU - Harkotte, M.* AU - Oyanedel, C.N.* AU - Niethard, N.* AU - Born, J. AU - Inostroza, M.* C1 - 65943 C2 - 52993 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Two distinct ways to form long-term object recognition memory during sleep and wakefulness. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 34 PB - Natl Acad Sciences PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Lipodystrophy syndromes (LDs) are characterized by loss of adipose tissue, metabolic complications such as dyslipidemia, insulin resistance, and fatty liver disease, as well as accelerated atherosclerosis. As a result of adipose tissue deficiency, the systemic concentration of the adipokine leptin is reduced. A current promising therapeutic option for patients with LD is treatment with recombinant leptin (metreleptin), resulting in reduced risk of mortality. Here, we investigate the effects of leptin on endothelial to mesenchymal transition (EndMT), which impair the functional properties of endothelial cells and promotes atherogenesis in LD. Leptin treatment reduced inflammation and TGF-β2-induced expression of mesenchymal genes and prevented impairment of endothelial barrier function. Treatment of lipodystrophic- and atherosclerosis-prone animals (Ldlr-/-; aP2-nSrebp1c-Tg) with leptin reduced macrophage accumulation in atherosclerotic lesions, vascular plaque protrusion, and the number of endothelial cells with mesenchymal gene expression, confirming a reduction in EndMT in LD after leptin treatment. Treatment with leptin inhibited LD-mediated induction of the proatherosclerotic cytokine growth/differentiation factor 15 (GDF15). Inhibition of GDF15 reduced EndMT induction triggered by plasma from patients with LD. Our study reveals that in addition to the effects on adipose tissue function, leptin treatment exerts beneficial effects protecting endothelial function and identity in LD by reducing GDF15. AU - Stürzebecher, P.E.* AU - Kralisch, S.* AU - Schubert, M.R.* AU - Filipova, V.* AU - Hoffmann, A.* AU - Oliveira, F. AU - Sheikh, B. AU - Blüher, M. AU - Kogel, A.* AU - Scholz, M.* AU - Kokot, K.E.* AU - Erbe, S.* AU - Kneuer, J.M.* AU - Ebert, T.* AU - Fasshauer, M.* AU - Miehle, K.* AU - Laufs, U.* AU - Tönjes, A.* AU - Boeckel, J.N.* C1 - 66294 C2 - 52771 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Leptin treatment has vasculo-protective effects in lipodystrophic mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 119 IS - 40 PB - Natl Acad Sciences PY - 2022 SN - 0027-8424 ER - TY - JOUR AB - Pollen exposure weakens the immunity against certain seasonal respiratory viruses by diminishing the antiviral interferon response. Here we investigate whether the same applies to the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is sensitive to antiviral interferons, if infection waves coincide with high airborne pollen concentrations. Our original hypothesis was that more airborne pollen would lead to increases in infection rates. To examine this, we performed a cross-sectional and longitudinal data analysis on SARS-CoV-2 infection, airborne pollen, and meteorological factors. Our dataset is the most comprehensive, largest possible worldwide from 130 stations, across 31 countries and five continents. To explicitly investigate the effects of social contact, we additionally considered population density of each study area, as well as lockdown effects, in all possible combinations: without any lockdown, with mixed lockdown−no lockdown regime, and under complete lockdown. We found that airborne pollen, sometimes in synergy with humidity and temperature, explained, on average, 44% of the infection rate variability. Infection rates increased after higher pollen concentrations most frequently during the four previous days. Without lockdown, an increase of pollen abundance by 100 pollen/m3 resulted in a 4% average increase of infection rates. Lockdown halved infection rates under similar pollen concentrations. As there can be no preventive measures against airborne pollen exposure, we suggest wide dissemination of pollen−virus coexposure dire effect information to encourage high-risk individuals to wear particle filter masks during high springtime pollen concentrations. AU - Damialis, A. AU - Gilles, S. AU - Sofiev, M.* AU - Sofieva, V.* AU - Kolek, F. AU - Bayr, D. AU - Plaza, M.P. AU - Leier-Wirtz, V. AU - Kaschuba, S. AU - Ziska, L.H.* AU - Bielory, L.* AU - Makra, L.* AU - del Mar Trigo, M.* AU - Traidl-Hoffmann, C. C1 - 61640 C2 - 50365 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Higher airborne pollen concentrations correlated with increased SARS-CoV-2 infection rates, as evidenced from 31 countries across the globe. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 12 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AU - Gilles, S. AU - Traidl-Hoffmann, C. AU - Damialis, A.* C1 - 62824 C2 - 51006 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Reply to Betsch and Sprengholz: Higher SARS-CoV-2 infection numbers related to more airborne pollen, regardless of testing frequency. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 34 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - Novel MRI techniques allow a noninvasive quantification of tissue sodium and reveal the skin as a prominent compartment of sodium storage in health and disease. Since multiple sclerosis (MS) immunopathology is initiated in the periphery and increased sodium concentrations induce proinflammatory immune cells, the skin represents a promising compartment linking high sodium concentrations and MS immunopathology. We used a 7-T sodium MRI (23Na-MRI) and inductively coupled plasma mass spectrometry to investigate the skin sodium content in two mouse models of MS. We additionally performed 3-T 23Na-MRI of calf skin and muscles in 29 male relapsing-remitting MS (RRMS) patients and 29 matched healthy controls. Demographic and clinical information was collected from interviews, and disease activity was assessed by expanded disability status scale scoring. 23Na-MRI and chemical analysis demonstrated a significantly increased sodium content in the skin during experimental autoimmune encephalomyelitis independent of active immunization. In male patients with RRMS, 23Na-MRI demonstrated a higher sodium signal in the area of the skin compared to age- and biological sex-matched healthy controls with higher sodium, predicting future disease activity in cranial MRI. In both studies, the sodium enrichment was specific to the skin, as we found no alterations of sodium signals in the muscle or other tissues. Our data add to the recently identified importance of the skin as a storage compartment of sodium and may further represent an important organ for future investigations on salt as a proinflammatory agent driving autoimmune neuroinflammation such as that in MS. AU - Huhn, K.* AU - Linz, P.* AU - Pemsel, F.* AU - Michalke, B. AU - Seyferth, S.* AU - Kopp, C.* AU - Chaudri, M.A.* AU - Rothhammer, V.* AU - Dörfler, A.* AU - Uder, M.* AU - Nagel, A.M.* AU - Müller, D.N.* AU - Waschbisch, A.* AU - Lee, D.H.* AU - Bäuerle, T.* AU - Linker, R.A.* AU - Haase, S.* C1 - 62537 C2 - 50814 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Skin sodium is increased in male patients with multiple sclerosis and related animal models. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 28 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista et al., Cell Stem Cell 15, 707-719 (2014); Y. Wang et al., Nat. Cell Biol. 16, 191-198 (2014); and S. Geula et al., Science 347, 1002-1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure. AU - Jin, K.X.* AU - Zuo, R.* AU - Anastassiadis, K.* AU - Klungland, A.* AU - Marr, C. AU - Filipczyk, A.A.* C1 - 63903 C2 - 51734 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 51 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - The mitochondrial thioredoxin/peroxiredoxin system encompasses NADPH, thioredoxin reductase 2 (TrxR2), thioredoxin 2, and peroxiredoxins 3 and 5 (Prx3 and Prx5) and is crucial to regulate cell redox homeostasis via the efficient catabolism of peroxides (TrxR2 and Trxrd2 refer to the mitochondrial thioredoxin reductase protein and gene, respectively). Here, we report that endothelial TrxR2 controls both the steady-state concentration of peroxynitrite, the product of the reaction of superoxide radical and nitric oxide, and the integrity of the vascular system. Mice with endothelial deletion of the Trxrd2 gene develop increased vascular stiffness and hypertrophy of the vascular wall. Furthermore, they suffer from renal abnormalities, including thickening of the Bowman's capsule, glomerulosclerosis, and functional alterations. Mechanistically, we show that loss of Trxrd2 results in enhanced peroxynitrite steady-state levels in both vascular endothelial cells and vessels by using a highly sensitive redox probe, fluorescein-boronate. High steady-state peroxynitrite levels were further found to coincide with elevated protein tyrosine nitration in renal tissue and a substantial change of the redox state of Prx3 toward the oxidized protein, even though glutaredoxin 2 (Grx2) expression increased in parallel. Additional studies using a mitochondria-specific fluorescence probe (MitoPY1) in vessels revealed that enhanced peroxynitrite levels are indeed generated in mitochondria. Treatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin [Mn(III)TMPyP], a peroxynitrite-decomposition catalyst, blunted intravascular formation of peroxynitrite. Our data provide compelling evidence for a yet-unrecognized role of TrxR2 in balancing the nitric oxide/peroxynitrite ratio in endothelial cells in vivo and thus establish a link between enhanced mitochondrial peroxynitrite and disruption of vascular integrity. AU - Kameritsch, P.* AU - Singer, M.* AU - Nuernbergk, C.* AU - Rios, N.* AU - Reyes, A.M.* AU - Schmidt, K.* AU - Kirsch, J.* AU - Schneider, H.* AU - Müller, S.* AU - Pogoda, K.* AU - Cui, R.* AU - Kirchner, T.* AU - de Wit, C.* AU - Lange-Sperandio, B.* AU - Pohl, U.* AU - Conrad, M. AU - Radi, R.* AU - Beck, H.* C1 - 61304 C2 - 50119 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - The mitochondrial thioredoxin reductase system (TrxR2) in vascular endothelium controls peroxynitrite levels and tissue integrity. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 7 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo-labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans. AU - Kemter, E.* AU - Müller, A.* AU - Neukam, M. AU - Ivanova, A. AU - Klymiuk, N.* AU - Renner, S.* AU - Yang, K. AU - Broichhagen, J.* AU - Kurome, M.* AU - Zakhartchenko, V.* AU - Kessler, B.* AU - Knoch, K.P.* AU - Bickle, M.* AU - Ludwig, B.* AU - Johnsson, K.* AU - Lickert, H. AU - Kurth, T.* AU - Wolf, E.* AU - Solimena, M. C1 - 62992 C2 - 51211 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 37 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AU - von Herrath, M.* AU - Bonifacio, E. C1 - 63444 C2 - 51321 TI - How benign autoimmunity becomes detrimental in type 1 diabetes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 44 PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in highrisk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 μm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers. AU - Yim, J.J.* AU - Harmsen, S.* AU - Flisikowski, K.* AU - Flisikowska, T.* AU - Namkoong, H.* AU - Garland, M.* AU - van den Berg, N.S.* AU - Vilches-Moure, J.G.* AU - Schnieke, A.* AU - Saur, D.* AU - Glasl, S. AU - Gorpas, D. AU - Habtezion, A.* AU - Ntziachristos, V. AU - Contag, C.H.* AU - Gambhir, S.S.* AU - Bogyo, M.* AU - Rogalla, S.* C1 - 60889 C2 - 49729 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa TI - A protease-activated, near-infrared fluorescent probe for early endoscopic detection of premalignant gastrointestinal lesions. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 118 IS - 1 PB - Natl Acad Sciences PY - 2021 SN - 0027-8424 ER - TY - JOUR AB - Posttranslational modifications (PTMs) are important physiological means to regulate the activities and structures of central regulatory proteins in health and disease. Small GTPases have been recognized as important molecules that are targeted by PTMs during infections of mammalian cells by bacterial pathogens. The enzymes DrrA/SidM and AnkX from Legionella pneumophila AMPylate and phosphocholinate Rab1b during infection, respectively. Cdc42 is AMPylated by IbpA from Histophilus somni at tyrosine 32 or by VopS from Vibrio parahaemolyticus at threonine 35. These modifications take place in the important regulatory switch I or switch II regions of the GTPases. Since Rab1b and Cdc42 are central regulators of intracellular vesicular trafficking and of the actin cytoskeleton, their modifications by bacterial pathogens have a profound impact on the course of infection. Here, we addressed the biochemical and structural consequences of GTPase AMPylation and phosphocholination. By combining biochemical experiments and NMR analysis, we demonstrate that AMPylation can overrule the activity state of Rab1b that is commonly dictated by binding to guanosine diphosphate or guanosine triphosphate. Thus, PTMs may exert conformational control over small GTPases and may add another previously unrecognized layer of activity control to this important regulatory protein family. AU - Barthelmes, K. AU - Ramcke, E.* AU - Kang, H.-S. AU - Sattler, M. AU - Itzen, A.* C1 - 58507 C2 - 48370 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 5772-5781 TI - Conformational control of small GTPases by AMPylation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 11 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging. AU - Degenhardt, K.* AU - Wagner, J.* AU - Skodras, A.* AU - Candlish, M.* AU - Koppelmann, A.J.* AU - Wild, K.* AU - Maxwell, R.* AU - Rotermund, C.* AU - von Zweydorf, F.* AU - Gloeckner, C.J.* AU - Davies, H.A.* AU - Madine, J.* AU - Del Turco, D.* AU - Feederle, R. AU - Lashley, T.* AU - Deller, T.* AU - Kahle, P.* AU - Hefendehl, J.K.* AU - Jucker, M.* AU - Neher, J.J.* C1 - 60175 C2 - 49233 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 23925-23931 TI - Medin aggregation causes cerebrovascular dysfunction in aging wild-type mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 38 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Land-use intensification can increase provisioning ecosystem services, such as food and timber production, but it also drives changes in ecosystem functioning and biodiversity loss, which may ultimately compromise human wellbeing. To understand how changes in land-use intensity affect the relationships between biodiversity, ecosystem functions, and services, we built networks from correlations between the species richness of 16 trophic groups, 10 ecosystem functions, and 15 ecosystem services. We evaluated how the properties of these networks varied across land-use intensity gradients for 150 forests and 150 grasslands. Land-use intensity significantly affected network structure in both habitats. Changes in connectance were larger in forests, while changes in modularity and evenness were more evident in grasslands. Our results show that increasing land-use intensity leads to more homogeneous networks with less integration within modules in both habitats, driven by the belowground compartment in grasslands, while forest responses to land management were more complex. Land-use intensity strongly altered hub identity and module composition in both habitats, showing that the positive correlations of provisioning services with biodiversity and ecosystem functions found at low land-use intensity levels, decline at higher intensity levels. Our approach provides a comprehensive view of the relationships between multiple components of biodiversity, ecosystem functions, and ecosystem services and how they respond to land use. This can be used to identify overall changes in the ecosystem, to derive mechanistic hypotheses, and it can be readily applied to further global change drivers. AU - Felipe-Lucia, M.R.* AU - Soliveres, S.* AU - Penone, C.* AU - Fischer, M.* AU - Ammer, C.* AU - Boch, S.* AU - Boeddinghaus, R.S.* AU - Bonkowski, M.* AU - Buscot, F.* AU - Fiore-Donno, A.M.* AU - Frank, K.* AU - Goldmann, K.* AU - Gossner, M.M.* AU - Hölzel, N.* AU - Jochum, M.* AU - Kandeler, E.* AU - Klaus, V.H.* AU - Kleinebecker, T.* AU - Leimer, S.* AU - Manning, P.* AU - Oelmann, Y.* AU - Saiz, H.* AU - Schall, P.* AU - Schloter, M. AU - Schöning, I.* AU - Schrumpf, M.* AU - Solly, E.F.* AU - Stempfhuber, B. AU - Weisser, W.W.* AU - Wilcke, W.* AU - Wubet, T.* AU - Allan, E.* C1 - 60552 C2 - 49426 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 28140-28149 TI - Land-use intensity alters networks between biodiversity, ecosystem functions, and services. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 45 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughput iCLIP approaches to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3' splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs. AU - Kang, H.-S. AU - Sanchez-Rico, C. AU - Ebersberger, S.* AU - Sutandy, F.X.R.* AU - Busch, A.* AU - Welte, T.* AU - Stehle, R.* AU - Hipp, C.* AU - Schulz, L.* AU - Buchbender, A.* AU - Zarnack, K.* AU - König, J.* AU - Sattler, M. C1 - 58641 C2 - 48284 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 7140-7149 TI - An autoinhibitory intramolecular interaction proof-reads RNA recognition by the essential splicing factor U2AF2. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 13 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Hybrid-poplar tree plantations provide a source for biofuel and biomass, but they also increase forest isoprene emissions. The consequences of increased isoprene emissions include higher rates of tropospheric ozone production, increases in the lifetime of methane, and increases in atmospheric aerosol production, all of which affect the global energy budget and/or lead to the degradation of air quality. Using RNA interference (RNAi) to suppress isoprene emission, we show that this trait, which is thought to be required for the tolerance of abiotic stress, is not required for high rates of photosynthesis and woody biomass production in the agroforest plantation environment, even in areas with high levels of climatic stress. Biomass production over 4 y in plantations in Arizona and Oregon was similar among genetic lines that emitted or did not emit significant amounts of isoprene. Lines that had substantially reduced isoprene emission rates also showed decreases in flavonol pigments, which reduce oxidative damage during extremes of abiotic stress, a pattern that would be expected to amplify metabolic dysfunction in the absence of isoprene production in stress-prone climate regimes. However, compensatory increases in the expression of other proteomic components, especially those associated with the production of protective compounds, such as carotenoids and terpenoids, and the fact that most biomass is produced prior to the hottest and driest part of the growing season explain the observed pattern of high biomass production with low isoprene emission. Our results show that it is possible to reduce the deleterious influences of isoprene on the atmosphere, while sustaining woody biomass production in temperate agroforest plantations. AU - Monson, R.K.* AU - Winkler, J.B. AU - Rosenstiel, T.N.* AU - Block, K. AU - Merl-Pham, J. AU - Strauss, S.H.* AU - Ault, K.* AU - Maxfield, J.* AU - Moore, D.J.P.* AU - Trahan, N.A.* AU - Neice, A.A.* AU - Shiach, I.* AU - Barron-Gafford, G.A.* AU - Ibsen, P.* AU - McCorkel, J.T.* AU - Bernhardt, J.* AU - Schnitzler, J.-P. C1 - 57767 C2 - 48121 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 1596-1605 TI - High productivity in hybrid-poplar plantations without isoprene emission to the atmosphere. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 3 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Infrared (IR) optoacoustic spectroscopy can separate a multitude of molecules based on their absorption spectra. However, the technique is limited when measuring target molecules in aqueous solution by strong water absorption at IR wavelengths, which reduces detection sensitivity. Based on the dependence of optoacoustic signal on the temperature of the probed medium, we introduce cooled IR optoacoustic spectroscopy (CIROAS) to mute water contributions in optoacoustic spectroscopy. We showcase that spectral measurements of proteins, lipids, and glucose in the short-wavelength IR region, performed at 4 degrees C, lead to marked sensitivity improvements over conventional optoacoustic or IR spectroscopy. We elaborate on the dependence of optoacoustic signals on water temperature and demonstrate polarity changes in the recorded signal at temperatures below 4 degrees C. We further elucidate the dependence of the optoacoustic signal and the muting temperature on sample concentration and demonstrate that changes in these dependences enable quantification of the solute concentration. We discuss how CIROAS may enhance abilities for molecular sensing in the IR. AU - Prakash, J. AU - Seyedebrahimi, M.M. AU - Ghazaryan, A. AU - Malekzadeh Najafabadi, J. AU - Gujrati, V. AU - Ntziachristos, V. C1 - 58107 C2 - 48072 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 4007-4014 TI - Short-wavelength optoacoustic spectroscopy based on water muting. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 8 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Every cell produces thousands of distinct lipid species, but insight into how lipid chemical diversity contributes to biological signaling is lacking, particularly because of a scarcity of methods for quantitatively studying lipid function in living cells. Using the example of diacylglycerols, prominent second messengers, we here investigate whether lipid chemical diversity can provide a basis for cellular signal specification. We generated photo-caged lipid probes, which allow acute manipulation of distinct diacylglycerol species in the plasma membrane. Combining uncaging experiments with mathematical modeling, we were able to determine binding constants for diacylglycerol-protein interactions, and kinetic parameters for diacylglycerol transbilayer movement and turnover in quantitative live-cell experiments. Strikingly, we find that affinities and kinetics vary by orders of magnitude due to diacylglycerol side-chain composition. These differences are sufficient to explain differential recruitment of diacylglycerol binding proteins and, thus, differing downstream phosphorylation patterns. Our approach represents a generally applicable method for elucidating the biological function of single lipid species on subcellular scales in quantitative live-cell experiments. AU - Schuhmacher, M.* AU - Grasskamp, A.T.* AU - Barahtjan, P.* AU - Wagner, N.* AU - Lombardot, B.* AU - Schuhmacher, J.S.* AU - Sala, P. AU - Lohmann, A.* AU - Henry, I.* AU - Shevchenko, A.* AU - Coskun, Ü. AU - Walter, A.M.* AU - Nadler, A.* C1 - 58716 C2 - 48291 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 7729-7738 TI - Live-cell lipid biochemistry reveals a role of diacylglycerol side-chain composition for cellular lipid dynamics and protein affinities. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 14 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis. AU - Sommermann, T.* AU - Yasuda, T.* AU - Ronen, J.* AU - Wirtz, T.* AU - Weber, T.* AU - Sack, U.* AU - Caeser, R.* AU - Zhang, J.* AU - Li, X.* AU - Chu, V.T.* AU - Jauch, A.* AU - Unger, K. AU - Hodson, D.J.* AU - Akalin, A.* AU - Rajewsky, K.* C1 - 59350 C2 - 48754 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 14421-14432 TI - Functional interplay of Epstein -Barr virus oncoproteins in a mouse model of B cell. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 25 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Functional connectivity (FC) is known to be individually unique and to reflect cognitive variability. Although FC can serve as a valuable correlate and potential predictor of (patho-) physiological nervous function in high-risk constellations, such as preterm birth, templates for individualized FC analysis are lacking, and knowledge about the capacity of the premature brain to develop FC variability is limited. In a cohort of prospectively recruited, preterm-born infants undergoing magnetic resonance imaging close to term equivalent age, we show that the overall pattern could be reliably detected with a broad range of interindividual FC variability in regions of higher-order cognitive functions (e.g., association cortices) and less interindividual variability in unimodal regions (e.g., visual and motor cortices). However, when comparing the preterm and adult brains, some brain regions showed a marked shift in variability toward adulthood. This shift toward greater variability was strongest in cognitive networks like the attention and frontoparietal networks and could be partially predicted by developmental cortical expansion. Furthermore, FC variability was reflected by brain tissue characteristics indicating cortical maturation. Brain regions with high functional variability (e.g., the inferior frontal gyrus and temporoparietal junction) displayed lower cortical maturation at birth compared with somatosensory cortices. In conclusion, the overall pattern of interindividual variability in FC is already present preterm; however, some brain regions show increased variability toward adulthood, identifying characteristic patterns, such as in cognitive networks. These changes are related to postnatal cortical expansion and maturation, allowing for environmental and developmental factors to translate into marked individual differences in FC. AU - Stoecklein, S.* AU - Hilgendorff, A. AU - Li, M.* AU - Förster, K. AU - Flemmer, A.W.* AU - Galiè, F.* AU - Wunderlich, S.* AU - Wang, D.* AU - Stein, S.* AU - Ehrhardt, H.* AU - Dietrich, O.* AU - Zou, Q.* AU - Zhou, S.* AU - Ertl-Wagner, B.* AU - Liu, H.* C1 - 57744 C2 - 47946 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 1201-1206 TI - Variable functional connectivity architecture of the preterm human brain: Impact of developmental cortical expansion and maturation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 2 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Systematic mappings of protein interactome networks have provided invaluable functional information for numerous model organisms. Here we develop PCR-mediated Linkage of barcoded Adapters To nucleic acid Elements for sequencing (PLATE-seq) that serves as a general tool to rapidly sequence thousands of DNA elements. We validate its utility by generating the ORFeome for Oryza sativa covering 2,300 genes and constructing a high-quality protein-protein interactome map consisting of 322 interactions between 289 proteins, expanding the known interactions in rice by roughly 50%. Our work paves the way for high-throughput profiling of protein-protein interactions in a wide range of organisms. AU - Wierbowski, S.D.* AU - Vo, T.V.* AU - Falter-Braun, P. AU - Jobe, T.O.* AU - Kruse, L.H.* AU - Wei, X.* AU - Liang, J.* AU - Meyer, M.J.* AU - Akturk, N.* AU - Rivera-Erick, C.A.* AU - Cordero, N.A.* AU - Paramo, M.I.* AU - Shayhidin, E.E.* AU - Bertolotti, M.* AU - Tippens, N.D.* AU - Akther, K.* AU - Sharma, R.* AU - Katayose, Y.* AU - Salehi-Ashtiani, K.* AU - Hao, T.* AU - Ronald, P.C.* AU - Ecker, J.R.* AU - Schweitzer, P.A.* AU - Kikuchi, S.* AU - Mizuno, H.* AU - Hill, D.E.* AU - Vidal, M.* AU - Moghe, G.D.* AU - McCouch, S.R.* AU - Yu, H.* C1 - 59101 C2 - 48572 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 11836-11842 TI - A massively parallel barcoded sequencing pipeline enables generation of the first ORFeome and interactome map for rice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 21 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Proton (H + ) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few pro- ton channels have been identified so far. Here, we identify a fam- ily of voltage -gated ?pacemaker ? channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpo- larization and conducts protons into the cytosol. Surprisingly, pro- tons permeate through the channel ?s voltage -sensing domain, whereas the pore domain is nonfunctional. Key to proton perme- ation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional do- mains, HCNL1 evolutionarily adapted to a low-Na + freshwater en- vironment to conserve sperm ?s ability to depolarize. AU - Wobig, L.* AU - Wolfenstetter, T.* AU - Fechner, S.* AU - Bönigk, W.* AU - Körschen, H.G.* AU - Jikeli, J.F.* AU - Trötschel, C.* AU - Feederle, R. AU - Kaupp, U.B.* AU - Seifert, R.* AU - Berger, T.K.* C1 - 59260 C2 - 48688 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 13783-13791 TI - A family of hyperpolarization-activated channels selective for. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 24 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Viral immune evasion is currently understood to focus on deflect-ing CD8 T cell recognition of infected cells by disrupting antigen presentation pathways. We evaluated viral interference with the ultimate step in cytotoxic T cell function, the death of infected cells. The viral inhibitor of caspase-8 activation (vICA) conserved in human cytomegalovirus (HCMV) and murine CMV (MCMV) pre-vents the activation of caspase-8 and proapoptotic signaling. We demonstrate the key role of vICA from either virus, in deflecting antigen-specific CD8 T cell-killing of infected cells. vICA-deficient mutants, lacking either UL36 or M36, exhibit greater susceptibility to CD8 T cell control than mutants lacking the set of immunoeva-sins known to disrupt antigen presentation via MHC class I. This difference is evident during infection in the natural mouse host infected with MCMV, in settings where virus-specific CD8 T cells are adoptively transferred. Finally, we identify the molecular mech-anism through which vICA acts, demonstrating the central contribu-tion of caspase-8 signaling at a point of convergence of death receptor-induced apoptosis and perforin/granzyme-dependent cytotoxicity. AU - Zeeshan Chaudhry, M.* AU - Casalegno-Garduno, R.* AU - Sitnik, K.M.* AU - Kasmapour, B.* AU - Pulm, A.K.* AU - Brizic, I.* AU - Eiz-Vesper, B.* AU - Moosmann, A. AU - Jonjic, S.* AU - Mocarski, E.S.* AU - Cicin-Sain, L.* C1 - 59383 C2 - 48792 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 12961-12968 TI - Cytomegalovirus inhibition of extrinsic apoptosis determines fitness and resistance to cytotoxic CD8 T cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 23 PB - Natl Acad Sciences PY - 2020 SN - 0027-8424 ER - TY - JOUR AB - Autoinflammatory syndromes are characterized by dysregulation of the innate immune response with subsequent episodes of acute spontaneous inflammation. Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder that presents with bone pain and localized swelling. Ali18 mice, isolated from a mutagenesis screen, exhibit a spontaneous inflammatory paw phenotype that includes sterile osteomyelitis and systemic reduced bone mineral density. To elucidate the molecular basis of the disease, positional cloning of the causative gene for Ali18 was attempted. Using a candidate gene approach, a missense mutation in the C-terminal region of Fgr, a member of Src family tyrosine kinases (SFKs), was identified. For functional confirmation, additional mutations at the N terminus of Fgr were introduced in Ali18 mice by CRISPR/Cas9-mediated genome editing. N-terminal deleterious mutations of Fgr abolished the inflammatory phenotype in Ali18 mice, but in-frame and missense mutations in the same region continue to exhibit the phenotype. The fact that Fgr null mutant mice are morphologically normal suggests that the inflammation in this model depends on Fgr products. Furthermore, the levels of C-terminal negative regulatory phosphorylation of Fgr(Ali18) are distinctly reduced compared with that of wild-type Fgr. In addition, whole-exome sequencing of 99 CRMO patients including 88 trios (proband and parents) identified 13 patients with heterozygous coding sequence variants in FGR, including two missense mutant proteins that affect kinase activity. Our results strongly indicate that gain-of-function mutations in Fgr are involved in sterile osteomyelitis, and thus targeting SFKs using specific inhibitors may allow for efficient treatment of the disease. AU - Abe, K.* AU - Cox, A.* AU - Takamatsu, N.* AU - Velez, G.* AU - Laxer, R.M.* AU - Tse, S.M.L.* AU - Mahajan, V.B.* AU - Bassuk, A.G.* AU - Fuchs, H. AU - Ferguson, P.J.* AU - Hrabě de Angelis, M. C1 - 56174 C2 - 46856 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 11872-11877 TI - Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 24 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis. AU - Belavgeni, A.* AU - Bornstein, S.R. AU - Krone, N.P. AU - von Mässenhausen, A.* AU - Tonnus, W.* AU - Stumpf, J.* AU - Meyer, C.* AU - Othmar, E.* AU - Latk, M.* AU - Kanczkowski, W.* AU - Kroiss, M.* AU - Hantel, C.* AU - Hugo, C.* AU - Fassnacht, M.* AU - Ziegler, C.G.* AU - Linkermann, A.* C1 - 57122 C2 - 47545 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 22269-22274 TI - Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 44 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Pancreatic β cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair β cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse β cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining β cell function and diabetes prevention. AU - Binger, K.J.* AU - Neukam, M. AU - Tattikota, S.G.* AU - Qadri, F.* AU - Puchkov, D.* AU - Willmes, D.M. AU - Wurmsee, S.* AU - Geisberger, S.* AU - Dechend, R.* AU - Raile, K.* AU - Kurth, T.* AU - Nguyen, G.* AU - Poy, M.N.* AU - Solimena, M. AU - Müller, D.N.* AU - Birkenfeld, A.L. C1 - 56920 C2 - 47365 SP - 19983-19988 TI - Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 40 PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Frizzled 3 receptor (FZD3) plays an important role in the homeo-stasis of the neural crest and its derivatives, which give rise to pigment-synthesizing cells, melanocytes. While the role for FZD3 in specification of the melanocytic lineage from neural crest is well established, its significance in the formation of melanoma, its associated malignancy, is less understood. In this study we identified FZD3 as a critical regulator of human melanoma tumorigenesis. Down-regulation of FZD3 abrogated growth, colony-forming potential, and invasive capacity of patient-derived melanoma cells. Xenotransplantation of tumor cells with down-regulated FZD3 levels originating from melanomas carrying the BRAF(V600) mutation uniformly suppressed their capacity for tumor and metastasis formation. FZD3 knockdown leads to the down-regulation of the core cell cycle protein components (cyclins D1, E2, B1, and CDKs 1, 2, and 4) in melanomas with a hyperactive BRAF oncogene, indicating a dominant role of this receptor during melanoma pathogenesis. Enriched pathway analysis revealed that FZD3 inhibits transcriptional networks controlled by CREB5, FOXD1, and ATF3, which suppress the activity of MAPK-mediated signaling. Thus, FZD3 establishes a positive-feedback mechanism that activates MAPK signal transduction network, critical to melanoma carcinogenesis. Importantly, high levels of FZD3 mRNA were found to be correlated with melanoma advancement to metastatic stages and limited patient survival. Changes in gene-expression patterns mediated by FZD3 activity occur in the absence of nuclear beta-catenin function, thus representing an important therapeutic target for the melanoma patients whose disease progresses independent of canonical WNT signaling. AU - Li, C.* AU - Nguyen, V.* AU - Clark, K.N.* AU - Zahed, T.* AU - Sharkas, S.* AU - Filipp, F.V. AU - Boiko, A.D.* C1 - 55679 C2 - 46455 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 4548-4557 TI - Down-regulation of FZD3 receptor suppresses growth and metastasis of human melanoma independently of canonical WNT signaling. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 10 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - The detection of microbes and damaged host cells by the innate immune system is essential for host defense against infection and tissue homeostasis. However, how distinct positive and negative regulatory signals from immune receptors are integrated to tailor specific responses in complex scenarios remains largely undefined. Clec12A is a myeloid cell-expressed inhibitory C-type lectin receptor that can sense cell death under sterile conditions. Clec12A detects uric acid crystals and limits proinflammatory pathways by counteracting the cell-activating spleen tyrosine kinase (Syk). Here, we surprisingly find that Clec12A additionally amplifies type I IFN (IFN-I) responses in vivo and in vitro. Using retinoic acid-inducible gene I (RIG-I) signaling as a model, we demonstrate that monosodium urate (MSU) crystal sensing by Clec12A enhances cytosolic RNA-induced IFN-I production and the subsequent induction of IFN-I-stimulated genes. Mechanistically, Clec12A engages Src kinase to positively regulate the TBK1-IRF3 signaling module. Consistently, Clec12A-deficient mice exhibit reduced IFN-I responses upon lymphocytic choriomeningitis virus (LCMV) infection, which affects the outcomes of these animals in acute and chronic virus infection models. Thus, our results uncover a previously unrecognized connection between an MSU crystal-sensing receptor and the IFN-I response, and they illustrate how the sensing of extracellular damage-associated molecular patterns (DAMPs) can shape the immune response. AU - Li, K.* AU - Neumann, K.* AU - Duhan, V.* AU - Namineni, S.* AU - Hansen, A.L.* AU - Wartewig, T.* AU - Kurgyis, Z.* AU - Holm, C.K.* AU - Heikenwälder, M. AU - Lang, K.S.* AU - Ruland, J.* C1 - 56808 C2 - 47322 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 18544-18549 TI - The uric acid crystal receptor Clec12A potentiates type I interferon responses. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 37 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - The presence of progenitor or stem cells in the adult pancreas and their potential involvement in homeostasis and cancer development remain unresolved issues. Here, we show that mouse centroacinar cells can be identified and isolated by virtue of the mitochondrial enzyme Aldh1b1 that they uniquely express. These cells are necessary and sufficient for the formation of self-renewing adult pancreatic organoids in an Aldh1b1-dependent manner. Aldh1b1-expressing centroacinar cells are largely quiescent, self-renew, and, as shown by genetic lineage tracing, contribute to all 3 pancreatic lineages in the adult organ under homeostatic conditions. Single-cell RNA sequencing analysis of these cells identified a progenitor cell population, established its molecular signature, and determined distinct differentiation pathways to early progenitors. A distinct feature of these progenitor cells is the preferential expression of small GTPases, including Kras, suggesting that they might be susceptible to Kras-driven oncogenic transformation. This finding and the overexpression of Aldh1b1 in human and mouse pancreatic cancers, driven by activated Kras, prompted us to examine the involvement of Aldh1b1 in oncogenesis. We demonstrated genetically that ablation of Aldh1b1 completely abrogates tumor development in a mouse model of Kras(G12D)-induced pancreatic cancer. AU - Mameishvili, E. AU - Serafimidis, I.* AU - Iwaszkiewicz, S. AU - Lesche, M. AU - Reinhardt, S.* AU - Bölicke, N. AU - Büttner, M. AU - Stellas, D.* AU - Papadimitropoulou, A.* AU - Szabolcs, M.* AU - Anastassiadis, K.* AU - Dahl, A.* AU - Theis, F.J. AU - Efstratiadis, A.* AU - Gavalas, A. C1 - 56963 C2 - 47367 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 20679-20688 TI - Aldh1b1 expression defines progenitor cells in the adult pancreas and is required for Kras-induced pancreatic cancer. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 41 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - In October 2017, most European countries reported unique atmospheric detections of aerosol-bound radioruthenium (Ru-106). The range of concentrations varied from some tenths of mu Bq.m(-3) to more than 150 mBq.m(-3). The widespread detection at such considerable (yet innocuous) levels suggested a considerable release. To compare activity reports of airborne Ru-106 with different sampling periods, concentrations were reconstructed based on the most probable plume presence duration at each location. Based on airborne concentration spreading and chemical considerations, it is possible to assume that the release occurred in the Southern Urals region (Russian Federation). The Ru-106 age was estimated to be about 2 years. It exhibited highly soluble and less soluble fractions in aqueous media, high radiopurity (lack of concomitant radionuclides), and volatility between 700 and 1,000 degrees C, thus suggesting a release at an advanced stage in the reprocessing of nuclear fuel. The amount and isotopic characteristics of the radioruthenium release may indicate a context with the production of a large Ce-144 source for a neutrino experiment. AU - Masson, O.* AU - Steinhauser, G.* AU - Zok, D.* AU - Saunier, O.* AU - Angelov, H.* AU - Babić, D.* AU - Bečková, V.* AU - Bieringer, J.* AU - Bruggeman, M.* AU - Burbidge, C.I.* AU - Conil, S.* AU - Dalheimer, A.* AU - de Geer, L.E.* AU - de Vismes Ott, A.* AU - Eleftheriadis, K.* AU - Estier, S.* AU - Fischer, H.* AU - Garavaglia, M.G.* AU - Gasco Leonarte, C.* AU - Gorzkiewicz, K.* AU - Hainz, D.* AU - Hoffman, I.* AU - Hýža, M.* AU - Isajenko, K.* AU - Karhunen, T.* AU - Kastlander, J.* AU - Katzlberger, C.* AU - Kierepko, R.* AU - Knetsch, G.J.* AU - Kövendiné Kónyi, J.* AU - Lecomte, M.* AU - Mietelski, J.W.* AU - Min, P.* AU - Møller, B.* AU - Nielsen, S.P.* AU - Nikolic, J.* AU - Nikolovska, L.* AU - Penev, I.* AU - Petrinec, B.* AU - Povinec, P.P.* AU - Querfeld, R.* AU - Raimondi, O.* AU - Ransby, D.* AU - Ringer, W.* AU - Romanenko, O.* AU - Rusconi, R.* AU - Saey, P.R.J.* AU - Samsonov, V.* AU - Šilobritienė, B.* AU - Simion, E.* AU - Söderström, C.* AU - Šoštarić, M.* AU - Steinkopff, T.* AU - Steinmann, P.* AU - Sýkora, I.* AU - Tabachnyi, L.* AU - Todorovic, D.* AU - Tomankiewicz, E.* AU - Tschiersch, J. AU - Tsibranski, R.* AU - Tzortzis, M.* AU - Ungar, K.* AU - Vidic, A.* AU - Weller, A.* AU - Wershofen, H.* AU - Zagyvai, P.* AU - Zalewska, T.* AU - Zapata García, D.* AU - Zorko, B.* C1 - 56649 C2 - 47240 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 16750-16759 TI - Airborne concentrations and chemical considerations of radioactive ruthenium from an undeclared major nuclear release in 2017. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 34 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a human tumor virus and a model of herpesviral latency. The virus efficiently infects resting human B lymphocytes and induces their continuous proliferation in vitro, which mimics certain aspects of EBV's oncogenic potential in vivo. How lymphoblastoid cell lines (LCLs) evolve from the infected lymphocytes is uncertain. We conducted a systematic time-resolved longitudinal study of cellular functions and transcriptional profiles of newly infected naive primary B lymphocytes. EBV reprograms the cells comprehensively and globally. Rapid and extensive transcriptional changes occur within 24 h and precede any metabolic and phenotypic changes. Within 72 h, the virus activates the cells, changes their phenotypes with respect to cell size, RNA, and protein content, and induces metabolic pathways to cope with the increased demand for energy, supporting an efficient cell cycle entry on day 3 postinfection. The transcriptional program that EBV initiates consists of 3 waves of clearly discernable clusters of cellular genes that peak on day 2, 3, or 4 and regulate RNA synthesis, metabolic pathways, and cell division, respectively. Upon onset of cell doublings on day 4, the cellular transcriptome appears to be completely reprogrammed to support the proliferating cells, but 3 additional clusters of EBV-regulated genes fine-tune cell signaling, migration, and immune response pathways, eventually. Our study reveals that more than 11,000 genes are regulated upon EBV infection as naive B cells exit quiescence to enter a germinal center-like differentiation program, which culminates in immortalized, proliferating cells that partially resemble plasmablasts and early plasma cells. AU - Mrozek-Gorska, P. AU - Buschle, A. AU - Pich, D. AU - Schwarzmayr, T. AU - Fechtner, R. AU - Scialdone, A. AU - Hammerschmidt, W. C1 - 56646 C2 - 47212 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 16046-16055 TI - Epstein-Barr virus reprograms human B lymphocytes immediately in the prelatent phase of infection. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 32 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - A common design principle of heteromeric signaling proteins is the use of shared subunits. This allows encoding of complex messages while maintaining evolutionary flexibility. How cells regulate and control assembly of such composite signaling proteins remains an important open question. An example of particular complexity and biological relevance is the interleukin 12 (IL-12) family. Four functionally distinct alpha beta heterodimers are assembled from only five subunits to regulate immune cell function and development. In addition, some subunits act as independent signaling molecules. Here we unveil key molecular mechanisms governing IL-27 biogenesis, an IL-12 family member that limits infections and autoimmunity. In mice, the IL-27 alpha subunit is secreted as a cytokine, whereas in humans only heterodimeric IL-27 is present. Surprisingly, we find that differences in a single amino acid determine if IL-27 alpha can be secreted autonomously, acting as a signaling molecule, or if it depends on hetero-dimerization for secretion. By combining computer simulations with biochemical experiments, we dissect the underlying structural determinants: a protein folding switch coupled to disulfide bond formation regulates chaperone-mediated retention versus secretion. Using these insights, we rationally change folding and assembly control for this protein. This provides the basis for a more human-like IL-27 system in mice and establishes a secretion-competent human IL-27 alpha that signals on its own and can regulate immune cell function. Taken together, our data reveal a close link between protein folding and immunoregulation. Insights into the underlying mechanisms can be used to engineer immune modulators. AU - Müller, S.I.* AU - Friedl, A. AU - Aschenbrenner, I.* AU - Esser-von Bieren, J. AU - Zacharias, M.* AU - Devergne, O.* AU - Feige, M.J.* C1 - 55249 C2 - 46299 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 1585-1590 TI - A folding switch regulates interleukin 27 biogenesis and secretion of its α-subunit as a cytokine. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 116 IS - 5 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Prenatal stress exposure is associated with risk for psychiatric disorders later in life. This may be mediated in part via enhanced exposure to glucocorticoids (GCs), which are known to impact neurogenesis. We aimed to identify molecular mediators of these effects, focusing on long-lasting epigenetic changes. In a human hippocampal progenitor cell (HPC) line, we assessed the short- and long-term effects of GC exposure during neurogenesis on messenger RNA (mRNA) expression and DNA methylation (DNAm) profiles. GC exposure induced changes in DNAm at 27,812 CpG dinucleotides and in the expression of 3,857 transcripts (false discovery rate [FDR] <= 0.1 and absolute fold change [FC] expression >= 1.15). HPC expression and GC-affected DNAm profiles were enriched for changes observed during human fetal brain development. Differentially methylated sites (DMSs) with GC exposure clustered into 4 trajectories over HPC differentiation, with transient as well as long-lasting DNAm changes. Lasting DMSs mapped to distinct functional pathways and were selectively enriched for poised and bivalent enhancer marks. Lasting DMSs had little correlation with lasting expression changes but were associated with a significantly enhanced transcriptional response to a second acute GC challenge. A significant subset of lasting DMSs was also responsive to an acute GC challenge in peripheral blood. These tissue-overlapping DMSs were used to compute a polyepigenetic score that predicted exposure to conditions associated with altered prenatal GCs in newborn's cord blood DNA. Overall, our data suggest that early exposure to GCs can change the set point of future transcriptional responses to stress by inducing lasting DNAm changes. Such altered set points may relate to differential vulnerability to stress exposure later in life. AU - Provençal, N.* AU - Knauer-Arloth, J. AU - Cattaneo, A.* AU - Anacker, C.* AU - Cattane, N.* AU - Wiechmann, T.* AU - Röh, S.* AU - Ködel, M.* AU - Klengel, T.* AU - Czamara, D.* AU - Müller, N.S.* AU - Lahti, J.* AU - Räikkönen, K.* AU - Pariante, C.M.* AU - Binder, E.B.* C1 - 56734 C2 - 47239 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 23280-23285 TI - Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 117 IS - 38 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-kappa B-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-kappa B regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-kappa B. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-kappa B through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-kappa B signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities. AU - Zannas, A.S.* AU - Jia, M.* AU - Hafner, K.* AU - Baumert, J.J. AU - Wiechmann, T.* AU - Pape, J.C.* AU - Knauer-Arloth, J. AU - Ködel, M.* AU - Martinelli, S.* AU - Roitman, M.* AU - Röh, S.* AU - Haehle, A.* AU - Emeny, R.T.* AU - Iurato, S.* AU - Carrillo-Roa, T.* AU - Lahti, J.* AU - Räikkönen, K.* AU - Eriksson, J.G.* AU - Drake, A.J.* AU - Waldenberger, M. AU - Wahl, S. AU - Kunze, S. AU - Lucae, S.* AU - Bradley, B.* AU - Gieger, C. AU - Hausch, F.* AU - Smith, A.K.* AU - Ressler, K.J.* AU - Müller-Myhsok, B.* AU - Ladwig, K.-H. AU - Rein, T.* AU - Gassen, N.C.* AU - Binder, E.B.* C1 - 56105 C2 - 46836 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 11370-11379 TI - Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-kappa B-driven inflammation and cardiovascular risk. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 166 IS - 23 PB - Natl Acad Sciences PY - 2019 SN - 0027-8424 ER - TY - JOUR AB - Recent technology developments have expanded the wavelength window for biological fluorescence imaging into the shortwave infrared. We show here a mechanistic understanding of how drastic changes in fluorescence imaging contrast can arise from slight changes of imaging wavelength in the shortwave infrared. We demonstrate, in 3D tissue phantoms and in vivo in mice, that light absorption by water within biological tissue increases image contrast due to attenuation of background and highly scattered light. Wavelengths of strong tissue absorption have conventionally been avoided in fluorescence imaging to maximize photon penetration depth and photon collection, yet we demonstrate that imaging at the peak absorbance of water (near 1,450 nm) results in the highest image contrast in the shortwave infrared. Furthermore, we show, through microscopy of highly labeled ex vivo biological tissue, that the contrast improvement from water absorption enables resolution of deeper structures, resulting in a higher imaging penetration depth. We then illustrate these findings in a theoretical model. Our results suggest that the wavelength-dependent absorptivity of water is the dominant optical property contributing to image contrast, and is therefore crucial for determining the optimal imaging window in the infrared. AU - Carr, J.A.* AU - Aellen, M.* AU - Franke, D.* AU - So, P.T.C.* AU - Bruns, O.T. AU - Bawendi, M.G.* C1 - 54198 C2 - 45437 CY - 2101 Constitution Ave Nw, Washington, Dc 20418 Usa SP - 9080-9085 TI - Absorption by water increases fluorescence image contrast of biological tissue in the shortwave infrared. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 115 IS - 37 PB - Natl Acad Sciences PY - 2018 SN - 0027-8424 ER - TY - JOUR AB - Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: (i) a physico-chemical characterization of the soil habitability after an exceptional rain event, (ii) identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], (iii) measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and (iv) microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity. AU - Schulze-Makuch, D.* AU - Wagner, D.* AU - Kounaves, S.P.* AU - Mangelsdorf, K.* AU - Devine, K.G.* AU - de Vera, J.P.* AU - Schmitt-Kopplin, P. AU - Grossart, H.P.* AU - Parro, V.* AU - Kaupenjohann, M.* AU - Galy, A.* AU - Schneider, B.* AU - Airo, A.* AU - Frösler, J.* AU - Davila, A.F.* AU - Arens, F.L.* AU - Cáceres, L.* AU - Cornejo, F.S.* AU - Carrizo, D.* AU - Dartnell, L.* AU - DiRuggiero, J.* AU - Flury, M.* AU - Ganzert, L.* AU - Gessner, M.O.* AU - Grathwohl, P.* AU - Guan, L. AU - Heinz, J.* AU - Hess, M.* AU - Keppler, F.* AU - Maus, D.* AU - McKay, C.P.* AU - Meckenstock, R.U.* AU - Montgomery, W.* AU - Oberlin, E.A.* AU - Probst, A.J.* AU - Sáenz, J.S. AU - Sattler, T.* AU - Schirmack, J.* AU - Sephton, M.A.* AU - Schloter, M. AU - Uhl, J. AU - Valenzuela, B.* AU - Vestergaard, G. AU - Wörmer, L.* AU - Zamorano, P.* C1 - 53062 C2 - 44483 CY - Washington SP - 2670-2675 TI - Transitory microbial habitat in the hyperarid Atacama Desert. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 115 IS - 11 PB - Natl Acad Sciences PY - 2018 SN - 0027-8424 ER - TY - JOUR AB - Advanced age is not only a major risk factor for a range of disorders within an aging individual but may also enhance susceptibility for disease in the next generation. In humans, advanced paternal age has been associated with increased risk for a number of diseases. Experiments in rodent models have provided initial evidence that paternal age can influence behavioral traits in offspring animals, but the overall scope and extent of paternal age effects on health and disease across the life span remain underexplored. Here, we report that old father offspring mice showed a reduced life span and an exacerbated development of aging traits compared with young father offspring mice. Genome-wide epigenetic analyses of sperm from aging males and old father offspring tissue identified differentially methylated promoters, enriched for genes involved in the regulation of evolutionarily conserved longevity pathways. Gene expression analyses, biochemical experiments, and functional studies revealed evidence for an overactive mTORC1 signaling pathway in old father offspring mice. Pharmacological mTOR inhibition during the course of normal aging ameliorated many of the aging traits that were exacerbated in old father offspring mice. These findings raise the possibility that inherited alterations in longevity pathways contribute to intergenerational effects of aging in old father offspring mice. AU - Xie, K.* AU - Ryan, D.P.* AU - Pearson, B.L.* AU - Henzel, K.S.* AU - Neff, F. AU - Vidal, R.O.* AU - Hennion, M.* AU - Lehmann, I.* AU - Schleif, M.* AU - Schröder, S.* AU - Adler, T. AU - Rathkolb, B. AU - Rozman, J. AU - Schütz, A.L.* AU - Prehn, C. AU - Mickael, M.E.* AU - Weiergräber, M.* AU - Adamski, J. AU - Busch, D.H.* AU - Ehninger, G.* AU - Matynia, A.* AU - Jackson, W.S.* AU - Wolf, E.* AU - Fuchs, H. AU - Gailus-Durner, V. AU - Bonn, S.* AU - Hrabě de Angelis, M. AU - Ehninger, D.* C1 - 53043 C2 - 44339 CY - Washington SP - E2348-E2357 TI - Epigenetic alterations in longevity regulators, reduced life span, and exacerbated aging-related pathology in old father offspring mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 115 IS - 10 PB - Natl Acad Sciences PY - 2018 SN - 0027-8424 ER - TY - JOUR AB - Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells. AU - Baldauf, H.M.* AU - Stegmann, L.* AU - Schwarz, S.M.* AU - Ambiel, I.* AU - Trotard, M.* AU - Martin, M.* AU - Burggraf, M.* AU - Lenzi, G.M.* AU - Lejk, H. AU - Pan, X.* AU - Fregoso, O.I.* AU - Lim, E.S.* AU - Abraham, L.* AU - Nguyen, L.A.* AU - Rutsch, F.* AU - König, R.* AU - Kim, B.* AU - Emerman, M.* AU - Fackler, O.T.* C1 - 53009 C2 - 44261 SP - 2729-2734 TI - Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 10 PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Aging is accompanied by major changes in adipose tissue distribution and function. In particular, with time, thermogenic-competent beige adipocytes progressively gain a white adipocyte morphology. However, the mechanisms controlling the age-related transition of beige adipocytes to white adipocytes remain unclear. Lysine-specific demethylase 1 (Lsd1) is an epigenetic eraser enzyme positively regulating differentiation and function of adipocytes. Here we show that Lsd1 levels decrease in aging inguinal white adipose tissue concomitantly with beige fat cell decline. Accordingly, adipocyte-specific increase of Lsd1 expression is sufficient to rescue the age-related transition of beige adipocytes to white adipocytes in vivo, whereas loss of Lsd1 precipitates it. Lsd1 maintains beige adipocytes by controlling the expression of peroxisome proliferator-activated receptor a (Ppara), and treatment with a Ppara agonist is sufficient to rescue the loss of beige adipocytes caused by Lsd1 ablation. In summary, our data provide insights into the mechanism controlling the age-related beige-to-white adipocyte transition and identify Lsd1 as a regulator of beige fat cell maintenance. AU - Duteil, D.* AU - Tosic, M.* AU - Willmann, D.* AU - Georgiadi, A. AU - Kanouni, T.* AU - Schuele, R.* C1 - 51214 C2 - 42923 CY - Washington SP - 5265-5270 TI - Lsd1 prevents age-programed loss of beige adipocytes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 20 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger signaling pathways independently of gene expression (noncanonical or type 3 TR signaling). To test the physiological relevance of noncanonical TR signaling, we generated knockin mice with a mutation in the TR DNA-binding domain that abrogates binding to DNA and leads to complete loss of canonical TH action. We show that several important physiological TH effects are preserved despite the disruption of DNA binding of TRα and TRβ, most notably heart rate, body temperature, blood glucose, and triglyceride concentration, all of which were regulated by noncanonical TR signaling. Additionally, we confirm that TRE-binding–defective TRβ leads to disruption of the hypothalamic–pituitary–thyroid axis with resistance to TH, while mutation of TRα causes a severe delay in skeletal development, thus demonstrating tissue- and TR isoform-specific canonical signaling. These findings provide in vivo evidence that noncanonical TR signaling exerts physiologically important cardiometabolic effects that are distinct from canonical actions. These data challenge the current paradigm that in vivo physiological TH action is mediated exclusively via regulation of gene transcription at the nuclear level. AU - Hönes, G.S.* AU - Rakov, H.* AU - Logan, J.* AU - Liao, X.-H.* AU - Werbenko, E.* AU - Pollard, A.S.* AU - Præstholm, S.M.* AU - Siersbæk, M.S.* AU - Rijntjes, E.* AU - Gassen, J.* AU - Latteyer, S.* AU - Engels, K.* AU - Strucksberg, K.-H.* AU - Kleinbongard, P.* AU - Zwanziger, D.* AU - Rozman, J. AU - Gailus-Durner, V. AU - Fuchs, H. AU - Hrabě de Angelis, M. AU - Klein-Hitpass, L.* AU - Köhrle, J.* AU - Armstrong, D.L.* AU - Grøntved, L.* AU - Basset, J.H.D.* AU - Williams, G.R.* AU - Refetoff, S.* AU - Führer, D.* AU - Moeller, L.C.* C1 - 52524 C2 - 44024 CY - Washington SP - E11323-E11332 TI - Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 52 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AU - Jastroch, M. C1 - 51644 C2 - 43320 CY - Washington SP - 7744-7746 TI - Uncoupling protein 1 controls reactive oxygen species in brown adipose tissue. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 30 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Amyloid-β (Aβ) is thought to play an essential pathogenic role in Alzheimer´s disease (AD). A key enzyme involved in the generation of Aβ is the β-secretase BACE, for which powerful inhibitors have been developed and are currently in use in human clinical trials. However, although BACE inhibition can reduce cerebral Aβ levels, whether it also can ameliorate neural circuit and memory impairments remains unclear. Using histochemistry, in vivo Ca(2+) imaging, and behavioral analyses in a mouse model of AD, we demonstrate that along with reducing prefibrillary Aβ surrounding plaques, the inhibition of BACE activity can rescue neuronal hyperactivity, impaired long-range circuit function, and memory defects. The functional neuronal impairments reappeared after infusion of soluble Aβ, mechanistically linking Aβ pathology to neuronal and cognitive dysfunction. These data highlight the potential benefits of BACE inhibition for the effective treatment of a wide range of AD-like pathophysiological and cognitive impairments. AU - Keskin, A.D.* AU - Kekuš, M.* AU - Adelsberger, H.* AU - Neumann, U.* AU - Shimshek, D.R.* AU - Song, B.* AU - Zott, B.* AU - Peng, T. AU - Förstl, H.* AU - Staufenbiel, M.* AU - Nelken, I.* AU - Sakmann, B.* AU - Konnerth, A.* AU - Busche, M.A.* C1 - 51583 C2 - 43312 CY - Washington SP - 8631-8636 TI - BACE inhibition-dependent repair of Alzheimer's pathophysiology. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 32 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - To elucidate the molecular basis of BMP4-induced differentiation of human pluripotent stem cells (PSCs) toward progeny with trophectoderm characteristics, we produced transcriptome, epigenome H3K4me3, H3K27me3, and CpG methylation maps of trophoblast progenitors, purified using the surface marker APA. We combined them with the temporally resolved transcriptome of the preprogenitor phase and of single APA+ cells. This revealed a circuit of bivalent TFAP2A, TFAP2C, GATA2, and GATA3 transcription factors, coined collectively the "trophectoderm four" (TEtra), which are also present in human trophectoderm in vivo. At the onset of differentiation, the TEtra factors occupy multiple sites in epigenetically inactive placental genes and in OCT4 Functional manipulation of GATA3 and TFAP2A indicated that they directly couple trophoblast-specific gene induction with suppression of pluripotency. In accordance, knocking down GATA3 in primate embryos resulted in a failure to form trophectoderm. The discovery of the TEtra circuit indicates how trophectoderm commitment is regulated in human embryogenesis. AU - Krendl, C. AU - Shaposhnikov, D. AU - Rishko, V. AU - Ori, C. AU - Ziegenhain, C.* AU - Sass, S. AU - Simon, L. AU - Müller, N.S. AU - Straub, T.* AU - Brooks, K.E.* AU - Chavez, S.L.* AU - Enard, W.* AU - Theis, F.J. AU - Drukker, M. C1 - 52233 C2 - 43883 CY - Washington SP - E9579-E9588 TI - GATA2/3-TFAP2A/C transcription factor network couples human pluripotent stem cell differentiation to trophectoderm with repression of pluripotency. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 45 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell-mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood-brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets. AU - Kyratsous, N.I.* AU - Bauer, I.J.* AU - Zhang, G.* AU - Pesic, M.* AU - Bartholomäus, I.* AU - Mues, M.* AU - Fang, P.* AU - Wörner, M.* AU - Everts, S.* AU - Ellwart, J.W. AU - Watt, J.M.* AU - Potter, B.V.L.* AU - Hohlfeld, R.* AU - Wekerle, H.* AU - Kawakami, N.* C1 - 51550 C2 - 43221 SP - E6381-E6389 TI - Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 31 PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macro-encapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions. AU - Ludwig, B. AU - Ludwig, S.* AU - Steffen, A. AU - Knauf, Y.* AU - Zimerman, B.* AU - Heinke, S.* AU - Lehmann, S.* AU - Schubert, U.* AU - Schmid, J.* AU - Bleyer, M.* AU - Schoenmann, U.* AU - Colton, C.K.* AU - Bonifacio, E.* AU - Solimena, M. AU - Reichel, A.* AU - Schally, A.V.* AU - Rotem, A.* AU - Barkai, U.* AU - Grinberg-Rashi, H.* AU - Kaup, F.* AU - Avni, Y.* AU - Jones, P.* AU - Bornstein, S.R. C1 - 52290 C2 - 43843 CY - Washington SP - 11745-11750 TI - Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 44 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Large artery atherosclerotic stroke (LAS) shows substantial heritability not explained by previous genome-wide association studies. Here, we explore the role of coding variation in LAS by analyzing variants on the HumanExome BeadChip in a total of 3,127 cases and 9,778 controls from Europe, Australia, and South Asia. We report on a nonsynonymous single-nucleotide variant in serpin family A member 1 (SERPINA1) encoding alpha-1 antitrypsin [AAT; p.V213A; P = 5.99E-9, odds ratio (OR) = 1.22] and confirm histone deacetylase 9 (HDAC9) as a major risk gene for LAS with an association in the 3'-UTR (rs2023938; P = 7.76E-7, OR = 1.28). Using quantitative microscale thermophoresis, we show that M1 (A213) exhibits an almost twofold lower dissociation constant with its primary target human neutrophil elastase (NE) in lipoprotein-containing plasma, but not in lipid-free plasma. Hydrogen/deuterium exchange combined with mass spectrometry further revealed a significant difference in the global flexibility of the two variants. The observed stronger interaction with lipoproteins in plasma and reduced global flexibility of the Val-213 variant most likely improve its local availability and reduce the extent of proteolytic inactivation by other proteases in atherosclerotic plaques. Our results indicate that the interplay between AAT, NE, and lipoprotein particles is modulated by the gate region around position 213 in AAT, far away from the unaltered reactive center loop (357-360). Collectively, our findings point to a functionally relevant balance between lipoproteins, proteases, and AAT in atherosclerosis. AU - Malik, R.* AU - Dau, T. AU - Gonik, M.* AU - Sivakumar, A.* AU - Deredge, D.J.* AU - Edeleva, E.V.* AU - Götzfried, J. AU - van der Laan, S.W.* AU - Pasterkamp, G.* AU - Beaufort, N.* AU - Seixas, S.* AU - Bevan, S.* AU - Lincz, L.F.* AU - Holliday, E.G.* AU - Burgess, A.I.* AU - Rannikmäe, K.* AU - Minnerup, J.* AU - Kriebel, J. AU - Waldenberger, M. AU - Müller-Nurasyid, M. AU - Lichtner, P. AU - Saleheen, D.* AU - Rothwell, P.M.* AU - Levi, C.* AU - Attia, J.* AU - Sudlow, C.L.* AU - Braun, D.* AU - Markus, H.S.* AU - Wintrode, P.L.* AU - Berger, K.* AU - Jenne, D. AU - Dichgans, M.* C1 - 50612 C2 - 42621 SP - 3613-3618 TI - Common coding variant in SERPINA1 increases the risk for large artery stroke. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 14 PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) orchestrates dynamic recruitment of specific cellular machines during different stages of transcription. Signature phosphorylation patterns of Y1S2P3T4S5P6S7 heptapeptide repeats of the CTD engage specific "readers." Whereas phospho-Ser5 and phospho-Ser2 marks are ubiquitous, phospho-Thr4 is reported to only impact specific genes. Here, we identify a role for phospho-Thr4 in transcription termination at noncoding small nucleolar RNA (snoRNA) genes. Quantitative proteomics reveals an interactome of known readers as well as protein complexes that were not known to rely on Thr4 for association with Pol II. The data indicate a key role for Thr4 in engaging the machinery used for transcription elongation and termination. We focus on Rtt103, a protein that binds phospho-Ser2 and phospho-Thr4 marks and facilitates transcription termination at protein-coding genes. To elucidate how Rtt103 engages two distinct CTD modifications that are differentially enriched at noncoding genes, we relied on NMR analysis of Rtt103 in complex with phospho-Thr4- or phospho-Ser2-bearing CTD peptides. The structural data reveal that Rtt103 interacts with phospho-Thr4 in a manner analogous to its interaction with phospho-Ser2-modified CTD. The same set of hydrogen bonds involving either the oxygen on phospho-Thr4 and the hydroxyl on Ser2, or the phosphate on Ser2 and the Thr4 hydroxyl, can be formed by rotation of an arginine side chain, leaving the intermolecular interface otherwise unperturbed. This economy of design enables Rtt103 to engage Pol II at distinct sets of genes with differentially enriched CTD marks. AU - Nemec, C.M.* AU - Yang, F.* AU - Gilmore, J.M.* AU - Hintermair, C. AU - Ho, Y.H.* AU - Tseng, S.C.* AU - Heidemann, M. AU - Zhang, Y.* AU - Florens, L.* AU - Gasch, A.P.* AU - Eick, D. AU - Washburn, M.P.* AU - Varani, G.* AU - Ansari, A.Z.* C1 - 51071 C2 - 43059 CY - Washington SP - E3944-E3953 TI - Different phosphoisoforms of RNA polymerase II engage the Rtt103 termination factor in a structurally analogous manner. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 20 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - The rich diversity and complexity of organic matter found in meteorites is rapidly expanding our knowledge and understanding of extreme environments from which the early solar system emerged and evolved. Here, we report the discovery of a hitherto unknown chemical class, dihydroxymagnesium carboxylates [(OH)2MgO2CR]-, in meteoritic soluble organic matter. High collision energies, which are required for fragmentation, suggest substantial thermal stability of these Mg-metalorganics (CHOMg compounds). This was corroborated by their higher abundance in thermally processed meteorites. CHOMg compounds were found to be present in a set of 61 meteorites of diverse petrological classes. The appearance of this CHOMg chemical class extends the previously investigated, diverse set of CHNOS molecules. A connection between the evolution of organic compounds and minerals is made, as Mg released from minerals gets trapped into organic compounds. These CHOMg metalorganic compounds and their relation to thermal processing in meteorites might shed new light on our understanding of carbon speciation at a molecular level in meteorite parent bodies. AU - Ruf, A. AU - Kanawati, B. AU - Hertkorn, N. AU - Yin, Q.Z.* AU - Moritz, F. AU - Harir, M. AU - Lucio, M. AU - Michalke, B. AU - Wimpenny, J.* AU - Shilobreeva, S.* AU - Bronsky, B.* AU - Saraykin, V.* AU - Gabelica, Z.* AU - Gougeon, R.D.* AU - Quirico, E.* AU - Ralew, S.* AU - Jakubowski, T.* AU - Haack, H.* AU - Gonsior, M.* AU - Jenniskens, P.* AU - Hinman, N.* AU - Schmitt-Kopplin, P. C1 - 50469 C2 - 42383 CY - Washington SP - 2819-2824 TI - Previously unknown class of metalorganic compounds revealed in meteorites. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 11 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - The activation mechanism of the classical transient receptor potential channels TRPC4 and -5 via the G(q/11) protein-phospholipase C (PLC) signaling pathway has remained elusive so far. In contrast to all other TRPC channels, the PLC product diacylglycerol (DAG) is not sufficient for channel activation, whereas TRPC4/5 channel activity is potentiated by phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. As a characteristic structural feature, TRPC4/5 channels contain a C-terminal PDZ-binding motif allowing for binding of the scaffolding proteins Na+/H+ exchanger regulatory factor (NHERF) 1 and 2. PKC inhibition or the exchange of threonine for alanine in the C-terminal PDZ-binding motif conferred DAG sensitivity to the channel. Altogether, we present a DAG-mediated activation mechanism for TRPC4/5 channels tightly regulated by NHERF1/2 interaction. PIP2 depletion evokes a C-terminal conformational change of TRPC5 proteins leading to dynamic dissociation of NHERF1/2 from the C terminus of TRPC5 as a prerequisite for DAG sensitivity. We show that NHERF proteins are direct regulators of ion channel activity and that DAG sensitivity is a distinctive hallmark of TRPC channels. AU - Storch, U.* AU - Forst, A.* AU - Pardatscher, F.* AU - Erdogmus, S.* AU - Philipp, M.* AU - Gregoritza, M.* AU - Mederos y Schnitzlera, M.* AU - Gudermann, T. C1 - 50335 C2 - 42312 CY - Washington SP - E37-E46 TI - Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 1 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondriatargeted opsinswere used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions. AU - Tkatch, T.* AU - Greotti, E.* AU - Baranauskas, G.* AU - Pendin, D.* AU - Roy, S.* AU - Nita, L.I.* AU - Wettmarshausen, J. AU - Prigge, M.* AU - Yizhar, O.* AU - Shirihai, O.S.* AU - Fishman, D.* AU - Hershfinkel, M.* AU - Fleidervish, I.A.* AU - Perocchi, F. AU - Pozzan, T.* AU - Sekler, I.* C1 - 51483 C2 - 43147 CY - Washington SP - E5167-E5176 TI - Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 26 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - The regeneration of organ morphology and function following tissue loss is critical to restore normal physiology, yet few cases are documented in mammalian postnatal life. Partial hepatectomy of the adult mammalian liver activates compensatory hepatocyte hypertrophy and cell division across remaining lobes, resulting in restitution of organ mass but with permanent alteration of architecture. Here, we identify a time window in early postnatal life wherein partial amputation culminates in a localized regeneration instead of global hypertrophy and proliferation. Quantifications of liver mass, enzymatic activity, and immunohistochemistry demonstrate that damaged lobes underwent multilineage regeneration, reforming a lobe often indistinguishable from undamaged ones. Clonal analysis during regeneration reveals local clonal expansions of hepatocyte stem/progenitors at injured sites that are lineage but not fate restricted. Tetrachimeric mice show clonal selection occurs during development with further selections following injury. Surviving progenitors associate mainly with central veins, in a pattern of selection different from that of normal development. These results illuminate a previously unknown program of liver regeneration after acute injury and allow for exploration of latent regenerative programs with potential applications to adult liver regeneration. AU - Tsai, J.M.* AU - Koh, P.W.* AU - Stefanska, A. AU - Xing, L.* AU - Walmsley, G.G.* AU - Poux, N.* AU - Weissman, I.L.* AU - Rinkevich, Y. C1 - 50785 C2 - 42814 SP - 3654-3659 TI - Localized hepatic lobular regeneration by central-vein-associated lineage-restricted progenitors. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 14 PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - IL-22 has been identified as a cancer-promoting cytokine that is secreted by infiltrating immune cells in several cancer models. We hypothesized that IL-22 regulation would occur at the interface between cancer cells and immune cells. Breast and lung cancer cells of murine and human origin induced IL-22 production from memory CD4 + T cells. In the present study, we found that IL-22 production in humans is dependent on activation of the NLRP3 inflammasome with the subsequent release of IL-1β from both myeloid and T cells. IL-1 receptor signaling via the transcription factors AhR and RORγt in T cells was necessary and sufficient for IL-22 production. In these settings, IL-1 induced IL-22 production from a mixed T helper cell population comprised of Th1, Th17, and Th22 cells, which was abrogated by the addition of anakinra. We confirmed these findings in vitro and in vivo in two murine tumor models, in primary human breast and lung cancer cells, and in deposited expression data. Relevant to ongoing clinical trials in breast cancer, we demonstrate here that the IL-1 receptor antagonist anakinra abrogates IL-22 production and reduces tumor growth in a murine breast cancer model. Thus, we describe here a previously unrecognized mechanism by which cancer cells induce IL-22 production from memory CD4 + T cells via activation of the NLRP3 inflammasome and the release of IL-1β to promote tumor growth. These findings may provide the basis for therapeutic interventions that affect IL-22 production by targeting IL-1 activity. AU - Voigt, C. AU - May, P.* AU - Gottschlich, A.* AU - Markota, A.* AU - Wenk, D.* AU - Gerlach, I.* AU - Voigt, S.* AU - Stathopoulos, G.T.* AU - Arendt, K.A.M. AU - Heise, C.* AU - Rataj, F.* AU - Janssen, K.P.* AU - Königshoff, M. AU - Winter, H.* AU - Himsl, I.* AU - Thasler, W.E.* AU - Schnurr, M.* AU - Rothenfußer, S.* AU - Endres, S.* AU - Kobold, S.* C1 - 52560 C2 - 44077 CY - Washington SP - 12994-12999 TI - Cancer cells induce interleukin-22 production from memory CD4+T cells via interleukin-1 to promote tumor growth. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 114 IS - 49 PB - Natl Acad Sciences PY - 2017 SN - 0027-8424 ER - TY - JOUR AB - The identification of tumor subpopulations that adversely affect patient outcomes is essential for a more targeted investigation into how tumors develop detrimental phenotypes, as well as for personalized therapy. Mass spectrometry imaging has demonstrated the ability to uncover molecular intratumor heterogeneity. The challenge has been to conduct an objective analysis of the resulting data to identify those tumor subpopulations that affect patient outcome. Here we introduce spatially mapped t-distributed stochastic neighbor embedding (t-SNE), a nonlinear visualization of the data that is able to better resolve the biomolecular intratumor heterogeneity. In an unbiased manner, t-SNE can uncover tumor subpopulations that are statistically linked to patient survival in gastric cancer and metastasis status in primary tumors of breast cancer. AU - Abdelmoula, W.M.* AU - Balluff, B.* AU - Englert, S. AU - Dijkstra, J.* AU - Reinders, M.J.T.* AU - Walch, A.K. AU - McDonnell, L.A.* AU - Lelieveldt, B.P.F.* C1 - 49870 C2 - 41824 CY - Washington SP - 12244-12249 TI - Data-driven identification of prognostic tumor subpopulations using spatially mapped t-SNE of mass spectrometry imaging data. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 43 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Infection with Epstein-Barr virus (EBV) affects most humans worldwide and persists life-long in the presence of robust virus-specific T-cell responses. In both immunocompromised and some immunocompetent people, EBV causes several cancers and lymphoproliferative diseases. EBV transforms B cells in vitro and encodes at least 44 microRNAs (miRNAs), most of which are expressed in EBV-transformed B cells, but their functions are largely unknown. Recently, we showed that EBV miRNAs inhibit CD4(+) T-cell responses to infected B cells by targeting IL-12, MHC class II, and lysosomal proteases. Here we investigated whether EBV miRNAs also counteract surveillance by CD8(+) T cells. We have found that EBV miRNAs strongly inhibit recognition and killing of infected B cells by EBV-specific CD8(+) T cells through multiple mechanisms. EBV miRNAs directly target the peptide transporter subunit TAP2 and reduce levels of the TAP1 subunit, MHC class I molecules, and EBNA1, a protein expressed in most forms of EBV latency and a target of EBV-specific CD8(+) T cells. Moreover, miRNA-mediated down-regulation of the cytokine IL-12 decreases the recognition of infected cells by EBV-specific CD8(+) T cells. Thus, EBV miRNAs use multiple, distinct pathways, allowing the virus to evade surveillance not only by CD4(+) but also by antiviral CD8(+) T cells. AU - Albanese, M. AU - Tagawa, T. AU - Bouvet, M. AU - Maliqi, L. AU - Lutter, D. AU - Hoser, J.D.S. AU - Hastreiter, M. AU - Hayes, M.* AU - Sugden, B.* AU - Martin, L. AU - Moosmann, A. AU - Hammerschmidt, W. C1 - 49682 C2 - 40875 CY - Washington SP - E6467-E6475 TI - Epstein-Barr virus microRNAs reduce immune surveillance by virus-specific CD8+ T cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 42 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Metabolic syndrome is characterized by insulin resistance, obesity, and dyslipidemia. It is the consequence of an imbalance between caloric intake and energy consumption. Adiponectin protects against metabolic syndrome. Insulin-induced signaling includes activation of PI3 kinase and protein kinase B (PKB)/Akt. PKB/Akt in turn inactivates glycogen synthase kinase (GSK) 3, a major regulator of metabolism. Here, we studied the significance of PI3K-dependent GSK3 inactivation for adiponectin formation in diet-induced metabolic syndrome. Mice expressing PI3K-insensitive GSK3 (gsk3(KI)) and wild-type mice (gsk3(WT)) were fed a high-fat diet. Compared with gsk3(WT) mice, gsk3(KI) mice were protected against the development of metabolic syndrome as evident from a markedly lower weight gain, lower total body and liver fat accumulation, better glucose tolerance, stronger hepatic insulin-dependent PKB/Akt phosphorylation, lower serum insulin, cholesterol, and triglyceride levels, as well as higher energy expenditure. Serum adiponectin concentration and the activity of transcription factor C/EBPα controlling the expression of adiponectin in adipose tissue was significantly higher in gsk3(KI) mice than in gsk3(WT) mice. Treatment with GSK3 inhibitor lithium significantly decreased the serum adiponectin concentration of gsk3(KI) mice and abrogated the difference in C/EBPα activity between the genotypes. Taken together, our data demonstrate that the expression of PI3K-insensitive GSK3 stimulates the production of adiponectin and protects from diet-induced metabolic syndrome. AU - Chen, H.* AU - Fajol, A.* AU - Hoene, M.* AU - Zhang, B.* AU - Schleicher, E. AU - Lin, Y.* AU - Calaminus, C.* AU - Pichler, B.J.* AU - Weigert, C. AU - Häring, H.-U. AU - Lang, F.* AU - Föller, M.* C1 - 48523 C2 - 41141 SP - 5754-5759 TI - PI3K-resistant GSK3 controls adiponectin formation and protects from metabolic syndrome. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 20 PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Treatment for fibrosis represents a critical unmet need, because fibrosis is the leading cause of death in industrialized countries, and there is no effective therapy to counteract the fibrotic process. The development of fibrosis relates to the interplay between vessel injury, immune cell activation, and fibroblast stimulation, which can occur in various tissues. Immunotherapies have provided a breakthrough in the treatment of immune diseases. The glycoprotein OX40-OX40 ligand (OX40L) axis offers the advantage of a targeted approach to costimulatory signals with limited impact on the whole immune response. Using systemic sclerosis (SSc) as a prototypic disease, we report compelling evidence that blockade of OX40L is a promising strategy for the treatment of inflammation-driven fibrosis. OX40L is overexpressed in the fibrotic skin and serum of patients with SSc, particularly in patients with diffuse cutaneous forms. Soluble OX40L was identified as a promising serum biomarker to predict the worsening of lung and skin fibrosis, highlighting the role of this pathway in fibrosis. In vivo, OX40L blockade prevents inflammation-driven skin, lung, and vessel fibrosis and induces the regression of established dermal fibrosis in different complementary mouse models. OX40L exerts potent profibrotic effects by promoting the infiltration of inflammatory cells into lesional tissues and therefore the release of proinflammatory mediators, thereafter leading to fibroblast activation. AU - Elhai, M.* AU - Avouac, J.* AU - Hoffmann-Vold, A.M.* AU - Ruzehaji, N.* AU - Amiar, O.* AU - Ruiz, B.* AU - Brahiti, H.* AU - Ponsoye, M.* AU - Fréchet, M.* AU - Burgevin, A.* AU - Pezet, S.* AU - Sadoine, J.* AU - Guilbert, T.* AU - Nicco, C.* AU - Akiba, H.* AU - Heissmeyer, V. AU - Subramaniam, A.* AU - Resnick, R.* AU - Molberg, O.* AU - Kahan, A.* AU - Chiocchia, G.* AU - Allanore, Y.* C1 - 48813 C2 - 41435 CY - Washington SP - E3901-E3910 TI - OX40L blockade protects against inflammation-driven fibrosis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 27 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Brassinosteroids (BRs) are growth-promoting plant hormones that play a role in abiotic stress responses, but molecular modes that enable this activity remain largely unknown. Here we show that BRs participate in the regulation of freezing tolerance. BR signaling-defective mutants of Arabidopsis thaliana were hypersensitive to freezing before and after cold acclimation. The constitutive activation of BR signaling, in contrast, enhanced freezing resistance. Evidence is provided that the BR-controlled basic helix-loop-helix transcription factor CESTA (CES) can contribute to the constitutive expression of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT BINDING FACTOR (CBF) transcriptional regulators that control cold responsive (COR) gene expression. In addition, CBF-independent classes of BR-regulated COR genes are identified that are regulated in a BR- and CES-dependent manner during cold acclimation. A model is presented in which BRs govern different cold-responsive transcriptional cascades through the posttranslational modification of CES and redundantly acting factors. This contributes to the basal resistance against freezing stress, but also to the further improvement of this resistance through cold acclimation. AU - Eremina, M.* AU - Unterholzner, S.J.* AU - Rathnayake, A.I.* AU - Castellanos, M.* AU - Khan, M.I.* AU - Kugler, K.G. AU - May, S.T.* AU - Mayer, K.F.X. AU - Rozhon, W.* AU - Poppenberger, B.* C1 - 49540 C2 - 40781 CY - Washington SP - E5982-E5991 TI - Brassinosteroids participate in the control of basal and acquired freezing tolerance of plants. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 40 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. AU - Goricanec, D. AU - Stehle, R. AU - Egloff, P.* AU - Grigoriu, S.* AU - Plückthun, A.* AU - Wagner, G.* AU - Hagn, F. C1 - 48814 C2 - 41455 CY - Washington SP - E3629-E3638 TI - Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 26 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD. AU - Guaitoli, G.* AU - Raimondi, F.* AU - Gilsbach, B.K.* AU - Gómez-Llorente, Y.* AU - Deyaert, E.* AU - Renzi, F.* AU - Li, X.* AU - Schaffner, A.* AU - Jagtap, P.K. AU - Boldt, K.* AU - von Zweydorf, F.* AU - Gotthardt, K.* AU - Lorimer, D.D.* AU - Yue, Z.* AU - Burgin, A.* AU - Janjic, N.* AU - Sattler, M. AU - Versées, W.* AU - Ueffing, M.* AU - Ubarretxena-Belandia, I.* AU - Kortholt, A. AU - Gloeckner, C.J.* C1 - 48977 C2 - 41505 CY - Washington SP - E4357-E4366 TI - Structural model of the dimeric Parkinson's protein LRRK2 reveals a compact architecture involving distant interdomain contacts. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 30 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Educational attainment is associated with many health outcomes, including longevity. It is also known to be substantially heritable. Here, we used data from three large genetic epidemiology cohort studies (Generation Scotland, n = ∼17,000; UK Biobank, n = ∼115,000; and the Estonian Biobank, n = ∼6,000) to test whether education-linked genetic variants can predict lifespan length. We did so by using cohort members’ polygenic profile score for education to predict their parents’ longevity. Across the three cohorts, meta-analysis showed that a 1 SD higher polygenic education score was associated with ∼2.7% lower mortality risk for both mothers (total ndeaths = 79,702) and ∼2.4% lower risk for fathers (total ndeaths = 97,630). On average, the parents of offspring in the upper third of the polygenic score distribution lived 0.55 y longer compared with those of offspring in the lower third. Overall, these results indicate that the genetic contributions to educational attainment are useful in the prediction of human longevity. AU - Marioni, R.E.* AU - Ritchie, S.J.* AU - Joshi, P.K.* AU - Hagenaars, S.P.* AU - Okbay, A.* AU - Fischer, K.* AU - Adams, M.* AU - Hill, W.* AU - Davies, G.* AU - Nagy, R.* AU - Amador, C.* AU - Läll, K.* AU - Metspalu, A.* AU - Liewald, D.C.* AU - Campbell, A.* AU - Wilson, J.F.* AU - Hayward, C.* AU - Esko, T.* AU - Porteous, D.J.* AU - Gale, C.R.* AU - Deary, I.J.* AU - EUMODIC Consortium (Baumbach, C. AU - Gieger, C. AU - Meisinger, C. AU - Lichtner, P. AU - Meitinger, T. AU - Strauch, K.) C1 - 50147 C2 - 42036 SP - 13366-13371 TI - Genetic variants linked to education predict longevity. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 47 PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Excessive alcohol consumption is a major public health problem worldwide. Although drinking habits are known to be inherited, few genes have been identified that are robustly linked to alcohol drinking. We conducted a genome-wide association metaanalysis and replication study among > 105,000 individuals of European ancestry and identified β-Klotho (KLB) as a locus associated with alcohol consumption (rs11940694; P = 9.2 × 10-12). β-Klotho is an obligate coreceptor for the hormone FGF21, which is secreted from the liver and implicated in macronutrient preference in humans. We show that brain-specific β-Klotho KO mice have an increased alcohol preference and that FGF21 inhibits alcohol drinking by acting on the brain. These data suggest that a liver-brain endocrine axis may play an important role in the regulation of alcohol drinking behavior and provide a unique pharmacologic target for reducing alcohol consumption. AU - Schumann, G.* AU - Liu, C.* AU - O'Reilly, P.F.* AU - Gao, H.* AU - Song, P.* AU - Xu, B.* AU - Ruggeri, B.* AU - Amin, N.* AU - Jia, T.* AU - Preis, S.R.* AU - Lepe, M.S.* AU - Akira, S.* AU - Barbieri, C.* AU - Baumeister, S.* AU - Cauchi, S.* AU - Clarke, T.K.* AU - Enroth, S.* AU - Fischer, K.* AU - Hällfors, J.* AU - Harris, S.E.* AU - Hieber, S.* AU - Hofer, E.* AU - Hottenga, J.J.* AU - Johansson, A.* AU - Joshi, P.K.* AU - Kaartinen, N.* AU - Laitinen, J.* AU - Lemaitre, R.* AU - Loukola, A.* AU - Luan, J.* AU - Lyytikäinen, L.-P.* AU - Mangino, M.* AU - Manichaikul, A.* AU - Mbarek, H.* AU - Milaneschi, Y.* AU - Moayyeri, A.* AU - Mukamal, K.J.* AU - Nelson, C.P.* AU - Nettleton, J.A.* AU - Partinen, E.* AU - Rawal, R. AU - Robino, A.* AU - Rose, L.* AU - Sala, C.* AU - Satoh, T.* AU - Schmidt, R.* AU - Schraut, K.* AU - Scott, R.* AU - Smith, A.V.* AU - Starr, J.M.* AU - Teumer, A.* AU - Trompet, S.* AU - Uitterlinden, A.G.* AU - Venturini, C.* AU - Gieger, C. AU - Elliott, P.* AU - Vuckovic, D.* AU - Wedenoja, J.* AU - Yengo, L.* AU - Yu, B.* AU - Zhang, W.* AU - Zhao, J.H.* AU - Boomsma, D.I.* AU - Chambers, J.* AU - Chasman, D.I.* AU - Daniela, T.* AU - de Geus, E.* AU - Deary, I.J.* AU - Eriksson, J.G.* AU - Esko, T.* AU - Eulenburg, V.* AU - Huth, C. C1 - 50188 C2 - 42156 CY - Washington SP - 14372-14377 TI - KLB is associated with alcohol drinking, and its gene product β-Klotho is necessary for FGF21 regulation of alcohol preference. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 50 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Aberrant immune activation mediated by T effector cell populations is pivotal in the onset of autoimmunity in type 1 diabetes (T1D). T follicular helper (TFH) cells are essential in the induction of high-affinity antibodies, and their precursor memory compartment circulates in the blood. The role of TFH precursors in the onset of islet autoimmunity and signaling pathways regulating their differentiation is incompletely understood. Here, we provide direct evidence that during onset of islet autoimmunity, the insulin-specific target T-cell population is enriched with a C-X-C chemokine receptor type 5 (CXCR5)(+)CD4(+) TFH precursor phenotype. During onset of islet autoimmunity, the frequency of TFH precursors was controlled by high expression of microRNA92a (miRNA92a). miRNA92a-mediated TFH precursor induction was regulated by phosphatase and tension homolog (PTEN) - phosphoinositol-3-kinase (PI3K) signaling involving PTEN and forkhead box protein O1 (Foxo1), supporting autoantibody generation and triggering the onset of islet autoimmunity. Moreover, we identify Krueppel-like factor 2 (KLF2) as a target of miRNA92a in regulating human TFH precursor induction. Importantly, a miRNA92a antagomir completely blocked induction of human TFH precursors in vitro. More importantly, in vivo application of a miRNA92a antagomir to nonobese diabetic (NOD) mice with ongoing islet autoimmunity resulted in a significant reduction of TFH precursors in peripheral blood and pancreatic lymph nodes. Moreover, miRNA92a antagomir application reduced immune infiltration and activation in pancreata of NOD mice as well as humanized NOD Scid IL2 receptor gamma chain knockout (NSG) human leucocyte antigen (HLA)-DQ8 transgenic animals. We therefore propose that miRNA92a and the PTEN-PI3K-KLF2 signaling network could function as targets for innovative precision medicines to reduce T1D islet autoimmunity. AU - Serr, I. AU - Fürst, R.W. AU - Ott, V.B. AU - Scherm, M.G. AU - Nikolaev, A.* AU - Gökmen, F. AU - Kälin, S. AU - Zillmer, S. AU - Bunk, M. AU - Weigmann, B.* AU - Kunschke, N.* AU - Loretz, B.* AU - Lehr, C.M.* AU - Kirchner, B.* AU - Haase, B.* AU - Pfaffl, M.W.* AU - Waisman, A.* AU - Willis, R.A.* AU - Ziegler, A.G. AU - Daniel, C. C1 - 49847 C2 - 40978 CY - Washington SP - E6659-E6668 TI - miRNA92a targets KLF2 and the phosphatase PTEN signaling to promote human T follicular helper precursors in T1D islet autoimmunity. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 43 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form andwhen bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein.RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants. AU - Von Voithenberg, L.V.* AU - Sanchez-Rico, C. AU - Kang, H.-S. AU - Madl, T. AU - Zanier, K.* AU - Barth, A.S.* AU - Warner, L. AU - Sattler, M. AU - Lamb, D.C.* C1 - 50029 C2 - 41983 CY - Washington SP - E7169-E7175 TI - Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 113 IS - 46 PB - Natl Acad Sciences PY - 2016 SN - 0027-8424 ER - TY - JOUR AB - Glycerides are of interest to the areas of food science and medicine because they are the main component of fat. From a chemical sensing perspective, glycerides are challenging analytes because they are structurally similar to one another and lack diversity in terms of functional groups. Furthermore, because animal and plant fat consists of a number of stereo- and regioisomeric acylglycerols, their components remain challenging analytes for chromatographic and mass spectrometric determination, particularly the quantitation of species in mixtures. In this study, we demonstrated the use of an array of cross-reactive serum albumins and fluorescent indicators with chemometric analysis to differentiate a panel of mono-, di-, and triglycerides. Due to the difficulties in identifying the regio- and stereochemistry of the unsaturated glycerides, a sample pretreatment consisting of olefin cross-metathesis with an allyl fluorescein species was used before array analysis. Using this simple assay, we successfully discriminated 20 glycerides via principal component analysis and linear discriminant analysis (PCA and LDA, respectively), including stereo- and regioisomeric pairs. The resulting chemometric patterns were used as a training space for which the structural characteristics of unknown glycerides were identified. In addition, by using our array to perform a standard addition analysis on a mixture of triglycerides and using a method introduced herein, we demonstrated the ability to quantitate glyceride components in a mixture. AU - Diehl, K.L.* AU - Ivy, M.A.* AU - Rabidoux, S.* AU - Petry, S.M.* AU - Müller, G. AU - Anslyn, E.V.* C1 - 46366 C2 - 37566 CY - Washington SP - E3977-E3986 TI - Differential sensing for the regio- and stereoselective identification and quantitation of glycerides. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 30 PB - Natl Acad Sciences PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Hundreds of organic chemicals are used during natural gas extraction via high-volume hydraulic fracturing (HVHF). However, it is unclear whether these chemicals, injected into deep shale horizons, reach shallow groundwater aquifers and affect local water quality, either from those deep HVHF injection sites or from the surface or shallow subsurface. Here, we report detectable levels of organic compounds in shallow groundwater samples from private residential wells overlying the Marcellus Shale in northeastern Pennsylvania. Analyses of purgeable and extractable organic compounds from 64 groundwater samples revealed trace levels of volatile organic compounds, well below the Environmental Protection Agency's maximum contaminant levels, and low levels of both gasoline range (0-8 ppb) and diesel range organic compounds (DRO; 0-157 ppb). A compound-specific analysis revealed the presence of bis(2-ethylhexyl) phthalate, which is a disclosed HVHF additive, that was notably absent in a representative geogenic water sample and field blanks. Pairing these analyses with (i) inorganic chemical fingerprinting of deep saline groundwater, (ii) characteristic noble gas isotopes, and (iii) spatial relationships between active shale gas extraction wells and wells with disclosed environmental health and safety violations, we differentiate between a chemical signature associated with naturally occurring saline groundwater and one associated with alternative anthropogenic routes from the surface (e.g., accidental spills or leaks). The data support a transport mechanism of DRO to groundwater via accidental release of fracturing fluid chemicals derived from the surface rather than subsurface flow of these fluids from the underlying shale formation. AU - Drollette, B.D.* AU - Hoelzer, K. AU - Warner, N.R.* AU - Darrah, T.H.* AU - Karatum, O.* AU - O'Connor, M.P.* AU - Nelson, R.K.* AU - Fernandez, L.A.* AU - Reddy, C.M.* AU - Vengosh, A.* AU - Jackson, R.B.* AU - Elsner, M. AU - Plata, D.L.* C1 - 47213 C2 - 39200 SP - 13184-13189 TI - Elevated levels of diesel range organic compounds in groundwater near Marcellus gas operations are derived from surface activities. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 43 PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Interleukin 4 (IL-4) can suppress delayed-type hypersensitivity reactions (DTHRs), including organ-specific autoimmune diseases in mice and humans. Despite the broadly documented antiinflammatory effect of IL-4, the underlying mode of action remains incompletely understood, as IL-4 also promotes IL-12 production by dendritic cells (DCs) and IFN-γ-producing TH1 cells in vivo. Studying the impact of IL-4 on the polarization of human and mouse DCs, we found that IL-4 exerts opposing effects on the production of either IL-12 or IL-23. While promoting IL-12-producing capacity of DCs, IL-4 completely abrogates IL-23. Bone marrow chimeras proved that IL-4-mediated suppression of DTHRs relies on the signal transducer and activator of transcription 6 (STAT6)-dependent abrogation of IL-23 in antigen-presenting cells. Moreover, IL-4 therapy attenuated DTHRs by STAT6- and activating transcription factor 3 (ATF3)-dependent suppression of the IL-23/TH17 responses despite simultaneous enhancement of IL-12/TH1 responses. As IL-4 therapy also improves psoriasis in humans and suppresses IL-23/TH17 responses without blocking IL-12/TH1, selective IL-4-mediated IL-23/TH17 silencing is promising as treatment against harmful inflammation, while sparing the IL-12-dependent TH1 responses. AU - Guenova, E.* AU - Skabytska, Y.* AU - Hoetzenecker, W.* AU - Weindl, G.* AU - Sauer, K.* AU - Tham, M.* AU - Kim, K.W.* AU - Park, J.H.* AU - Seo, J.H.* AU - Ignatova, D.* AU - Cozzio, A.* AU - Levesque, M.P.* AU - Volz, T.* AU - Köberle, M.* AU - Kaesler, S.* AU - Thomas, P.* AU - Mailhammer, R. AU - Ghoreschi, K.* AU - Schäkel, K.* AU - Amarov, B.* AU - Eichner, M.* AU - Schaller, M.* AU - Clark, R.A.* AU - Röcken, M.* AU - Biedermann, T.* C1 - 43220 C2 - 36350 CY - Washington SP - 2163-2168 TI - IL-4 abrogates TH17 cell-mediated inflammation by selective silencing of IL-23 in antigen-presenting cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 7 PB - Natl Acad Sciences PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Insulin secretion is key for glucose homeostasis. Insulin secretory granules (SGs) exist in different functional pools, with young SGs being more mobile and preferentially secreted. However, the principles governing the mobility of age-distinct SGs remain undefined. Using the time-reporter insulin-SNAP to track age-distinct SGs we now show that their dynamics can be classified into three components: highly dynamic, restricted, and nearly immobile. Young SGs display all three components, whereas old SGs are either restricted or nearly immobile. Both glucose stimulation and F-actin depolymerization recruit a fraction of nearly immobile young, but not old, SGs for highly dynamic, microtubule-dependent transport. Moreover, F-actin marks multigranular bodies/lysosomes containing aged SGs. These data demonstrate that SGs lose their responsiveness to glucose stimulation and competence for microtubule-mediated transport over time while changing their relationship with F-actin. AU - Hoboth, P. AU - Müller, A. AU - Ivanova, A. AU - Mziaut, H. AU - Dehghany, J.* AU - Sönmez, A. AU - Lachnit, M. AU - Meyer-Hermann, M.* AU - Kalaidzidis, Y.* AU - Solimena, M. C1 - 43221 C2 - 36351 CY - Washington SP - E667-E676 TI - Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 7 PB - Natl Acad Sciences PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Archaeochemistry as the application of the most recent analytical techniques to ancient samples now provides an unprecedented understanding of human culture throughout history. In this paper, we report on a multiplatform analytical investigation of 170-y-old champagne bottles found in a shipwreck at the bottom of the Baltic Sea, which provides insight into winemaking practices used at the time. Organic spectroscopy-based nontargeted metabolomics and metallomics give access to the detailed composition of these wines, revealing, for instance, unexpected chemical characteristics in terms of small ion, sugar, and acid contents as well as markers of barrel aging and Maillard reaction products. The distinct aroma composition of these ancient champagne samples, first revealed during tasting sessions, was later confirmed using state-of-the-art aroma analysis techniques. After 170 y of deep sea aging in close-to-perfect conditions, these sleeping champagne bottles awoke to tell us a chapter of the story of winemaking and to reveal their extraordinary archaeometabolome and elemental diversity in the form of chemical signatures related to each individual step of champagne production. AU - Jeandet, P.* AU - Heinzmann, S.S. AU - Roullier-Gall, C. AU - Cilindre, C.* AU - Aron, A.* AU - Deville, M.A.* AU - Moritz, F. AU - Karbowiak, T.* AU - Demarville, D.* AU - Brun, C.* AU - Moreau, F.* AU - Michalke, B. AU - Liger-Belair, G.* AU - Witting, M. AU - Lucio, M. AU - Steyer, D.* AU - Gougeon, R.D.* AU - Schmitt-Kopplin, P. C1 - 44457 C2 - 36868 CY - Washington SP - 5893-5898 TI - Chemical messages in 170-year-old champagne bottles from the Baltic Sea: Revealing tastes from the past. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 19 PB - Natl Acad Sciences PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - The epidermal growth factor receptor (EGFR) regulates several critical cellular processes and is an important target for cancer therapy. In lieu of a crystallographic structure of the complete receptor, atomistic molecular dynamics (MD) simulations have recently shown that they can excel in studies of the full-length receptor. Here we present atomistic MD simulations of the monomeric N-glycosylated human EGFR in biomimetic lipid bilayers that are, in parallel, also used for the reconstitution of full-length receptors. This combination enabled us to experimentally validate our simulations, using ligand binding assays and antibodies to monitor the conformational properties of the receptor reconstituted into membranes. We find that N-glycosylation is a critical determinant of EGFR conformation, and specifically the orientation of the EGFR ectodomain relative to the membrane. In the absence of a structure for full-length, posttranslationally modified membrane receptors, our approach offers new means to structurally define and experimentally validate functional properties of cell surface receptors in biomimetic membrane environments. AU - Kaszuba, K.* AU - Grzybek, M. AU - Orłowski, A.* AU - Danne, R.* AU - Róg, T.* AU - Simons, K.* AU - Coskun, Ü. AU - Vattulainen, I.* C1 - 43976 C2 - 36702 CY - Washington SP - 4334–4339 TI - N-Glycosylation as determinant of epidermal growth factor receptor conformation in membranes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 14 PB - Natl Acad Sciences PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Here, we show CRISPR/Cas9-based targeted somatic multiplex-mutagenesis and its application for high-throughput analysis of gene function in mice. Using hepatic single guide RNA (sgRNA) delivery, we targeted large gene sets to induce hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). We observed Darwinian selection of target genes, which suppress tumorigenesis in the respective cellular/tissue context, such as Pten or Cdkn2a, and conversely found low frequency of Brca1/2 alterations, explaining mutational spectra in human ICC/HCC. Our studies show that multiplexed CRISPR/Cas9 can be used for recessive genetic screening or high-throughput cancer gene validation in mice. The analysis of CRISPR/Cas9-induced tumors provided support for a major role of chromatin modifiers in hepatobiliary tumorigenesis, including that of ARID family proteins, which have recently been reported to be mutated in ICC/HCC. We have also comprehensively characterized the frequency and size of chromosomal alterations induced by combinatorial sgRNA delivery and describe related limitations of CRISPR/Cas9 multiplexing, as well as opportunities for chromosome engineering in the context of hepatobiliary tumorigenesis. Our study describes novel approaches to model and study cancer in a high-throughput multiplexed format that will facilitate the functional annotation of cancer genomes. AU - Weber, J.* AU - Öllinger, R.* AU - Friedrich, M.* AU - Ehmer, U.* AU - Barenboim, M.* AU - Steiger, K.* AU - Heid, I.M.* AU - Mueller, S.* AU - Maresch, R.* AU - Engleitner, T.* AU - Gross, N.* AU - Geumann, U.* AU - Fu, B.* AU - Segler, A.* AU - Yuan, D.T. AU - Lange, S.* AU - Strong, A.* AU - de la Rosa, J.* AU - Esposito, I.* AU - Liu, P.* AU - Cadiñanos, J.* AU - Vassiliou, G.S.* AU - Schmid, R.M.* AU - Schneider, G.* AU - Unger, K. AU - Yang, F.* AU - Braren, R.* AU - Heikenwälder, M. AU - Varela, I.* AU - Saur, D.* AU - Bradley, A.* AU - Rad, R.* C1 - 47232 C2 - 39352 SP - 13982-13987 TI - CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 112 IS - 45 PY - 2015 SN - 0027-8424 ER - TY - JOUR AB - Cell-to-cell variations in gene regulation occur in a number of biological contexts, such as development and cancer. Discovering regulatory heterogeneities in an unbiased manner is difficult owing to the population averaging that is required for most global molecular methods. Here, we show that we can infer single-cell regulatory states by mathematically deconvolving global measurements taken as averages from small groups of cells. This averaging-and-deconvolution approach allows us to quantify single-cell regulatory heterogeneities while avoiding the measurement noise of global single-cell techniques. Our method is particularly relevant to solid tissues, where single-cell dissociation and molecular profiling is especially problematic. AU - Bajikar, S.S.* AU - Fuchs, C. AU - Roller, A. AU - Theis, F.J. AU - Janes, K.A.* C1 - 29043 C2 - 33604 CY - Washington SP - E626-E635 TI - Parameterizing cell-to-cell regulatory heterogeneities via stochastic transcriptional profiles. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 5 PB - Natl Acad Sciences PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis. AU - Beaufort, N.* AU - Scharrer, E.* AU - Kremmer, E. AU - Lux, V.* AU - Ehrmann, M.* AU - Huber, R.* AU - Houlden, H.* AU - Werring, D.* AU - Haffner, C.* AU - Dichgans, M.* C1 - 34368 C2 - 35237 SP - 16496-16501 TI - Cerebral small vessel disease-related protease HtrA1 processes latent TGF-β binding protein 1 and facilitates TGF-β signaling. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 46 PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein–protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G–associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy–lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms. AU - Beilina, A.* AU - Rudenko, I.N.* AU - Kaganovich, A.* AU - Civiero, L.* AU - Chau, H.* AU - Kalia, S.K.* AU - Kalia, L.V.* AU - Lobbestael, E.* AU - Chia, R.* AU - Ndukwe, K.* AU - Ding, J.* AU - Nalls, M.A.* AU - EUMODIC Consortium (Illig, T. AU - Lichtner, P. AU - *) AU - Olszewski, M.* AU - Hauser, D.N.* AU - Kumaran, R.* AU - Lozano, A.M.* AU - Baekelandt, V.* AU - Greene, L.E.* AU - Taymans, J.-M.* AU - Greggio, E.* AU - Cookson, M.R.* C1 - 30836 C2 - 33940 SP - 2626-2631 TI - Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 7 PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules. AU - Feige, M.J.* AU - Gräwert, M.A.* AU - Marcinowski, M.* AU - Hennig, J. AU - Behnke, J.* AU - Ausländer, D.* AU - Herold, E.M.* AU - Peschek, J.* AU - Castro, C.D.* AU - Flajnik, M.* AU - Hendershot, L.M.* AU - Sattler, M. AU - Groll, M.* AU - Buchner, J.* C1 - 31294 C2 - 34319 CY - Washington SP - 8155-8160 TI - The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 22 PB - Natl Acad Sciences PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - The mechanisms by which gene expression is regulated by oxygen are of considerable interest from basic science and therapeutic perspectives. Using mass spectrometric analyses of Saccharomyces cerevisiae ribosomes, we found that the amino acid residue in closest proximity to the decoding center, Pro-64 of the 40S subunit ribosomal protein Rps23p (RPS23 Pro-62 in humans) undergoes posttranslational hydroxylation. We identify RPS23 hydroxylases as a highly conserved eukaryotic subfamily of Fe(II) and 2-oxoglutarate dependent oxygenases; their catalytic domain is closely related to transcription factor prolyl trans-4-hydroxylases that act as oxygen sensors in the hypoxic response in animals. The RPS23 hydroxylases in S. cerevisiae (Tpa1p), Schizosaccharomyces pombe and green algae catalyze an unprecedented dihydroxylation modification. This observation contrasts with higher eukaryotes, where RPS23 is monohydroxylated; the human Tpa1p homolog OGFOD1 catalyzes prolyl trans-3-hydroxylation. TPA1 deletion modulates termination efficiency up to ∼10-fold, including of pathophysiologically relevant sequences; we reveal Rps23p hydroxylation as its molecular basis. In contrast to most previously characterized accuracy modulators, including antibiotics and the prion state of the S. cerevisiae translation termination factor eRF3, Rps23p hydroxylation can either increase or decrease translational accuracy in a stop codon context-dependent manner. We identify conditions where Rps23p hydroxylation status determines viability as a consequence of nonsense codon suppression. The results reveal a direct link between oxygenase catalysis and the regulation of gene expression at the translational level. They will also aid in the development of small molecules altering translational accuracy for the treatment of genetic diseases linked to nonsense mutations. AU - Loenarz, C.* AU - Sekirnik, R.* AU - Thalhammer, A.* AU - Ge, W.* AU - Spivakovsky, E.* AU - Mackeen, M.M.* AU - McDonough, M.A.* AU - Cockman, M.E.* AU - Kessler, B.M.* AU - Ratcliffe, P.J.* AU - Wolf, A. AU - Schofield, C.J.* C1 - 30810 C2 - 33886 SP - 4019-4024 TI - Hydroxylation of the eukaryotic ribosomal decoding center affects translational accuracy. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 11 PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - Peroxisome proliferator-activated receptor gamma (PPARG) is a master transcriptional regulator of adipocyte differentiation and a canonical target of antidiabetic thiazolidinedione medications. In rare families, loss-of-function (LOF) mutations in PPARG are known to cosegregate with lipodystrophy and insulin resistance; in the general population, the common P12A variant is associated with a decreased risk of type 2 diabetes (T2D). Whether and how rare variants in PPARG and defects in adipocyte differentiation influence risk of T2D in the general population remains undetermined. By sequencing PPARG in 19,752 T2D cases and controls drawn from multiple studies and ethnic groups, we identified 49 previously unidentified, nonsynonymous PPARG variants (MAF < 0.5%). Considered in aggregate (with or without computational prediction of functional consequence), these rare variants showed no association with T2D (OR = 1.35; P = 0.17). The function of the 49 variants was experimentally tested in a novel high-throughput human adipocyte differentiation assay, and nine were found to have reduced activity in the assay. Carrying any of these nine LOF variants was associated with a substantial increase in risk of T2D (OR = 7.22; P = 0.005). The combination of large-scale DNA sequencing and functional testing in the laboratory reveals that approximately 1 in 1,000 individuals carries a variant in PPARG that reduces function in a human adipocyte differentiation assay and is associated with a substantial risk of T2D. AU - Majithia, A.R.* AU - Flannick, J.* AU - Shahinian, P.* AU - Guo, M.* AU - Bray, M.A.* AU - Fontanillas, P.* AU - Gabriel, S.B.* AU - GoT2D Consortium (Gieger, C. AU - Hrabě de Angelis, M. AU - Huth, C. AU - Kriebel, J. AU - Meisinger, C. AU - Meitinger, T. AU - Peters, A. AU - Rathmann, W. AU - Ried, J.S. AU - Strauch, K. AU - Strom, T.M.) AU - NHGRI JHS/FHS Allelic Spectrum Project (*) AU - SIGMA T2D Consortium (*) AU - T2D-GENES Consortium (*) AU - Rosen, E.D.* AU - Altshuler, D.* C1 - 32079 C2 - 34952 SP - 13127-13132 TI - Rare variants in PPARG with decreased activity in adipocyte differentiation are associated with increased risk of type 2 diabetes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 36 PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - There has been accumulating evidence for a regionalized organization of the cerebellum, which was mostly deduced from anatomical mapping of axonal projections of cerebellar afferents. A likewise regionalization of the cerebellar output has been suggested from lesion studies and dye-tracer experiments, but its physiological targets as well as the functional relevance of such an output regionalization are less clear. Ideally, such functional regionalization should be proven noninvasively in vivo. We here provide evidence for such a regionalization of the output from the cerebellar cortex by genetically encoded transneuronal mapping of efferent circuits of zebrafish Purkinje neurons. These identified circuits correspond to distinct regionalized Purkinje cell activity patterns in freely behaving zebrafish larvae during the performance of cerebellar-dependent behaviors. Furthermore, optogenetic interrogation of selected Purkinje cell regions during animal behavior confirms the functional regionalization of Purkinje cell efferents and reveals their contribution to behavior control as well as their function in controlling lateralized behavioral output. Our findings reveal how brain compartments serve to fulfill a multitude of functions by dedicating specialized efferent circuits to distinct behavioral tasks. AU - Matsui, H.* AU - Namikawa, K. AU - Babaryka, A. AU - Köster, R.W.* C1 - 31743 C2 - 34701 CY - Washington SP - 11846-11851 TI - Functional regionalization of the teleost cerebellum analyzed in vivo. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 32 PB - Natl Acad Sciences PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein implicated in Parkinson disease (PD); however, the molecular mechanism and mode of action of this protein remain elusive. cAMP-dependent protein kinase (PKA), along with other kinases, has been suggested to be an upstream kinase regulating LRRK2 function. Using MS, we detected several sites phosphorylated by PKA, including phosphorylation sites within the Ras of complex proteins (ROC) GTPase domain as well as some previously described sites (S910 and S935). We systematically mapped those sites within LRRK2 and investigated their functional consequences. S1444 in the ROC domain was confirmed as a target for PKA phosphorylation using ROC single-domain constructs and through site-directed mutagenesis. Phosphorylation at S1444 is strikingly reduced in the major PD-related LRRK2 mutations R1441C/G/H, which are part of a consensus PKA recognition site ((1441)RASpS(1444)). Furthermore, our work establishes S1444 as a PKA-regulated 14-3-3 docking site. Experiments of direct binding to the three 14-3-3 isotypes gamma, theta, and zeta with phosphopeptides encompassing pS910, pS935, or pS1444 demonstrated the highest affinities to phospho-S1444. Strikingly, 14-3-3 binding to phospho-S1444 decreased LRRK2 kinase activity in vitro. Moreover, substitution of S1444 by alanine or by introducing the mutations R1441C/G/H, abrogating PKA phosphorylation and 14-3-3 binding, resulted in increased LRRK2 kinase activity. In conclusion, these data clearly demonstrate that LRRK2 kinase activity is modulated by PKA-mediated binding of 14-3-3 to S1444 and suggest that 14-3-3 interaction with LRRK2 is hampered in R1441C/G/H-mediated PD pathogenesis. AU - Muda, K.* AU - Bertinetti, D.* AU - Gesellchen, F.* AU - Hermann, J.S.* AU - von Zweydorf, F.* AU - Geerlof, A. AU - Jacob, A.* AU - Ueffing, M. AU - Gloeckner, C.J. AU - Herberg, F.W.* C1 - 28984 C2 - 33598 SP - E34-E43 TI - Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 1 PB - Natl Acad Sciences PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - GF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which-together with patch-clamp and flow cytometry studies-identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs. AU - Peters, D.M.* AU - Vadász, I.* AU - Wujak, L.* AU - Wygrecka, M.* AU - Olschewski, A.* AU - Becker, C.* AU - Herold, S.* AU - Papp, R.* AU - Mayer, K.* AU - Rummel, S.* AU - Brandes, R.P.* AU - Günther, A.* AU - Waldegger, S.* AU - Eickelberg, O. AU - Seeger, W.* AU - Morty, R.E.* C1 - 29301 C2 - 33643 SP - E374-E383 TI - TGF-β directs trafficking of the epithelial sodium channel ENaC which has implications for ion and fluid transport in acute lung injury. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 3 PB - Natl Acad Sciences PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - 2-Oxoglutarate (2OG) and Fe(II)-dependent oxygenase domain-containing protein 1 (OGFOD1) is predicted to be a conserved 2OG oxygenase, the catalytic domain of which is related to hypoxia-inducible factor prolyl hydroxylases. OGFOD1 homologs in yeast are implicated in diverse cellular functions ranging from oxygen-dependent regulation of sterol response genes (Ofd1, Schizosaccharomyces pombe) to translation termination/mRNA polyadenylation (Tpa1p, Saccharomyces cerevisiae). However, neither the biochemical activity of OGFOD1 nor the identity of its substrate has been defined. Here we show that OGFOD1 is a prolyl hydroxylase that catalyzes the posttranslational hydroxylation of a highly conserved residue (Pro-62) in the small ribosomal protein S23 (RPS23). Unusually OGFOD1 retained a high affinity for, and forms a stable complex with, the hydroxylated RPS23 substrate. Knockdown or inactivation of OGFOD1 caused a cell type-dependent induction of stress granules, translational arrest, and growth impairment in a manner complemented by wild-type but not inactive OGFOD1. The work identifies a human prolyl hydroxylase with a role in translational regulation. AU - Singleton, R.S.* AU - Liu-Yi, P.* AU - Formenti, F.* AU - Ge, W.* AU - Sekirnik, R.* AU - Fischer, R.* AU - Adam, J.* AU - Pollard, P.J.* AU - Wolf, A. AU - Thalhammer, A.* AU - Loenarz, C.* AU - Flashman, E.* AU - Yamamoto, A.* AU - Coleman, M.L.* AU - Kessler, B.M.* AU - Wappner, P.* AU - Schofield, C.J.* AU - Ratcliffe, P.J.* AU - Cockman, M.E.* C1 - 30811 C2 - 33887 SP - 4031-4036 TI - OGFOD1 catalyzes prolyl hydroxylation of RPS23 and is involved in translation control and stress granule formation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 111 IS - 11 PY - 2014 SN - 0027-8424 ER - TY - JOUR AB - Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR. AU - Collin, R.W.* AU - Nikopoulos, K.* AU - Dona, M.* AU - Gilissen, C.* AU - Hoischen, A.* AU - Boonstra, F.N.* AU - Poulter, J.A.* AU - Kondo, H.* AU - Berger, W.* AU - Toomes, C.* AU - Tahira, T.* AU - Mohn, L.R.* AU - Blokland, E.A.* AU - Hetterschijt, L.* AU - Ali, M.* AU - Groothuismink, J.M.* AU - Duijkers, L.* AU - Inglehearn, C.F.* AU - Sollfrank, L.* AU - Strom, T.M. AU - Uchio, E.* AU - van Nouhuys, C.E.* AU - Kremer, H.* AU - Veltman, J.A.* AU - van Wijk, E.* AU - Cremers, F.P.* C1 - 26104 C2 - 32071 SP - 9856-9861 TI - ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 24 PB - Natl. Acad. Sci. PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-beta receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-beta receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling. AU - Dagnell, M.* AU - Frijhoff, J.* AU - Pader, I.* AU - Augsten, M.* AU - Boivin, B.* AU - Xu, J.Q.* AU - Mandal, P.K.* AU - Tonks, N.K.* AU - Hellberg, C.* AU - Conrad, M. AU - Arner, E.S.J.* AU - Östman, A.* C1 - 27189 C2 - 32577 SP - 13398-13403 TI - Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-β receptor tyrosine kinase signaling. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 33 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Identifying the connectome of adult-generated neurons is essential for understanding how the preexisting circuitry is refined by neurogenesis. Changes in the pattern of connectivity are likely to control the differentiation process of newly generated neurons and exert an important influence on their unique capacity to contribute to information processing. Using a monosynaptic rabies virus-based tracing technique, we studied the evolving presynaptic connectivity of adult-generated neurons in the dentate gyrus (DG) of the hippocampus and olfactory bulb (OB) during the first weeks of their life. In both neurogenic zones, adult-generated neurons first receive local connections from multiple types of GABAergic interneurons before long-range projections become established, such as those originating from cortical areas. Interestingly, despite fundamental similarities in the overall pattern of evolution of presynaptic connectivity, there were notable differences with regard to the development of cortical projections: although DG granule neuron input originating from the entorhinal cortex could be traced starting only from 3 to 5 wk on, newly generated neurons in the OB received input from the anterior olfactory nucleus and piriform cortex already by the second week. This early glutamatergic input onto newly generated interneurons in the OB was matched in time by the equally early innervations of DG granule neurons by glutamatergic mossy cells. The development of connectivity revealed by our study may suggest common principles for incorporating newly generated neurons into a preexisting circuit. AU - Deshpande, A.* AU - Bergami, M.* AU - Ghanem, A.* AU - Conzelmann, K.-H.* AU - Lepier, A.* AU - Götz, M. AU - Berninger, B.* C1 - 23249 C2 - 31017 SP - 1152-1161 TI - Retrograde monosynaptic tracing reveals the temporal evolution of inputs onto new neurons in the adult dentate gyrus and olfactory bulb. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 12 PB - National Academy of Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - The current limitations in genome sequencing technology require the construction of physical maps for high-quality draft sequences of large plant genomes, such as that of Aegilops tauschii, the wheat D-genome progenitor. To construct a physical map of the Ae. tauschii genome, we fingerprinted 461,706 bacterial artificial chromosome clones, assembled contigs, designed a 10K Ae. tauschii Infinium SNP array, constructed a 7,185-marker genetic map, and anchored on the map contigs totaling 4.03 Gb. Using whole genome shotgun reads, we extended the SNP marker sequences and found 17,093 genes and gene fragments. We showed that collinearity of the Ae. tauschii genes with Brachypodium distachyon, rice, and sorghum decreased with phylogenetic distance and that structural genome evolution rates have been high across all investigated lineages in subfamily Pooideae, including that of Brachypodieae. We obtained additional information about the evolution of the seven Triticeae chromosomes from 12 ancestral chromosomes and uncovered a pattern of centromere inactivation accompanying nested chromosome insertions in grasses. We showed that the density of noncollinear genes along the Ae. tauschii chromosomes positively correlates with recombination rates, suggested a cause, and showed that new genes, exemplified by disease resistance genes, are preferentially located in high-recombination chromosome regions. AU - Luo, M.C.* AU - Gu, Y.Q.* AU - You, F.M.* AU - Deal, K.R.* AU - Ma, Y.* AU - Hu, Y.* AU - Huo, N.* AU - Wang, Y.* AU - Wang, J.* AU - Chen, S.* AU - Jorgensen, C.M.* AU - Zhang, Y.* AU - McGuire, P.E.* AU - Pasternak, S.* AU - Stein, J.C.* AU - Ware, D.* AU - Kramer, M.* AU - McCombie, W.R.* AU - Kianian, S.F.* AU - Martis, M.M. AU - Mayer, K.F.X. AU - Sehgal, S.K.* AU - Li, W.* AU - Gill, B.S.* AU - Bevan, M.W.* AU - Šimková, H.* AU - Dolezel, J.* AU - Weining, S.* AU - Lazo, G.R.* AU - Anderson, O.D.* AU - Dvorak, J.* C1 - 24141 C2 - 31333 SP - 7940-7945 TI - A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 19 PB - National Academy of Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - IgM is the first antibody produced during the humoral immune response. Despite its fundamental role in the immune system, IgM is structurally only poorly described. In this work we used X-ray crystallography and NMR spectroscopy to determine the atomic structures of the constant IgM Fc domains (Cµ2, Cµ3, and Cµ4) and to address their roles in IgM oligomerization. Although the isolated domains share the typical Ig fold, they differ substantially in dimerization properties and quaternary contacts. Unexpectedly, the Cµ4 domain and its C-terminal tail piece are responsible and sufficient for the specific polymerization of Cµ4 dimers into covalently linked hexamers of dimers. Based on small angle X-ray scattering data, we present a model of the ring-shaped Cµ4 structure, which reveals the principles of IgM oligomerization. AU - Müller, R.* AU - Gräwert, M.A.* AU - Kern, T. AU - Madl, T. AU - Peschek, J.* AU - Sattler, M. AU - Groll, M.* AU - Buchner, J.* C1 - 26204 C2 - 32121 SP - 10183-10188 TI - High-resolution structures of the IgM Fc domains reveal principles of its hexamer formation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 25 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Directional transport of mRNA is a universal feature in eukaryotes, requiring the assembly of motor-dependent RNA-transport particles. The cytoplasmic transport of mRNAs is preceded by the nuclear assembly of pre-messenger ribonucleoprotein particles (mRNPs). In budding yeast, the asymmetric synthesis of HO 1 (ASH1) pre-mRNP originates already cotranscriptionally and passes through the nucleolus before its nuclear export. The nucleolar localization of ASH1 mRNA protein 1 (Loc1p) is required for efficient ASH1 mRNA localization. Immunoprecipitation experiments have revealed that Loc1p forms cocomplexes with other components of the ASH1 transport complex. However, it remains unclear how Loc1p is recruited into this mRNP and why Loc1p is important for ASH1 mRNA localization. Here we demonstrate that Loc1p undergoes a direct and specific interaction with the ASH1 mRNA-binding Swi5p-dependent HO expression protein 2 (She2p). This cocomplex shows higher affinity and specificity for RNA bearing localization elements than the individual proteins. It also stabilizes the otherwise transient binding of She2p to ASH1 mRNA, suggesting that cooperative mRNA binding of Loc1p with She2p is the required nuclear function of Loc1p for ASH1 mRNA localization. After nuclear export, myosin-bound She3p joins the ASH1 mRNP to form a highly specific cocomplex with She2p and ASH1 mRNA. Because Loc1p is found only in the nucleus, it must be removed from the complex directly before or after export. In vitro and in vivo experiments indicate that the synergistic interaction of She2p and She3p displaces Loc1p from the ASH1 complex, allowing free Loc1p to rapidly reenter the nucle(ol)us. Together these findings suggest an ordered process of nuclear assembly and reorganization for the maturation of localizing ASH1 mRNPs. AU - Niedner, A. AU - Müller, M. AU - Moorthy, B.T.* AU - Jansen, R.-P.* AU - Niessing, D. C1 - 28727 C2 - 33532 SP - E5049–E5058 TI - Role of Loc1p in assembly and reorganization of nuclear ASH1 messenger ribonucleoprotein particles in yeast. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 52 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa. AU - Pfaff, J. AU - Hennig, J. AU - Herzog, F.* AU - Aebersold, R.* AU - Sattler, M. AU - Niessing, D. AU - Meister, G.* C1 - 27266 C2 - 32593 SP - 3770-3779 TI - Structural features of Argonaute–GW182 protein interactions. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 40 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Multinucleated Reed-Sternberg (RS) cells are pathognomonic for classical Hodgkin lymphoma (HL), and their presence is essential for diagnosis. How these giant tumor cells develop is controversial, however. It has been postulated that RS cells arise from mononucleated Hodgkin cells via endomitosis. Conversely, continuous single-cell tracking of HL cell lines by long-term time-lapse microscopy has identified cell fusion as the main route of RS cell formation. In contrast to growth-induced formation of giant Hodgkin cells, fusion of small mononuclear cells followed by a size increase gives rise to giant RS cells. Of note, fusion of cells originating from the same ancestor, termed re-fusion, is seen nearly exclusively. In the majority of cases, re-fusion of daughter cells is preceded by incomplete cytokinesis, as demonstrated by microtubule bonds among the cells. We confirm at the level of individual tracked cells that giant Hodgkin and RS cells have little proliferative capacity, further supporting small mononuclear Hodgkin cells as the proliferative compartment of the HL tumor clone. In addition, sister cells show a shared propensity for re-fusion, providing evidence of early RS cell fate commitment. Thus, RS cell generation is related neither to cell fusion of unrelated Hodgkin cells nor to endomitosis, but rather is mediated by re-fusion of daughter cells that underwent mitosis. This surprising finding supports the existence of a unique mechanism for the generation of multinuclear RS cells that may have implications beyond HL, given that RS-like cells are frequently observed in several other lymphoproliferative diseases as well. AU - Rengstl, B.* AU - Newrzela, S.* AU - Heinrich, T.* AU - Weiser, C.* AU - Thalheimer, F.B.* AU - Schmid, F.* AU - Warner, K.* AU - Hartmann, S.* AU - Schroeder, T. AU - Küppers, R.* AU - Rieger, M.A.* AU - Hansmann, M.L.* C1 - 28739 C2 - 33534 SP - 20729-20734 TI - Incomplete cytokinesis and re-fusion of small mononucleated Hodgkin cells lead to giant multinucleated Reed-Sternberg cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 51 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Mutations in the Tar DNA binding protein of 43 kDa (TDP-43; TARDBP) are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43(+) inclusions (FTLD-TDP). To determine the physiological function of TDP-43, we knocked out zebrafish Tardbp and its paralogue Tardbp (TAR DNA binding protein-like), which lacks the glycine-rich domain where ALS- and FTLD-TDP-associated mutations cluster. tardbp mutants show no phenotype, a result of compensation by a unique splice variant of tardbpl that additionally contains a C-terminal elongation highly homologous to the glycine-rich domain of tardbp. Double-homozygous mutants of tardbp and tardbpl show muscle degeneration, strongly reduced blood circulation, mispatterning of vessels, impaired spinal motor neuron axon outgrowth, and early death. In double mutants the muscle-specific actin binding protein Filamin Ca is up-regulated. Strikingly, Filamin C is similarly increased in the frontal cortex of FTLD-TDP patients, suggesting aberrant expression in smooth muscle cells and TDP-43 loss-of-function as one underlying disease mechanism. AU - Schmid, B.* AU - Hruscha, A.* AU - Högl, S.* AU - Banzhaf-Strathmann, J.* AU - Strecker, K.* AU - van der Zee, J.* AU - Teucke, M.* AU - Eimer, S.* AU - Hegermann, J.* AU - Kittelmann, M.* AU - Kremmer, E. AU - Cruts, M.* AU - Solchenberger, B.* AU - Hasenkamp, L.* AU - van Bebber, F.* AU - van Broeckhoven, C.* AU - Edbauer, D.* AU - Lichtenthaler, S.F.* AU - Haass, C.* C1 - 24691 C2 - 31635 SP - 4986-4991 TI - Loss of ALS-associated TDP-43 in zebrafish causes muscle degeneration, vascular dysfunction, and reduced motor neuron axon outgrowth. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 13 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Alveolar fluid clearance driven by active epithelial Na(+) and secondary Cl(-) absorption counteracts edema formation in the intact lung. Recently, we showed that impairment of alveolar fluid clearance because of inhibition of epithelial Na(+) channels (ENaCs) promotes cardiogenic lung edema. Concomitantly, we observed a reversal of alveolar fluid clearance, suggesting that reversed transepithelial ion transport may promote lung edema by driving active alveolar fluid secretion. We, therefore, hypothesized that alveolar ion and fluid secretion may constitute a pathomechanism in lung edema and aimed to identify underlying molecular pathways. In isolated perfused lungs, alveolar fluid clearance and secretion were determined by a double-indicator dilution technique. Transepithelial Cl(-) secretion and alveolar Cl(-) influx were quantified by radionuclide tracing and alveolar Cl(-) imaging, respectively. Elevated hydrostatic pressure induced ouabain-sensitive alveolar fluid secretion that coincided with transepithelial Cl(-) secretion and alveolar Cl(-) influx. Inhibition of either cystic fibrosis transmembrane conductance regulator (CFTR) or Na(+)-K(+)-Cl(-) cotransporters (NKCC) blocked alveolar fluid secretion, and lungs of CFTR(-/-) mice were protected from hydrostatic edema. Inhibition of ENaC by amiloride reproduced alveolar fluid and Cl(-) secretion that were again CFTR-, NKCC-, and Na(+)-K(+)-ATPase-dependent. Our findings show a reversal of transepithelial Cl(-) and fluid flux from absorptive to secretory mode at hydrostatic stress. Alveolar Cl(-) and fluid secretion are triggered by ENaC inhibition and mediated by NKCC and CFTR. Our results characterize an innovative mechanism of cardiogenic edema formation and identify NKCC1 as a unique therapeutic target in cardiogenic lung edema. AU - Solymosi, E.A.* AU - Kaestle-Gembardt, S.M.* AU - Vadász, I.* AU - Wang, L.* AU - Neye, N.* AU - Chupin, C.J.* AU - Rozowsky, S.* AU - Ruehl, R.* AU - Tabuchi, A.* AU - Schulz, H. AU - Kapus, A.* AU - Morty, R.E.* AU - Kuebler, W.M.* C1 - 26244 C2 - 32141 SP - E2308-E2316 TI - Chloride transport-driven alveolar fluid secretion is a major contributor to cardiogenic lung edema. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 25 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - We reported earlier the delivery of antiangiogenic single chain antibodies by using oncolytic vaccinia virus strains to enhance their therapeutic efficacy. Here, we provide evidence that gene-evoked production of melanin can be used as a therapeutic and diagnostic mediator, as exemplified by insertion of only one or two genes into the genome of an oncolytic vaccinia virus strain. We found that produced melanin is an excellent reporter for optical imaging without addition of substrate. Melanin production also facilitated deep tissue optoacoustic imaging as well as MRI. In addition, melanin was shown to be a suitable target for laser-induced thermotherapy and enhanced oncolytic viral therapy. In conclusion, melanin as a mediator for thermotherapy and reporter for different imaging modalities may soon become a versatile alternative to replace fluorescent proteins also in other biological systems. After ongoing extensive preclinical studies, melanin overproducing oncolytic virus strains might be used in clinical trials in patients with cancer. AU - Stritzker, J.* AU - Kirscher, L.* AU - Scadeng, M.* AU - Deliolanis, N.C. AU - Morscher, S. AU - Symvoulidis, P. AU - Schaefer, K. AU - Zhang, Q.* AU - Buckel, L.* AU - Hess, M.* AU - Donat, U.* AU - Bradley, W.G.* AU - Ntziachristos, V. AU - Szalay, A.A.* C1 - 23302 C2 - 31021 SP - 3316-3320 TI - Vaccinia virus-mediated melanin production allows MR and optoacoustic deep tissue imaging and laser-induced thermotherapy of cancer. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 9 PB - National Academy of Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions. AU - Wefers, B. AU - Meyer, M. AU - Ortiz, O. AU - Hrabě de Angelis, M. AU - Hansen, J. AU - Wurst, W. AU - Kühn, R. C1 - 23626 C2 - 31219 SP - 3782-3787 TI - Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 10 PB - National Academy of Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species. AU - Windheim, M.* AU - Southcombe, J.H.* AU - Kremmer, E. AU - Chaplin, L.* AU - Urlaub, D.* AU - Falk, C.S.* AU - Claus, M.* AU - Mihm, J.* AU - Braithwaite, M.* AU - Dennehy, K.* AU - Renz, H.* AU - Sester, M.* AU - Watzl, C.* AU - Burgert, H.G.* C1 - 28877 C2 - 33559 SP - E4884-E4893 TI - A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 50 PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Psoriasis is an autoinflammatory skin disease of unknown etiology. Topical application of Aldara cream containing the Toll-like receptor (TLR)7 agonist Imiquimod (IMQ) onto patients induces flares of psoriasis. Likewise, in mice IMQ triggers pathological changes closely resembling psoriatic plaque formation. Key cytokines like IL-23 and type-I IFN (IFN-I), both being produced mainly by dendritic cells (DCs), have been implicated in psoriasis. Although plasmacytoid DCs (pDCs) are the main source of IFNα and thought to initiate disease, conventional DCs (cDCs) appear to maintain the psoriatic lesions. Any role of cDCs during lesion formation remains elusive. Here, we report that selective activation of TLR7 signaling specifically in CD11c(+) DCs was sufficient to induce psoriasiform skin disease in mice. Intriguingly, both pDCs and the IFN-I pathway were dispensable for the development of local skin inflammation. Selective TLR7 triggering of Langerin(+) DCs resulted in attenuated disease, whereas their depletion did not alter the severity of skin lesions. Moreover, after IMQ-painting, IL-23 was exclusively produced by Langerin(neg) DCs in vivo. In conclusion, TLR7-activated Langerin(neg) cDCs trigger psoriatic plaque formation via IL-23-mediated activation of innate IL-17/IL-22-producing lymphocytes, independently of pDCs or IFN-I. These results suggest therapeutic targeting of IL-23 production by cDCs to refine current treatment strategies for psoriasis. AU - Wohn, C.* AU - Ober-Blöbaum, J.L.* AU - Haak, S. AU - Pantelyushin, S.* AU - Cheong, C.* AU - Zahner, S.P.* AU - Onderwater, S.* AU - Kant, M.* AU - Weighardt, H.* AU - Holzmann, B.* AU - Reizis, B.* AU - Becher, B.* AU - Prens, E.P.* AU - Clausen, B.E.* C1 - 26262 C2 - 32150 SP - 10723-10728 TI - Langerinneg conventional dendritic cells produce IL-23 to drive psoriatic plaque formation in mice. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 110 IS - 26 PB - Natl. Acad. Sciences PY - 2013 SN - 0027-8424 ER - TY - JOUR AB - Expanding the repertoire of molecularly diverse neurons in the human nervous system is paramount to characterizing the neuronal networks that underpin sensory processing. Defining neuronal identities is particularly timely in the human olfactory system, whose structural differences from nonprimate macrosmatic species have recently gained momentum. Here, we identify clusters of bipolar neurons in a previously unknown outer "shell" domain of the human olfactory tract, which express secretagogin, a cytosolic Ca(2+) binding protein. These "shell" neurons are wired into the olfactory circuitry because they can receive mixed synaptic inputs. Unexpectedly, secretagogin is often coexpressed with polysialylated-neural cell adhesion molecule, β-III-tubulin, and calretinin, suggesting that these neurons represent a cell pool that might have escaped terminal differentiation into the olfactory circuitry. We hypothesized that secretagogin-containing "shell" cells may be eliminated from the olfactory axis under neurodegenerative conditions. Indeed, the density, but not the morphological or neurochemical integrity, of secretagogin-positive neurons selectively decreases in the olfactory tract in Alzheimer's disease. In conclusion, secretagogin identifies a previously undescribed cell pool whose cytoarchitectonic arrangements and synaptic connectivity are poised to modulate olfactory processing in humans. AU - Attems, J.* AU - Alpar, A.* AU - Spence, L.* AU - McParland, S. AU - Heikenwälder, M. AU - Uhlén, M.* AU - Tanila, H.* AU - Hökfelt, T.G.* AU - Harkany, T.* C1 - 7492 C2 - 29752 SP - 6259-6264 TI - Clusters of secretagogin-expressing neurons in the aged human olfactory tract lack terminal differentiation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 16 PB - National Academy of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - During early pancreatic development, Notch signaling represses differentiation of endocrine cells and promotes proliferation of Nkx6-1(+)Ptf1a(+) multipotent progenitor cells (MPCs). Later, antagonistic interactions between Nkx6 transcription factors and Ptf1a function to segregate MPCs into distal Nkx6-1(-)Ptf1a(+) acinar progenitors and proximal Nkx6-1(+)Ptf1a(-) duct and beta-cell progenitors. Distal cells are initially multipotent, but evolve into unipotent, acinar cell progenitors. Conversely, proximal cells are bipotent and give rise to duct cells and late-born endocrine cells, including the insulin producing beta-cells. However, signals that regulate proximodistal (P-D) patterning and thus formation of beta-cell progenitors are unknown. Here we show that Mind bomb 1 (Mib1) is required for correct P-D patterning of the developing pancreas and beta-cell formation. We found that endoderm-specific inactivation of Mib1 caused a loss of Nkx6-1(+)Ptf1a(-) and Hnf1 beta(+) cells and a corresponding loss of Neurog3(+) endocrine progenitors and beta-cells. An accompanying increase in Nkx6-1(-)Ptf1a(+) and amylase(+) cells, occupying the proximal domain, suggests that proximal cells adopt a distal fate in the absence of Mib1 activity. Impeding Notch-mediated transcriptional activation by conditional expression of dominant negative Mastermind-like 1 (Maml1) resulted in a similarly distorted P-D patterning and suppressed beta-cell formation, as did conditional inactivation of the Notch target gene Hes1. Our results reveal iterative use of Notch in pancreatic development to ensure correct P-D patterning and adequate beta-cell formation. AU - Horn, S.* AU - Kobberup, S.* AU - Jorgensen, M.C.* AU - Kalisz, M.* AU - Klein, T.* AU - Kageyama, R.* AU - Gegg, M. AU - Lickert, H. AU - Lindner, J.* AU - Magnuson, M.A.* AU - Kong, Y.Y.* AU - Serup, P.* AU - Ahnfelt-Ronne, J.* AU - Jensen, J.N.* C1 - 7998 C2 - 29992 SP - 7356-7361 TI - Mind bomb 1 is required for pancreatic β-cell formation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 19 PB - Natl. Acad. of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - Herpesviruses are dsDNA viruses, but their virions may additionally contain RNAs that can be transduced to recipient cells. The biological functions of herpes virion RNA species are unknown. Here we address this issue for EBV, a widespread human herpesvirus with oncogenic potential. We show that EBV-derived particles that include virions, virus-like particles, and subviral vesicles contain viral mRNAs, microRNAs, and other noncoding RNAs. Viral RNAs were transduced during infection and deployed immediate functions that enhanced EBV's capacity to transform primary B cells. Among these transduced viral RNAs, BZLF1 transcripts transactivated viral promoters triggering the prelatent phase of EBV infection, noncoding EBV-encoded RNA transcripts induced cellular cytokine synthesis, and BNLF2a mRNA led to immune evasion that prevented T-cell responses to newly infected B cells. Hence, transduced viral RNAs govern critical processes immediately after infection of B cells with EBV and likely play important roles in herpesviral infection in general. AU - Jochum, S. AU - Ruiss, R.* AU - Moosmann, A. AU - Hammerschmidt, W. AU - Zeidler, R.* C1 - 8023 C2 - 29972 SP - E1396-E1404 TI - RNAs in Epstein-Barr virions control early steps of infection. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 21 PB - Natl. Acad. of Sci. PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology. AU - Lange, O.F. AU - Rossi, P.* AU - Sgourakis, N.G.* AU - Song, Y.F.* AU - Lee, H.-W.* AU - Aramini, J.M.* AU - Ertekin, A.* AU - Xiao, R.* AU - Acton, T.B.* AU - Montelione, G.T.* AU - Baker, D.* C1 - 8313 C2 - 30102 SP - 10873-10878 TI - Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 27 PB - Natl. Acad. Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated similar to 1.1-1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats. AU - Martis, M.M. AU - Klemme, S.* AU - Banaei-Moghaddam, A.M.* AU - Blattner, F.R.* AU - Macasc, J.* AU - Schmutzer, T.* AU - Scholz, U.* AU - Gundlach, H. AU - Wicker, T.* AU - Šimková, H.* AU - Novak, P.* AU - Neumann, P.* AU - Kubaláková, M.* AU - Bauer, E.* AU - Haseneyer, G.* AU - Fuchs, J.* AU - Dolezel, J.* AU - Stein, N.* AU - Mayer, K.F.X. AU - Houben, A.* C1 - 8533 C2 - 30177 SP - 13343-13346 TI - Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 33 PB - Natl. Acad. of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - Gene targeting by zinc-finger nucleases in one-cell embryos provides an expedite mutagenesis approach in mice, rats, and rabbits. This technology has been recently used to create knockout and knockin mutants through the deletion or insertion of nucleotides. Here we apply zinc-finger nucleases in one-cell mouse embryos to generate disease-related mutants harboring single nucleotide or codon replacements. Using a gene-targeting vector or a synthetic oligodesoxynucleotide as template for homologous recombination, we introduced missense and silent mutations into the Rab38 gene, encoding a small GTPase that regulates intracellular vesicle trafficking. These results demonstrate the feasibility of seamless gene editing in one-cell embryos to create genetic disease models and establish synthetic oligodesoxynucleotides as a simplified mutagenesis tool. AU - Meyer, M. AU - Ortiz, O. AU - Hrabě de Angelis, M. AU - Wurst, W. AU - Kühn, R. C1 - 7500 C2 - 29760 SP - 9354-9359 TI - Modeling disease mutations by gene targeting in one-cell mouse embryos. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 24 PB - National Academy of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - The concept of DNA "repair centers" and the meaning of radiation-induced foci (RIF) in human cells have remained controversial. RIFs are characterized by the local recruitment of DNA damage sensing proteins such as p53 binding protein (53BP1). Here, we provide strong evidence for the existence of repair centers. We used live imaging and mathematical fitting of RIF kinetics to show that RIF induction rate increases with increasing radiation dose, whereas the rate at which RIFs disappear decreases. We show that multiple DNA double-strand breaks (DSBs) 1 to 2 μm apart can rapidly cluster into repair centers. Correcting mathematically for the dose dependence of induction/resolution rates, we observe an absolute RIF yield that is surprisingly much smaller at higher doses: 15 RIF/Gy after 2 Gy exposure compared to approximately 64 RIF/Gy after 0.1 Gy. Cumulative RIF counts from time lapse of 53BP1-GFP in human breast cells confirmed these results. The standard model currently in use applies a linear scale, extrapolating cancer risk from high doses to low doses of ionizing radiation. However, our discovery of DSB clustering over such large distances casts considerable doubts on the general assumption that risk to ionizing radiation is proportional to dose, and instead provides a mechanism that could more accurately address risk dose dependency of ionizing radiation. AU - Neumaier, T. AU - Swenson, J.* AU - Pham, C.* AU - Polyzos, A.* AU - Lo, A.T.* AU - Yang, P.* AU - Dyball, J.* AU - Asaithamby, A.* AU - Chen, D.J.* AU - Bissell, M.J.* AU - Thalhammer, S. AU - Costes, S.V.* C1 - 6838 C2 - 29342 SP - 443-448 TI - Evidence for formation of DNA repair centers and dose-response nonlinearity in human cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 2 PB - National Academy of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - Neutrophil serine proteases (NSPs) in cytoplasmic granules of neutrophils are regarded as important antimicrobial defense weapons after engulfment and exposure of pathogens to the content of primary granules. Despite intensive studies on neutrophils during the last three decades, only three active serine proteases, neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3) have been identified in these short-lived cells. Here, we report on the identification of a fourth serine protease (NSP4) with 39% identity to NE and PR3, but arginine specificity, yet sharing features like propeptide processing by dipeptidyl peptidase I, storage, and release as an active enzyme with the three active proteases. We established monoclonal antibodies against NSP4, excluded cross-reactivity to human granzymes, NE, CG, PR3, and azurocidin, and screened for NSP4 protein expression in various human tissues and blood leukocyte populations. Only granulocyte precursors and neutrophil populations from peripheral blood were positive. The content of NSP4 in neutrophil lysates, however, was about 20-fold lower compared with CG. Upon neutrophil activation, NSP4 was released into the supernatant. Profiling its specificity with peptide libraries from Escherichia coli revealed a preference for arginine in P1; it cleaved Tyr-Arg-Phe-Arg-AMC and Ala-Pro-Nva-thiobenzyl esters. NSP4 was inhibited by α(1)-proteinase inhibitor (α(1)-antitrypsin), C1 inhibitor, and most efficiently by antithrombin-heparin, but not by elafin, secretory leukocyte protease inhibitor, α(1)-antichymotrypsin, and monocyte-neutrophil elastase inhibitor. Functional specialization and preferred natural substrates of NSP4 remain to be determined to understand the biological interplay of all four NSPs during neutrophil responses. AU - Perera, N.C.* AU - Schilling, O.* AU - Kittel, H.* AU - Back, W.* AU - Kremmer, E. AU - Jenne, D. C1 - 7515 C2 - 29774 SP - 6229-6234 TI - NSP4, an elastase-related protease in human neutrophils with arginine specificity. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 16 PB - National Academy of Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - In early stages of various pulmonary diseases, such as emphysema and fibrosis, the change in X-ray attenuation is not detectable with absorption-based radiography. To monitor the morphological changes that the alveoli network undergoes in the progression of these diseases, we propose using the dark-field signal, which is related to small-angle scattering in the sample. Combined with the absorption-based image, the dark-field signal enables better discrimination between healthy and emphysematous lung tissue in a mouse model. All measurements have been performed at 36 keV using a monochromatic laser-driven miniature synchrotron X-ray source (Compact Light Source). In this paper we present grating-based dark-field images of emphysematous vs. healthy lung tissue, where the strong dependence of the dark-field signal on mean alveolar size leads to improved diagnosis of emphysema in lung radiographs. AU - Schleede, S.* AU - Meinel, F.G.* AU - Bech, M.* AU - Herzen, J.* AU - Achterhold, K.* AU - Potdevin, G.* AU - Malecki, A.* AU - Adam-Neumair, S.* AU - Thieme, S.F.* AU - Bamberg, F.* AU - Nikolaou, K.* AU - Bohla, A. AU - Yildirim, A.Ö. AU - Loewen, R.* AU - Gifford, M.* AU - Ruth, R.* AU - Eickelberg, O. AU - Reiser, M.* AU - Pfeiffer, F.* C1 - 11265 C2 - 30576 SP - 17880-17885 TI - Emphysema diagnosis using X-ray dark-field imaging at a laser-driven compact synchrotron light source. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 44 PB - Natl. Acad. Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - The lung surface is an ideal pathway to the bloodstream for nanoparticle-based drug delivery. Thus far, research has focused on the lungs of adults, and little is known about nanoparticle behavior in the immature lungs of infants. Here, using nonlinear dynamical systems analysis and in vivo experimentation in developing animals, we show that nanoparticle deposition in postnatally developing lungs peaks at the end of bulk alveolation. This finding suggests a unique paradigm, consistent with the emerging theory that as alveoli form through secondary septation, alveolar flow becomes chaotic and chaotic mixing kicks in, significantly enhancing particle deposition. This finding has significant implications for the application of nanoparticle-based inhalation therapeutics in young children with immature lungs from birth to 2 y of age. AU - Semmler-Behnke, M. AU - Kreyling, W.G. AU - Schulz, H. AU - Takenaka, S. AU - Butler, J.P.* AU - Henry, F.S.* AU - Tsuda, A.* C1 - 7298 C2 - 29659 SP - 5092-5097 TI - Nanoparticle delivery in infant lungs. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 109 IS - 13 PB - Natl. Acad. Sciences PY - 2012 SN - 0027-8424 ER - TY - JOUR AB - Soil pH is a major determinant of microbial ecosystem processes and potentially a major driver of evolution, adaptation, and diversity of ammonia oxidizers, which control soil nitrification. Archaea are major components of soil microbial communities and contribute significantly to ammonia oxidation in some soils. To determine whether pH drives evolutionary adaptation and community structure of soil archaeal ammonia oxidizers, sequences of amoA, a key functional gene of ammonia oxidation, were examined in soils at global, regional, and local scales. Globally distributed database sequences clustered into 18 well-supported phylogenetic lineages that dominated specific soil pH ranges classified as acidic (pH <5), acido-neutral (5≤ pH <7), or alkalinophilic (pH ≥7). To determine whether patterns were reproduced at regional and local scales, amoA gene fragments were amplified from DNA extracted from 47 soils in the United Kingdom (pH 3.5-8.7), including a pH-gradient formed by seven soils at a single site (pH 4.5-7.5). High-throughput sequencing and analysis of amoA gene fragments identified an additional, previously undiscovered phylogenetic lineage and revealed similar pH-associated distribution patterns at global, regional, and local scales, which were most evident for the five most abundant clusters. Archaeal amoA abundance and diversity increased with soil pH, which was the only physicochemical characteristic measured that significantly influenced community structure. These results suggest evolution based on specific adaptations to soil pH and niche specialization, resulting in a global distribution of archaeal lineages that have important consequences for soil ecosystem function and nitrogen cycling. AU - Gubry-Rangin, C.* AU - Hai, B. AU - Quince, C.* AU - Engel, M. AU - Thomson, B.C.* AU - James, P.* AU - Schloter, M. AU - Griffiths, R.I.* AU - Prosser, J.I.* AU - Nicol, G.W.* C1 - 6854 C2 - 29357 SP - 21206-21211 TI - Niche specialization of terrestrial archaeal ammonia oxidizers. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 52 PB - National Academy of Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - The main consequence of the Chernobyl accident has been an increase in papillary thyroid carcinomas (PTCs) in those exposed to radioactive fallout as young children. Our aim was to identify genomic alterations that are associated with exposure to radiation. We used array comparative genomic hybridization to analyze a main (n = 52) and a validation cohort (n = 28) of PTC from patients aged <25 y at operation and matched for age at diagnosis and residency. Both cohorts consisted of patients exposed and not exposed to radioiodine fallout. The study showed association of a gain on chromosome 7 (7q11.22-11.23) with exposure (false discovery rate = 0.035). Thirty-nine percent of the exposed group showed the alteration; however, it was not found in a single case from the unexposed group. This was confirmed in the validation set. Because only a subgroup of cases in the exposed groups showed gain of 7q11.22-11.23, it is likely that different molecular subgroups and routes of radiation-induced carcinogenesis exist. The candidate gene CLIP2 was specifically overexpressed in the exposed cases. In addition, the expression of the genes PMS2L11, PMS2L3, and STAG3L3 correlated with gain of 7q11.22-11.23. An enrichment of Gene Ontology terms "DNA repair" (PMS2L3, PMS2L5), "response to DNA damage stimulus" (BAZ1B, PMS2L3, PMS2L5, RFC2), and "cell-cell adhesion" (CLDN3, CLDN4) was found. This study, using matched exposed and unexposed cohorts, provides insights into the radiation-related carcinogenesis of young-onset PTC and, with the exposure-specific gain of 7q11 and overexpression of the CLIP2 gene, radiation-specific molecular markers. AU - Hess J. AU - Thomas, G.* AU - Braselmann, H. AU - Bauer, V. AU - Bogdanova, T.* AU - Wienberg, J.* AU - Zitzelsberger, H. AU - Unger, K. C1 - 5969 C2 - 28722 SP - 9595-9600 TI - Gain of chromosome band 7q11 in papillary thyroid carcinomas of young patients is associated with exposure to low-dose irradiation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 23 PB - Natl. Acad. Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Neural stem cells (NSCs) generate new granule cells throughout life in the mammalian hippocampus. Canonical Wnt signaling regulates the differentiation of NSCs towards the neuronal lineage. Here we identified the prospero-related homeodomain transcription factor Prox1 as a target of β-catenin-TCF/LEF signaling in vitro and in vivo. Prox1 overexpression enhanced neuronal differentiation whereas shRNA-mediated knockdown of Prox1 impaired the generation of neurons in vitro and within the hippocampal niche. In contrast, Prox1 was not required for survival of adult-generated granule cells after they had matured, suggesting a role for Prox1 in initial granule cell differentiation but not in the maintenance of mature granule cells. The data presented here characterize a molecular pathway from Wnt signaling to a transcriptional target leading to granule cell differentiation within the adult brain and identify a stage-specific function for Prox1 in the process of adult neurogenesis. AU - Karalay, O.* AU - Doberauer, K. AU - Vadodaria, K.C.* AU - Knobloch, M.* AU - Berti, L. AU - Miquelajauregui, A.* AU - Schwark, M.* AU - Jagasia, R.* AU - Taketo, MM.* AU - Tarabykin, V.* AU - Lie, D.C. AU - Jessberger, S.* C1 - 5046 C2 - 28811 SP - 5807-5812 TI - Prospero-related homeobox 1 gene (Prox1) is regulated by canonical Wnt signaling and has a stage-specific role in adult hippocampal neurogenesis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 14 PB - Natl Acad Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) represents a very aggressive human lymphoma entity. Constitutive NF-κB activation caused by chronic active B-cell receptor (BCR) signaling is common feature of many ABC DLBCL cells; however, the pathways linking BCR signaling to the NF-κB prosurvival network are largely unknown. Here we report that constitutive activity of PI3K and the downstream kinase PDK1 are essential for the viability of two ABC DLBCL cell lines that carry mutations in the BCR proximal signaling adaptor CD79B. In these cells, PI3K inhibition reduces NF-κB activity and decreases the expression of NF-κB target genes. Furthermore, PI3K and PDK1 are required for maintaining MALT1 protease activity, which promotes survival of the affected ABC DLBCL cells. These results demonstrate a critical function of PI3K-PDK1 signaling upstream of MALT1 protease and NF-κB in distinct ABC DLBCL cells and provide a rationale for the pharmacologic use of PI3K inhibitors in DLBCL therapy. AU - Kloo, B. AU - Nagel, D. AU - Pfeifer, M.* AU - Grau, M.* AU - Düwel, M. AU - Vincendeau, M. AU - Dörken, B.* AU - Lenz, P.* AU - Lenz, G.* AU - Krappmann, D. C1 - 6087 C2 - 28042 SP - 272-277 TI - Critical role of PI3K signaling for NF-κB-dependent survival in a subset of activated B-cell-like diffuse large B-cell lymphoma cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 1 PB - Natl. Acad. Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Stress has been identified as a major causal factor for many mental disorders. However, our knowledge about the chain of molecular and cellular events translating stress experience into altered behavior is still rather scant. Here, we have characterized a murine ortholog of the putative tumor suppressor gene DRR1 as a unique stress-induced protein in brain. It binds to actin, promotes bundling and stabilization of actin filaments, and impacts on actin-dependent neurite outgrowth. Endogenous DRR1 localizes to some, but not all, synapses, with preference for the presynaptic region. Hippocampal virus-mediated enhancement of DRR1 expression reduced spine density, diminished the probability of synaptic glutamate release, and altered cognitive performance. DRR1 emerges as a protein to link stress with actin dynamics, which in addition is able to act on synaptic function and cognition. AU - Schmidt, M.V.* AU - Schülke, .J.-P.* AU - Liebl, C.* AU - Stiess, M.* AU - Avrabos, C.* AU - Bock, J.* AU - Wochnik, GM.* AU - Davies, H.A.* AU - Zimmermann, N.* AU - Scharf, S.H.* AU - Trümbach, D. AU - Wurst, W. AU - Zieglgänsberger, W.* AU - Turck, C.* AU - Holsboer, F.* AU - Stewart, M.G.* AU - Bradke, F.* AU - Eder, M.* AU - Müller, M.B.* AU - Rein, T. C1 - 6115 C2 - 29002 CY - Washington, DC, USA SP - 17213-17218 TI - Tumor suppressor down-regulated in renal cell carcinoma 1 (DRR1) is a stress-induced actin bundling factor that modulates synaptic efficacy and cognition. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 41 PB - National Academy of Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Alcohol consumption is a moderately heritable trait, but the genetic basis in humans is largely unknown, despite its clinical and societal importance. We report a genome-wide association study meta-analysis of ∼2.5 million directly genotyped or imputed SNPs with alcohol consumption (gram per day per kilogram body weight) among 12 population-based samples of European ancestry, comprising 26,316 individuals, with replication genotyping in an additional 21,185 individuals. SNP rs6943555 in autism susceptibility candidate 2 gene (AUTS2) was associated with alcohol consumption at genome-wide significance (P = 4 × 10(-8) to P = 4 × 10(-9)). We found a genotype-specific expression of AUTS2 in 96 human prefrontal cortex samples (P = 0.026) and significant (P < 0.017) differences in expression of AUTS2 in whole-brain extracts of mice selected for differences in voluntary alcohol consumption. Down-regulation of an AUTS2 homolog caused reduced alcohol sensitivity in Drosophila (P < 0.001). Our finding of a regulator of alcohol consumption adds knowledge to our understanding of genetic mechanisms influencing alcohol drinking behavior. AU - Schumann, G.* AU - Coin, L.J.* AU - Lourdusamy, A.* AU - Charoen, P.* AU - Berger, K.H.* AU - Stacey, D.* AU - Desrivières, S.* AU - Aliev, F.A.* AU - Khan, A.A.* AU - Amin, N.* AU - Aulchenko, Y.S.* AU - Bakalkin, G.* AU - Bakker, S.J.* AU - Balkau, B.* AU - Beulens, J.W.* AU - Bilbao, A.* AU - de Boer, R.A.* AU - Beury, D.* AU - Bots, M.L.* AU - Breetvelt, E.J.* AU - Cauchi, S.* AU - Cavalcanti-Proença, C.* AU - Chambers, J.C.* AU - Clarke, T.K.* AU - Dahmen, N.* AU - de Geus, E.J.* AU - Dick, D.* AU - Ducci, F.* AU - Easton, A.* AU - Edenberg, H.J.* AU - Esko, T.* AU - Fernández-Medarde, A.* AU - Foroud, T.* AU - Freimer, N.B.* AU - Girault, J.A.* AU - Grobbee, DE.* AU - Guarrera, S.* AU - Gudbjartsson, D.F.* AU - Hartikainen, A.L.* AU - Heath, A.C.* AU - Hesselbrock, V.* AU - Hofman, A.* AU - Hottenga, J.J.* AU - Isohanni, M.K.* AU - Kaprio, J.* AU - Khaw, K.T.* AU - Kuehnel, B. AU - Laitinen, J.* AU - Lobbens, S.* AU - Luan, J.* AU - Mangino, M.* AU - Maroteaux, M.* AU - Matullo, G.* AU - McCarthy, M.I.* AU - Mueller, C.* AU - Navis, G.* AU - Numans, M.E.* AU - Núñez, A.* AU - Nyholt, D.R.* AU - Onland-Moret, C.N.* AU - Oostra, B.A.* AU - O'Reilly, P.F.* AU - Palkovits, M.* AU - Penninx, B.W.* AU - Polidoro, S.* AU - Pouta, A.* AU - Prokopenko, I.* AU - Ricceri, F.* AU - Santos, E.* AU - Smit, J.H.* AU - Soranzo, N.* AU - Song, K.* AU - Sovio, U.* AU - Stumvoll, M.* AU - Surakk, I.* AU - Thorgeirsson, TE.* AU - Thorsteinsdottir, U.* AU - Troakes, C.* AU - Tyrfingsson, T.* AU - Tönjes, A.* AU - Uiterwaal, C.S.* AU - Uitterlinden, A.G.* AU - van der Harst, P.* AU - van der Schouw, Y.T.* AU - Staehlin, O.* AU - Vogelzangs, N.* AU - Vollenweider, P.* AU - Waeber, G.* AU - Wareham, N.J.* AU - Waterworth, D.M.* AU - Whitfield, J.B.* AU - Wichmann, H.-E. AU - Willemsen, G.* AU - Witteman, J.C.* AU - Yuan, X.* AU - Zhai, G.* AU - Zhao, J.H.* AU - Zhang, W.* AU - Martin, N.G.* AU - Metspalu, A.* AU - Döring, A. AU - Scott, J.* AU - Spector, T.D.* AU - Loos, R.J.* AU - Boomsma, D.I.* AU - Mooser, V.* AU - Peltonen, L.* AU - Stefansson, K.* AU - van Duijn, C.M.* AU - Vineis, P.* AU - Sommer, W.H.* AU - Kooner, J.S.* AU - Spanagel, R.* AU - Heberlein, U.A.* AU - Jarvelin, M.R.* AU - Elliott, P. C1 - 6327 C2 - 28509 CY - Washington, USA SP - 7119-7124 TI - Genome-wide association and genetic functional studies identify autism susceptibility candidate 2 gene (AUTS2) in the regulation of alcohol consumption. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 17 PB - Natl. Acad. Of Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed "TKD," comprising amino acids 450-461 (aa(450-461)) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1 mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors. Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70(+) mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70. AU - Stangl, S.* AU - Gehrmann, M.* AU - Riegger, J.* AU - Kuhs, K.* AU - Riederer, I.* AU - Sievert, W.* AU - Hube, K.* AU - Mocikat, R. AU - Dressel, R.* AU - Kremmer, E. AU - Pockley, A.G.* AU - Friedrich, L.* AU - Vigh, L.* AU - Skerra, A.* AU - Multhoff, G.* C1 - 6097 C2 - 27861 SP - 733-738 TI - Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 2 PB - Natl. Acad. Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Genes of archaea encoding homologues of ammonia monooxygenases have been found on a widespread basis and in large amounts in almost all terrestrial and marine environments, indicating that ammonia oxidizing archaea (AOA) might play a major role in nitrification on Earth. However, only one pure isolate of this group from a marine environment has so far been obtained, demonstrating archaeal ammonia oxidation coupled with autotrophic growth similar to the bacterial counterparts. Here we describe the cultivation and isolation of an AOA from soil. It grows on ammonia or urea as an energy source and is capable of using higher ammonia concentrations than the marine isolate, Nitrosopumilus maritimus. Surprisingly, although it is able to grow chemolithoautotrophically, considerable growth rates of this strain are obtained only upon addition of low amounts of pyruvate or when grown in coculture with bacteria. Our findings expand the recognized metabolic spectrum of AOA and help explain controversial results obtained in the past on the activity and carbon assimilation of these globally distributed organisms. AU - Tourna, M.* AU - Stieglmeier, M.* AU - Spang, A.* AU - Könneke, M.* AU - Schintlmeister, A.* AU - Urich, T.* AU - Engel, M. AU - Schloter, M. AU - Wagner, M.* AU - Richter, A.* AU - Schleper, C. C1 - 6200 C2 - 28563 SP - 8420-8425 TI - Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 108 IS - 20 PB - Natl Acad Sciences PY - 2011 SN - 0027-8424 ER - TY - JOUR AB - Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation. AU - Conrad, M. AU - Sandin, A.* AU - Förster, H. AU - Seiler, A. AU - Frijhoff, J.* AU - Dagnell, M.* AU - Bornkamm, G.W. AU - Radmark, O.* AU - van Huijsduijnen, R.H.* AU - Aspenström, P.* AU - Böhmer, F.* AU - Östman, A.* C1 - 4473 C2 - 27974 SP - 15774-15779 TI - 12/15-lipoxygenase-derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 36 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Plants respond to low levels of UV-B radiation with a coordinated photomorphogenic response that allows acclimation to this environmental stress factor. The key players in this UV-B response are COP1 (an E3 ubiquitin ligase), UVR8 (a β-propeller protein), and HY5 (a bZIP transcription factor). We have shown previously that an elevated UV-B-specific response is associated with dwarf growth, indicating the importance of balancing UV-B-specific signaling. Negative regulators of this pathway are not known, however. Here, we describe two highly related WD40-repeat proteins, REPRESSOR OF UV-B PHOTOMORPHOGENESIS 1 (RUP1) and RUP2, that interact directly with UVR8 as potent repressors of UV-B signaling. Both genes were transcriptionally activated by UV-B in a COP1-, UVR8-, and HY5-dependent manner. rup1 rup2 double mutants showed an enhanced response to UV-B and elevated UV-B tolerance after acclimation. Overexpression of RUP2 resulted in reduced UV-B-induced photomorphogenesis and impaired acclimation, leading to hypersensitivity to UV-B stress. These results are consistent with an important regulatory role for RUP1 and RUP2, which act downstream of UVR8-COP1 in a negative feedback loop impinging on UVR8 function, balancing UV-B defense measures and plant growth. AU - Gruber, H.* AU - Heijde, M.* AU - Heller, W. AU - Albert, A. AU - Seidlitz, H.K. AU - Ulm, R.* C1 - 5312 C2 - 27614 SP - 20132-20137 TI - Negative feedback regulation of UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 46 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1, related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1's initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV's epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host's mode of methylating DNA for its own epigenetic regulation. AU - Kalla, M. AU - Schmeinck, A. AU - Bergbauer, M. AU - Pich, D. AU - Hammerschmidt, W. C1 - 422 C2 - 27244 SP - 850-855 TI - AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 2 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Pancreatic cancer is one of the most fatal malignancies lacking effective therapies. Notch signaling is a key regulator of cell fate specification and pancreatic cancer development; however, the role of individual Notch receptors and downstream signaling is largely unknown. Here, we show that Notch2 is predominantly expressed in ductal cells and pancreatic intraepithelial neoplasia (PanIN) lesions. Using genetically engineered mice, we demonstrate the effect of conditional Notch receptor ablation in KrasG12D-driven pancreatic carcinogenesis. Deficiency of Notch2 but not Notch1 stops PanIN progression, prolongs survival, and leads to a phenotypical switch toward anaplastic pancreatic cancer with epithelial-mesenchymal transition. By expression profiling, we identified increased Myc signaling regulated by Notch2 during tumor development, placing Notch2 as a central regulator of PanIN progression and malignant transformation. Our study supports the concept of distinctive roles of individual Notch receptors in cancer development. AU - Mazur, P.K.* AU - Einwächter, H.* AU - Lee, M.* AU - Sipos, B.* AU - Nakhai, H.* AU - Rad, R.* AU - Zimber-Strobl, U. AU - Strobl, L.J. AU - Radtke, F.* AU - Klöppel, G.* AU - Schmid, R.M.* AU - Siveke, J.T.* C1 - 3422 C2 - 27622 SP - 13438-13443 TI - Notch2 is required for progression of pancreatic intraepithelial neoplasia and development of pancreatic ductal adenocarcinoma. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 30 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Gene targeting by homologous recombination in embryonic stem cells is extensively used to generate specific mouse mutants. However, most mammalian species lack tools for targeted gene manipulation. Since double-strand breaks strongly increase the rate of homologous recombination at genomic loci, we explored whether gene targeting can be directly performed in zygotes by the use of zinc-finger nucleases. Here we report that gene targeting is achieved in 1.7-4.5% of murine one-cell embryos upon the coinjection of targeting vectors with zinc-finger nucleases, without preselection. These findings enable the manipulation of the mammalian germ line in a single step in zygotes, independent of ES cells. AU - Meyer, M. AU - Hrabě de Angelis, M. AU - Wurst, W. AU - Kühn, R. C1 - 5570 C2 - 27606 SP - 15022-15026 TI - Gene targeting by homologous recombination in mouse zygotes mediated by zinc-finger nucleases. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 34 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Pheochromocytomas are rare neoplasias of neural crest origin arising from chromaffin cells of the adrenal medulla and sympathetic ganglia (extra-adrenal pheochromocytoma). Pheochromocytoma that develop in rats homozygous for a loss-of-function mutation in p27Kip1 (MENX syndrome) show a clear progression from hyperplasia to tumor, offering the possibility to gain insight into tumor pathobiology. We compared the gene-expression signatures of both adrenomedullary hyperplasia and pheochromocytoma with normal rat adrenal medulla. Hyperplasia and tumor show very similar transcriptome profiles, indicating early determination of the tumorigenic signature. Overrepresentation of developmentally regulated neural genes was a feature of the rat lesions. Quantitative RT-PCR validated the up-regulation of 11 genes, including some involved in neural development: Cdkn2a, Cdkn2c, Neurod1, Gal, Bmp7, and Phox2a. Overexpression of these genes precedes histological changes in affected adrenal glands. Their presence at early stages of tumorigenesis indicates they are not acquired during progression and may be a result of the lack of functional p27Kip1. Adrenal and extra-adrenal pheochromocytoma development clearly follows diverged molecular pathways in MENX rats. To correlate these findings to human pheochromocytoma, we studied nine genes overexpressed in the rat lesions in 46 sporadic and familial human pheochromocytomas. The expression of GAL, DGKH, BMP7, PHOX2A, L1CAM, TCTE1, EBF3, SOX4, and HASH1 was up-regulated, although with different frequencies. Immunohistochemical staining detected high L1CAM expression selectively in 27 human pheochromocytomas but not in 140 nonchromaffin neuroendocrine tumors. These studies reveal clues to the molecular pathways involved in rat and human pheochromocytoma and identify previously unexplored biomarkers for clinical use. AU - Molatore, S. AU - Liyanarachchi, S.* AU - Irmler, M. AU - Perren, A.* AU - Mannelli, M.* AU - Ercolino, T.* AU - Beuschlein, F.* AU - Jarzab, B.* AU - Wloch, J.* AU - Ziaja, J.* AU - Zoubaa, S. AU - Neff, F. AU - Beckers, J. AU - Höfler, H. AU - Atkinson, M.J. AU - Pellegata, N.S. C1 - 5657 C2 - 27736 CY - Washington SP - 18493-18498 TI - Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 43 PB - Natl. Acad. Science PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Recent data indicate that a variety of regulatory molecules active in embryonic development may also play a role in the regulation of early hematopoiesis. Here we report that the human Vent-like homeobox gene VENTX, a putative homolog of the Xenopus xvent2 gene, is a unique regulatory hematopoietic gene that is aberrantly expressed in CD34(+) leukemic stem-cell candidates in human acute myeloid leukemia (AML). Quantitative RT-PCR documented expression of the gene in lineage positive hematopoietic subpopulations, with the highest expression in CD33(+) myeloid cells. Notably, expression levels of VENTX were negligible in normal CD34(+)/CD38(-) or CD34(+) human progenitor cells. In contrast to this, leukemic CD34(+)/CD38(-) cells from AML patients with translocation t(8,21) and normal karyotype displayed aberrantly high expression of VENTX. Gene expression and pathway analysis demonstrated that in normal CD34(+) cells enforced expression of VENTX initiates genes associated with myeloid development and down-regulates genes involved in early lymphoid development. Functional analyses confirmed that aberrant expression of VENTX in normal CD34(+) human progenitor cells perturbs normal hematopoietic development, promoting generation of myeloid cells and impairing generation of lymphoid cells in vitro and in vivo. Stable knockdown of VENTX expression inhibited the proliferation of human AML cell lines. Taken together, these data extend our insights into the function of embryonic mesodermal factors in human postnatal hematopoiesis and indicate a role for VENTX in normal and malignant myelopoiesis. AU - Rawat, V.P.S.* AU - Arseni, N. AU - Ahmed, F. AU - Mulaw, M.A. AU - Thoene, S.* AU - Heilmeier, B. AU - Sadlon, T.* AU - D'Andrea, R.J.* AU - Hiddemann, W. AU - Bohlander, S.K. AU - Buske, C.* AU - Feuring-Buske, M.* C1 - 5304 C2 - 27810 SP - 16946-16951 TI - The vent-like homeobox gene VENTX promotes human myeloid differentiation and is highly expressed in acute myeloid leukemia. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 39 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - The propensity of helminths, such as schistosomes, to immunomodulate the host's immune system is an essential aspect of their survival. Previous research has demonstrated how soluble schistosomal egg antigens (SEA) dampen TLR-signaling during innate immune responses. We show here that the suppressive effect by SEA on TLR signaling is simultaneously coupled to the activation of the Nlrp3 (NLR family, pyrin domain containing 3) inflammasome and thus IL-1β production. Therefore, the responsible protein component of SEA contains the second signal that is required to trigger proteolytic pro-IL-1β processing. Moreover, the SEA component binds to the Dectin-2/FcRγ (Fc receptor γ chain) complex and activates the Syk kinase signaling pathway to induce reactive oxygen species and potassium efflux. As IL-1β has been shown to be an essential orchestrator against several pathogens we studied the in vivo consequences of Schistosoma mansoni infection in mice deficient in the central inflammasome adapter ASC and Nlrp3 molecule. These mice failed to induce local IL-1β levels in the liver and showed decreased immunopathology. Interestingly, antigen-specific Th1, Th2, and Th17 responses were down-regulated. Overall, these data imply that component(s) within SEA induce IL-1β production and unravel a crucial role of Nlrp3 during S. mansoni infection. AU - Ritter, M.* AU - Gross, O.* AU - Kays, S.* AU - Ruland, J. AU - Nimmerjahn, F.* AU - Saijo, S.* AU - Tschopp, J.* AU - Layland, L.E.* AU - da Costa, C.P.* C1 - 3902 C2 - 28028 SP - 20459-20464 TI - Schistosoma mansoni triggers Dectin-2, which activates the Nlrp3 inflammasome and alters adaptive immune responses. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 47 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Numerous descriptions of organic molecules present in the Murchison meteorite have improved our understanding of the early interstellar chemistry that operated at or just before the birth of our solar system. However, all molecular analyses were so far targeted toward selected classes of compounds with a particular emphasis on biologically active components in the context of prebiotic chemistry. Here we demonstrate that a nontargeted ultrahigh-resolution molecular analysis of the solvent-accessible organic fraction of Murchison extracted under mild conditions allows one to extend its indigenous chemical diversity to tens of thousands of different molecular compositions and likely millions of diverse structures. This molecular complexity, which provides hints on heteroatoms chronological assembly, suggests that the extraterrestrial chemodiversity is high compared to terrestrial relevant biological- and biogeochemical-driven chemical space. AU - Schmitt-Kopplin, P. AU - Gabelica, Z.* AU - Gougeon, R.D.* AU - Fekete, A. AU - Kanawati, B. AU - Harir, M. AU - Gebefügi, I.L. AU - Eckel, G.* AU - Hertkorn, N. C1 - 25 C2 - 27039 SP - 2763-2768 TI - High molecular diversity of extraterrestrial organic matter in Murchison meteorite revealed 40 years after its fall. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 7 PB - Natl. Acad. Sciences PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Polyploidy, the presence of more than two complete sets of chromosomes in an organism, has significantly shaped the genomes of angiosperms during evolution. Two forms of polyploidy are often considered: allopolyploidy, which originates from interspecies hybrids, and autopolyploidy, which originates from intraspecies genome duplication events. Besides affecting genome organization, polyploidy generates other genetic effects. Synthetic allopolyploid plants exhibit considerable transcriptome alterations, part of which are likely caused by the reunion of previously diverged regulatory hierarchies. In contrast, autopolyploids have relatively uniform genomes, suggesting lower alteration of gene expression. To evaluate the impact of intraspecies genome duplication on the transcriptome, we generated a series of unique Arabidopsis thaliana autotetraploids by using different ecotypes. A. thaliana autotetraploids show transcriptome alterations that strongly depend on their parental genome composition and include changed expression of both new genes and gene groups previously described from allopolyploid Arabidopsis. Alterations in gene expression are stable, nonstochastic, developmentally specific, and associated with changes in DNA methylation. We propose that Arabidopsis possesses an inherent and heritable ability to sense and respond to elevated, yet balanced chromosome numbers. The impact of natural variation on alteration of autotetraploid gene expression stresses its potential importance in the evolution and breeding of plants. AU - Yu, Z.* AU - Haberer, G. AU - Matthes, M.* AU - Rattei, T.* AU - Mayer, K.F.X. AU - Gierl, A.* AU - Torres-Ruiz, R.A.* C1 - 3733 C2 - 28008 SP - 17809-17814 TI - Impact of natural genetic variation on the transcriptome of autotetraploid Arabidopsis thaliana. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 107 IS - 41 PB - Natl. Acad. Science PY - 2010 SN - 0027-8424 ER - TY - JOUR AB - Combinatorial genetics for conditional transgene activation allows studying gene function with temporal and tissue specific control like the Gal4-UAS system, which has enabled sophisticated genetic studies in Drosophila. Recently this system was adapted for zebrafish and promising applications have been introduced. Here, we report a systematic optimization of zebrafish Gal4-UAS genetics by establishing an optimized Gal4-activator (KalTA4). We provide quantitative data for KalTA4-mediated transgene activation in dependence of UAS copy numbers to allow for studying dosage effects of transgene expression. Employing a Tol2 transposon-mediated KalTA4 enhancer trap screen biased for central nervous system expression, we present a collection of self-reporting red fluorescent KalTA4 activator strains. These strains reliably transactivate UAS-dependent transgenes and can be rendered homozygous. Furthermore, we have characterized the transactivation kinetics of tissue-specific KalTA4 activation, which led to the development of a self-maintaining effector strain "Kaloop.'' This strain relates transient KalTA4 expression during embryogenesis via a KalTA4-mediated autoregulatory mechanism to live adult structures. We demonstrate its use by showing that the secondary octaval nucleus in the adult hindbrain is likely derived from egr2b-expressing cells in rhombomere 5 during stages of early embryogenesis. These data demonstrate prolonged and maintained expression by Kalooping, a technique that can be used for permanent spatiotemporal genetic fate mapping and targeted transgene expression in zebrafish. AU - Distel, M. AU - Wullimann, M.F.* AU - Köster, R.W. C1 - 2511 C2 - 26995 SP - 13365-13370 TI - Optimized Gal4 genetics for permanent gene expression mapping in zebrafish. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 32 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - Wine chemical compositions, which result from a complex interplay between environmental factors, genetic factors, and viticultural practices, have mostly been studied using targeted analyses of selected families of metabolites. Detailed studies have particularly concerned volatile and polyphenolic compounds because of their acknowledged roles in the organoleptic and therapeutic properties. However, we show that an unprecedented chemical diversity of wine composition can be unraveled through a nontargeted approach by ultrahigh-resolution mass spectrometry, which provides an instantaneous image of complex interacting processes, not easily or possibly resolvable into their unambiguous individual contributions. In particular, the statistical analysis of a series of barrel-aged wines revealed that 10-year-old wines still express a metabologeographic signature of the forest location where oaks of the barrel in which they were aged have grown. AU - Gougeon, R.D.* AU - Lucio, M. AU - Frommberger, M. AU - Peyron, D.* AU - Chassagne, D.* AU - Alexandre, H.* AU - Feuillat, F.* AU - Voilley, A.* AU - Cayot, P.* AU - Gebefügi, I. AU - Hertkorn, N. AU - Schmitt-Kopplin, P. C1 - 2467 C2 - 26237 SP - 9174-9179 TI - The chemodiversity of wines can reveal a metabologeography expression of cooperage oak wood. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 23 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - The PUR protein family is a distinct and highly conserved class that is characterized by its sequence-specific RNA-and DNA-binding. Its best-studied family member, Pur-alpha, acts as a transcriptional regulator, as host factor for viral replication, and as cofactor for mRNP localization in dendrites. Pur-alpha-deficient mice show severe neurologic defects and die after birth. Nucleic-acid binding by Pur-alpha is mediated by its central core region, for which no structural information is available. We determined the x-ray structure of residues 40 to 185 from Drosophila melanogaster Pur-alpha, which constitutes a major part of the core region. We found that this region contains two almost identical structural motifs, termed "PUR repeats,'' which interact with each other to form a PUR domain. DNA-and RNA-binding studies confirmed that PUR domains are indeed functional nucleic-acid binding domains. Database analysis show that PUR domains share a fold with the Whirly class of nucleic-acid binding proteins. Structural analysis combined with mutational studies suggest that a PUR domain binds nucleic acids through two independent surface regions involving concave beta-sheets. Structure- based sequence alignment revealed that the core region harbors a third PUR repeat at its C terminus. Subsequent characterization by small-angle x-ray scattering (SAXS) and size-exclusion chromatography indicated that PUR repeat III mediates dimerization of Pur-alpha. Surface envelopes calculated from SAXS data show that the Pur-alpha dimer consisting of repeats I to III is arranged in a Z-like shape. This unexpected domain organization of the entire core domain of Pur-alpha has direct implications for ssDNA/ssRNA and dsDNA binding. AU - Graebsch, A. AU - Roche, S. AU - Niessing, D. C1 - 1297 C2 - 26482 CY - Washington SP - 18521-18526 TI - X-ray structure of pur-α reveals a whirly-like fold and an unusual nucleic-acid binding surface. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 44 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNF alpha in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNF alpha levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) beta-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause. AU - Khasawneh, J.* AU - Schulz, M.D.* AU - Walch, A.K. AU - Rozman, J. AU - Hrabě de Angelis, M. AU - Klingenspor, M.* AU - Buck, A.* AU - Schwaiger, M.* AU - Saur, D.* AU - Schmid, R.M.* AU - Klöppel, G.* AU - Sipos, B.* AU - Greten, F.R.* AU - Arkan, M.C.* C1 - 809 C2 - 26213 SP - 3354-3359 TI - Inflammation and mitochondrial fatty acid beta-oxidation link obesity to early tumor promotion. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 9 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - Prions are infectious, self-propagating amyloid-like protein aggregates of mammals and fungi. We have studied aggregation propensities of a yeast prion domain in cell culture to gain insights into general mechanisms of prion replication in mammalian cells. Here, we report the artificial transmission of a yeast prion across a phylogenetic kingdom. HA epitope-tagged yeast Sup35p prion domain NM was stably expressed in murine neuroblastoma cells. Although cytosolically expressed NM-HA remained soluble, addition of fibrils of bacterially produced Sup35NM to the medium efficiently induced appearance of phenotypically and biochemically distinct NM- HA aggregates that were inherited by daughter cells. Importantly, NM-HA aggregates also were infectious to recipient mammalian cells expressing soluble NM-HA and, to a lesser extent, to yeast. The fact that the yeast Sup35NM domain can propagate as a prion in neuroblastoma cells strongly argues that cellular mechanisms support prion-like inheritance in the mammalian cytosol. AU - Krammer, C.* AU - Kryndushkin, D.* AU - Suhre, M.H.* AU - Kremmer, E. AU - Hofmann, A.* AU - Pfeifer, A.* AU - Scheibel, T.* AU - Wickner, R.B.* AU - Schatzl, H.M.* AU - Vorberg, I.* C1 - 2900 C2 - 26518 SP - 462-467 TI - The yeast Sup35NM domain propagates as a prion in mammalian cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 2 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - As champagne or sparkling wine is poured into a glass, the myriad of ascending bubbles collapse and radiate a multitude of tiny droplets above the free surface into the form of very characteristic and refreshing aerosols. Ultrahigh-resolution MS was used as a nontargeted approach to discriminate hundreds of surface active compounds that are preferentially partitioning in champagne aerosols; thus, unraveling different chemical fingerprints between the champagne bulk and its aerosols. Based on accurate exact mass analysis and database search, tens of these compounds overconcentrating in champagne aerosols were unambiguously discriminated and assigned to compounds showing organoleptic interest or being aromas precursors. By drawing a parallel between the fizz of the ocean and the fizz in Champagne wines, our results closely link bursting bubbles and flavor release; thus, supporting the idea that rising and collapsing bubbles act as a continuous paternoster lift for aromas in every glass of champagne. AU - Liger-Belair, G.* AU - Cilindre, C.* AU - Gougeon, R.D.* AU - Lucio, M. AU - Gebefügi, I. AU - Jeandet, P.* AU - Schmitt-Kopplin, P. C1 - 2513 C2 - 26413 CY - Washington SP - 16545-16549 TI - Unraveling different chemical fingerprints between a champagne wine and its aerosols. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 39 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains sternness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2(-/-)gamma c(-/-) mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated sternness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor. AU - Richter, G.H.S.* AU - Plehm, S.* AU - Fasan, A.* AU - Rössler, S.* AU - Unland, R.* AU - Bennani-Baiti, I.M.* AU - Hotfilder, M.* AU - Löwel, D.* AU - von Luettichau, I.* AU - Mossbrugger, I. AU - Quintanilla-Martinez, L. AU - Kovar, H.* AU - Staege, M.S.* AU - Müller-Tidow, C.* AU - Burdach, S.* C1 - 881 C2 - 26633 SP - 5324-5329 TI - EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 106 IS - 13 PB - Natl Acad Sciences PY - 2009 SN - 0027-8424 ER - TY - JOUR AB - New neurons in the adult dentate gyrus are widely held to incorporate into hippocampal circuitry via a stereotypical sequence of morphological and physiological transitions, yet the molecular control over this process remains unclear. We studied the role of brain-derived neurotrophic factor (BDNF)/TrkB signaling in adult neurogenesis by deleting the full-length TrkB via Cre expression within adult progenitors in TrkB(lox/lox) mice. By 4 weeks after deletion, the growth of dendrites and spines is reduced in adult-born neurons demonstrating that TrkB is required to create the basic organization of synaptic connections. Later, when new neurons normally display facilitated synaptic plasticity and become preferentially recruited into functional networks, lack of TrkB results in impaired neurogenesis-dependent long-term potentiation and cell survival becomes compromised. Because of the specific lack of TrkB signaling in recently generated neurons a remarkably increased anxiety-like behavior was observed in mice carrying the mutation, emphasizing the contribution of adult neurogenesis in regulating mood-related behavior. AU - Bergami, M.* AU - Rimondini, R.* AU - Santi, S.* AU - Blum, R.* AU - Götz, M. AU - Canossa, M.* C1 - 1697 C2 - 25917 SP - 15570-15575 TI - Deletion of TrkB in adult progenitors alters newborn neuron integration into hippocampal circuits and increases anxiety-like behavior. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 40 PB - National Academy of Sciences PY - 2008 SN - 0027-8424 ER - TY - JOUR AB - Reactive gliosis is the universal reaction to brain injury, but the precise origin and subsequent fate of the glial cells reacting to injury are unknown. Astrocytes react to injury by hypertrophy and up-regulation of the glial-fibrillary acidic protein (GFAP). Whereas mature astrocytes do not normally divide, a subpopulation of the reactive GFAP(+) cells does so, prompting the question of whether the proliferating GFAP(+) cells arise from endogenous glial progenitors or from mature astrocytes that start to proliferate in response to brain injury. Here we show by genetic fate mapping and cell type-specific viral targeting that quiescent astrocytes start to proliferate after stab wound injury and contribute to the reactive gliosis and proliferating GFAP(+) cells. These proliferating astrocytes remain within their lineage in vivo, while a more favorable environment in vitro revealed their multipotency and capacity for self-renewal. Conversely, progenitors present in the adult mouse cerebral cortex labeled by NG2 or the receptor for the platelet-derived growth factor (PDGFRalpha) did not form neurospheres after (or before) brain injury. Taken together, the first fate-mapping analysis of astrocytes in the adult mouse cerebral cortex shows that some astrocytes acquire stem cell properties after injury and hence may provide a promising cell type to initiate repair after brain injury. AU - Buffo, A. AU - Rite, I. AU - Tripathi, P. AU - Lepier, A.* AU - Colak, D. AU - Horn, A.P. AU - Mori, T. AU - Götz, M. C1 - 3257 C2 - 25399 SP - 3581-3586 TI - Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 9 PB - Natl. Acad. Sciences PY - 2008 SN - 0027-8424 ER - TY - JOUR AB - Two case/control studies with different phenotypes, marker densities, and microarrays were examined for the most significant single markers in defined regions. They show a pronounced bias toward exaggerated significance that increases with the number of observed markers and would increase further with imputed markers. This bias is eliminated by Bonferroni adjustment, thereby allowing combination by principal component analysis with a Malecot model composite likelihood evaluated by a permutation procedure to allow for multiple dependent markers. This intermediate value identifies the only demonstrated causal locus as most significant even in the preliminary analysis and clearly recognizes the strongest candidate in the other sample. Because the three metrics (most significant single marker, composite likelihood, and their principal component) are correlated, choice of the n smallest P values by each test gives <3n regions for follow-up in the next stage. In this way, methods with different response to marker selection and density are given approximately equal weight and economically compared, without expressing an untested prejudice or sacrificing the most significant results for any of them. Large numbers of cases, controls, and markers are by themselves insufficient to control type 1 and 2 errors, and so efficient use of multiple metrics with Bonferroni adjustment promises to be valuable in identifying causal variants and optimal design simultaneously. AU - Gibson, J.* AU - Tapper, W.* AU - Cox, D.* AU - Zhang, W.* AU - Pfeufer, A.* AU - Gieger, C. AU - Wichmann, H.-E. AU - Kääb, S.* AU - Collins, A.R.* AU - Meitinger, T.* AU - Morton, N.* C1 - 2480 C2 - 25175 SP - 2592-2597 TI - A multimetric approach to analysis of genome-wide association by single markers and composite likelihood. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 7 PB - National Academy of Sciences PY - 2008 SN - 0027-8424 ER - TY - JOUR AB - Imaging of targeted fluorescent probes offers significant advantages for investigating disease and tissue function in animal models in vivo. Conversely, macroscopic tomographic imaging is challenging because of the high scatter of light in biological tissue and the ill-posed nature of the reconstruction mathematics. In this work, we use the earliest-transmitted photons through Lewis Lung Carcinoma bearing mice, thereby dramatically reducing the effect of tissue scattering. By using a fluorescent probe sensitive to cysteine proteases, the method yielded outstanding imaging performance compared with conventional approaches. Accurate visualization of biochemical abnormalities was achieved, not only in the primary tumor, but also in the surrounding tissue related to cancer progression and inflammatory response at the organ level. These findings were confirmed histologically and with ex vivo fluorescence microscopy. The imaging fidelity demonstrated underscores a method that can use a wide range of fluorescent probes to accurately visualize cellular- and molecular-level events in whole animals in vivo. AU - Niedre, M.J.* AU - de Kleine, R.H.* AU - Aikawa, E.* AU - Kirsch, D.G.* AU - Weissleder, R.* AU - Ntziachristos, V. C1 - 1920 C2 - 25946 SP - 19126-19131 TI - Early photon tomography allows fluorescence detection of lung carcinomas and disease progression in mice in vivo. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 49 PB - National Academy of Sciences PY - 2008 SN - 0027-8424 ER - TY - JOUR AB - In all eukaryotic cells, origins of DNA replication are characterized by the binding of the origin recognition complex (ORC). How ORC is positioned to sites where replication initiates is unknown, because metazoan ORC binds DNA without apparent sequence specificity. Thus, additional factors might be involved in ORC positioning. Our experiments indicate that a family member of the high-mobility group proteins, HMGA1a, can specifically target ORC to DNA. Coimmunoprecipitations and imaging studies demonstrate that HMGA1a interacts with different ORC subunits in vitro and in vivo. This interaction occurs mainly in AT-rich heterochromatic regions to which HMGA1a localizes. Fusion proteins of HMGA1a and the DNA-binding domain of the viral factor EBNA1 or the prokaryotic tetracycline repressor, TetR, can recruit ORC to cognate operator sites forming functional origins of DNA replication. When HMGA1a is targeted to plasmid DNA, the prereplicative complex is assembled during G(1) and the amount of ORC correlates with the local concentration of HMGA1a. Nascent-strand abundance assays demonstrate that DNA replication initiates at or near HMGA1a-rich sites. Our experiments indicate that chromatin proteins can target ORC to DNA, suggesting they might specify origins of DNA replication in metazoan cells. AU - Thomae, A.W. AU - Pich, D. AU - Brocher, J.* AU - Spindler, M.-P. AU - Berens, C.* AU - Hock, R.* AU - Hammerschmidt, W. AU - Schepers, A. C1 - 1279 C2 - 25305 SP - 1692-1697 TI - Interaction between HMGA1a and the origin recognition complex creates site-specific replication origins. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 5 PB - National Academy of Sciences PY - 2008 SN - 0027-8424 ER - TY - JOUR AB - Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters in vitro. Its association with TBP prevents the binding of TFIIB and leads to inhibition of preinitiation complex formation. Here, we investigate the association of NC2 subunit-alpha with human RNA polymerase II promoter regions by using gene-specific ChIP and genome-wide promoter ChIPchip analyses. We find NC2alpha associated with a large number of human promoters, where it peaks close to the core regions. NC2 occupancy in vivo positively correlates with mRNA levels, which perhaps reflects its capacity to stabilize TBP on promoter regions. In single gene analyses, we confirm core promoter binding and in addition map the NC2 complex to enhancer proximal regions. High-occupancy histone genes display a stable NC2/TFIIB ratio during the cell cycle, which otherwise varies markedly from one gene to another. The latter is at least in part explained by an observed negative correlation of NC2 occupancy with the presence of the TFIIB recognition element in core promoter regions. Our data establish the genome-wide basis for general and gene-specific functions of NC2 in mammalian cells. AU - Albert, T.K. AU - Grote, K.* AU - Boeing, S. AU - Stelzer, G. AU - Schepers, A. AU - Meisterernst, M. C1 - 4289 C2 - 24882 SP - 10000-10005 TI - Global distribution of negative cofactor 2 subunit-alpha on human promoters. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 24 PB - National Academy of Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - Myosin-motors are conserved from yeast to human and transport a great variety of cargoes. Most plus-end directed myosins, which constitute the vast majority of all myosin motors, form stable dimers and interact constitutively with their cargo complexes. To date, little is known about regulatory mechanisms for cargo-complex assembly. In this study, we show that the type V myosin Myo4p binds to its cargo via two distinct binding regions, the C-terminal tail and a coiled-coil domain-containing fragment. Furthermore, we find that Myo4p is strictly monomeric at physiologic concentrations. Because type V myosins are thought to require dimerization for processive movement, a mechanism must be in place to ensure that oligomeric Myo4p is incorporated into cargo-translocation complexes. Indeed, we find that artificial dimerization of the Myo4p C-terminal tail promotes stabilization of myosin-cargo complexes, suggesting that full-length Myo4p dimerizes in the cocomplex as well. We also combined the Myo4p C-terminal tail with the coiled-coil region, lever arm, and motor domain from a different myosin to form constitutively dimeric motor proteins. This heterologous motor successfully translocates its cargo in vivo, suggesting that wild-type Myo4p may also function as a dimer during cargo-complex transport. AU - Heuck, A. AU - Du, T.G. AU - Jellbauer, S.* AU - Richter, K.* AU - Kruse, C.* AU - Jaklin, S. AU - Müller, M. AU - Buchner, J.* AU - Jansen, R.P. AU - Niessing, D. C1 - 1922 C2 - 24999 SP - 19778-19783 TI - Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 105 IS - 50 PB - Natl. Acad. Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - Acute lymphoblastic leukemia (ALL) is the most common childhood cancer, whereas acute myeloid leukemia (AML) is the most common acute leukemia in adults. In general, ALL has a better prognosis than AML. To understand the distinct mechanisms in leukemogenesis between ALL and AML and to identify markers for diagnosis and treatment, we performed a large-scale genome-wide microRNA (miRNA, miR) expression profiling assay and identified 27 miRNAs that are differentially expressed between ALL and AML. Among them, miR-128a and -128b are significantly overexpressed, whereas let-7b and miR-223 are significantly down-regulated in ALL compared with AML. They are the most discriminatory miRNAs between ALL and AML. Using the expression signatures of a minimum of two of these miRNAs resulted in an accuracy rate of >95% in the diagnosis of ALL and AML. The differential expression patterns of these four miRNAs were validated further through large-scale real-time PCR on 98 acute leukemia samples covering most of the common cytogenetic subtypes, along with 10 normal control samples. Furthermore, we found that overexpression of miR-128 in ALL was at least partly associated with promoter hypomethylation and not with an amplification of its genomic locus. Taken together, we showed that expression signatures of as few as two miRNAs could accurately discriminate ALL from AML, and that epigenetic regulation might play an important role in the regulation of expression of miRNAs in acute leukemias. AU - Mi, S.* AU - Lu, J.* AU - Sun, M.* AU - Li, Z.* AU - Zhang, H.* AU - Neilly, M.B.* AU - Wang, Y.* AU - Qian, Z.* AU - Jin, J.* AU - Zhang, Y.* AU - Bohlander, S.K. AU - Le Beau, M.M.* AU - Larson, R.A.* AU - Golub, T.R.* AU - Rowley, J.D.* AU - Chen, J.* C1 - 2043 C2 - 25038 SP - 19971-19976 TI - MicroRNA expression signatures accurately discriminate acute lymphoblastic leukemia from acute myeloid leukemia. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 50 PB - Natl. Acad. Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - i.v. infusion of native autoantigen or its altered peptide variants is an important therapeutic option for the treatment of autoimmune diseases, because it selectively targets the disease-inducing T cells. To learn more about the mechanisms and kinetics of this approach, we visualized the crucial initial effects of i.v. infusion of peptides or intact protein on GFP-tagged autoaggressive CD4(+) effector T cells using live-video and two-photon in situ imaging of spleens in living animals. We found that the time interval between i.v. injection of intact protein to first changes in T cell behavior was extremely short; within 10 min after protein application, the motility of the T cells changed drastically. They slowed down and became tethered to local sessile stromal cells. A part of the cells aggregated to form clusters. Within the following 20 min, IFN-gamma mRNA was massively (>100-fold) up-regulated; surface IL-2 receptor and OX-40 (CD 134) increased 1.5 h later. These processes depleted autoimmune T cells in the blood circulation, trapping the cells in the peripheral lymphoid organs and thus preventing them from invading the CNS. This specific blockage almost completely abrogated CNS inflammation and clinical disease. These findings highlight the speed and efficiency of antigen recognition in vivo and add to our understanding of T cell-mediated autoimmunity. AU - Odoardi, F.* AU - Kawakami, N.* AU - Li, Z.* AU - Cordiglieri, C.* AU - Streyl, K.* AU - Nosov, M.* AU - Klinkert, WE.* AU - Ellwart, J.W. AU - Bauer, J.* AU - Lassmann, H.* AU - Wekerle, H.* AU - Flügel, A.* C1 - 1430 C2 - 24798 SP - 920-925 TI - Instant effect of soluble antigen on effector T cells in peripheral immune organs during immunotherapy of autoimmune encephalomyelitis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 3 PB - National Academy of Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - In contrast to a detailed understanding of antiviral cellular immune responses, the impact of neutralizing antibodies for the resolution of acute hepatitis C is poorly defined. The analysis of neutralizing responses has been hampered by the fact that patient cohorts as well as hepatitis C virus (HCV) strains are usually heterogeneous, and that clinical data from acute-phase and long-term follow-up after infection are not readily available. Using an infectious retroviral HCV pseudoparticle model system, we studied a cohort of women accidentally exposed to the same HCV strain of known sequence. In this single-source outbreak of hepatitis C, viral clearance was associated with a rapid induction of neutralizing antibodies in the early phase of infection. Neutralizing antibodies decreased or disappeared after recovery from HCV infection. In contrast, chronic HCV infection was characterized by absent or low-titer neutralizing antibodies in the early phase of infection and the persistence of infection despite the induction of cross-neutralizing antibodies in the late phase of infection. These data suggest that rapid induction of neutralizing antibodies during the early phase of infection may contribute to control of HCV infection. This finding may have important implications for understanding the pathogenesis of HCV infection and for the development of novel preventive and therapeutic antiviral strategies. AU - Pestka, J.M.* AU - Zeisel, M.B.* AU - Bläser, E.* AU - Schürmann, P.* AU - Bartosch, B.* AU - Cosset, F.L.* AU - Patel, A.H.* AU - Meisel, H.* AU - Baumert, J.J. AU - Viazov, S.* AU - Rispeter, K.* AU - Blum, H.E.* AU - Roggendorf, M.* AU - Baumert, T.F.* C1 - 2448 C2 - 24606 SP - 6025-6030 TI - Rapid induction of virus-neutralizing antibodies and viral clearance in a single-source outbreak of hepatitis C. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 14 PB - Natl. Acad. Sci. PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - Notch-mediated induction of Nodal at the vertebrate node is a critical step in initiating left-right (LR) asymmetry. In mice and zebrafish we show that Baf60c, a subunit of the Swi/Snf-like BAF chromatin remodeling complex, is essential for establishment of LR asymmetry. Baf60c knockdown mouse embryos fail to activate Nodal at the node and also have abnormal node morphology with mixing of crown and pit cells. In cell culture, Baf60c is required for Notch-dependent transcriptional activation and functions to stabilize interactions between activated Notch and its DNA-binding partner, RBP-J. Brg1 is also required for these processes, suggesting that BAF complexes are key components of nuclear Notch signaling. We propose a critical role for Baf60c in Notch-dependent transcription and LR asymmetry. AU - Takeuchi, J.K.* AU - Lickert, H. AU - Bisgrove, B.W.* AU - Sun, X.* AU - Yamamoto, M.* AU - Chawengsaksophak, K.* AU - Hamada, H.* AU - Yost, H.J.* AU - Rossant, J.* AU - Bruneau, BG.* C1 - 2526 C2 - 24695 SP - 846-851 TI - Baf60c is a nuclear Notch signaling component required for the establishment of left-right asymmetry. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 3 PB - Natl. Acad. Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - Small intestine plasmacytoid dendritic cells (pDC) are poorly characterized. Here, we demonstrate that intestinal pDC show the characteristic plasma cell-like morphology, and are recognized by antibodies against B220, Ly6c, 120G8, and PDCA-1, markers that are typically expressed by pDC. Furthermore, intestinal pDC carry high levels of CCR9 and are largely absent in the intestine, but not in lung, liver, or secondary lymphoid organs of CCR9-deficient animals. Competitive adoptive transfers reveal that CCR9-deficient pDC are impaired in homing to the small intestine after i.v. transfer. In a model of cholera toxin-induced gut inflammation, pDC are recruited to the intestine in WT but not CCR9-deficient animals. Furthermore, after oral application of a Toll-like receptor (TLR) 7/8 ligand, myeloid DC of the lamina propria are rapidly mobilized in WT but not in CCR9-deficient animals. Mobilization of myeloid DC can be completely rescued by adoptively transferred WT pDC to CCR9-deficient mice before oral challenge. Together, our data reveal an essential role for CCR9 in the homing of pDC to the intestine under homeostatic and inflammatory conditions and demonstrate an important role for intestinal pDC for the rapid mobilization of lamina propria DC. AU - Wendland, M.* AU - Czeloth, N.* AU - Mach, N.* AU - Malissen, B.* AU - Kremmer, E. AU - Pabst, O.* AU - Forster, R.* C1 - 3002 C2 - 24800 SP - 6347-6352 TI - CCR9 is a homing receptor for plasmacytoid dendritic cells to the small intestine. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 15 PB - National Academy of Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - Purkinje cells are one of the major types of neurons that form the neural circuitry in the cerebellum essential for fine control of movement and posture. During development, Purkinje cells also are critically involved in the regulation of proliferation of progenitors of granule cells, the other major type of neurons in the cerebellum. The process that controls differentiation of Purkinje cells from their early precursors is poorly understood. Here we report that two closely related LIM-homeobox genes, Lhx1 and Lhx5, were expressed in the developing Purkinje cells soon after they exited the cell cycle and migrated out of the cerebellar ventricular zone. Double-mutant mice lacking function of both Lhx1 and Lhx5 showed a severe reduction in the number of Purkinje cells. In addition, targeted inactivation of Ldb1, which encodes a cofactor for all LIM-homeodomain proteins, resulted in a similar phenotype. Our studies thus provide evidence that these transcription regulators are essential for controlling Purkinje cell differentiation in the developing mammalian cerebellum. AU - Zhao, Y.* AU - Kwan, K.M.* AU - Mailloux, C.M.* AU - Lee, W.K.* AU - Grinberg, A.* AU - Wurst, W. AU - Behringer, R.R.* AU - Westphal, H.* C1 - 3743 C2 - 24851 SP - 13182-13186 TI - LIM-homeodomain proteins Lhx1 and Lhx5, and their cofactor Ldb1, control Purkinje cell differentiation in the developing cerebellum. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 104 IS - 32 PB - National Academy of Sciences PY - 2007 SN - 0027-8424 ER - TY - JOUR AB - EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes. AU - Altmann, M. AU - Pich, D. AU - Ruiss, R. AU - Wang, J.* AU - Sugden, B.* AU - Hammerschmidt, W. C1 - 5121 C2 - 24014 SP - 14188-14193 TI - Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 38 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - Somatic hypermutation of Ig genes is initiated by transcription-coupled cytidine deamination in Ig loci. Error-prone processing of the resultant DNA lesions is thought to cause extensive mutagenesis, but it is presently an enigma how and why error-prone rather than error-free repair pathways are recruited. During DNA replication, recruitment of error-prone translesion polymerases may be mediated by Rad6/Rad18-mediated ubiquitination of proliferating cell nuclear antigen, a major switchboard controlling the fidelity of DNA lesion bypass in eukaryotes. By inactivation of Rad18 in the DT40 B cell line, we show that the Rad6 pathway is involved in somatic hypermutation in these cells. Our findings imply that targeted recruitment of mutagenic polymerases by the Rad6 pathway contributes to the complex process of somatic hypermutation and provide a framework for more detailed mechanistic studies of the mutagenesis phase of secondary Ig diversification. AU - Bachl, J. AU - Ertongur, I. AU - Jungnickel, B. C1 - 3575 C2 - 23734 SP - 12081-12086 TI - Involvement of Rad18 in somatic hypermutation. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 32 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - Comment on: Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells. [Proc. Natl. Acad. Sci. U. S. A. 2006] AU - Bornkamm, G.W. AU - Behrends, U.* AU - Mautner, J. C1 - 5347 C2 - 23740 SP - 7201-7202 TI - The infectious kiss: Newly infected B cells deliver Epstein-Barr virus to epithelial cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 19 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - Genome sequencing of the model legumes, Medicago truncatula and Lotus japonicus, provides an opportunity for large-scale sequence-based comparison of two genomes in the same plant family. Here we report synteny comparisons between these species, including details about chromosome relationships, large-scale synteny blocks, microsynteny within blocks, and genome regions lacking clear correspondence. The Lotus and Medicago genomes share a minimum of 10 large-scale synteny blocks, each with substantial collinearity and frequently extending the length of whole chromosome arms. The proportion of genes syntenic and collinear within each synteny block is relatively homogeneous. Medicago-Lotus comparisons also indicate similar and largely homogeneous gene densities, although gene-containing regions in Mt occupy 20-30% more space than Lj counterparts, primarily because of larger numbers of Mt retrotransposons. Because the interpretation of genome comparisons is complicated by large-scale genome duplications, we describe synteny, synonymous substitutions and phylogenetic analyses to identify and date a probable whole-genome duplication event. There is no direct evidence for any recent large-scale genome duplication in either Medicago or Lotus but instead a duplication predating speciation. Phylogenetic comparisons place this duplication within the Rosid I clade, clearly after the split between legumes and Salicaceae (poplar). AU - Cannon, S.B.* AU - Sterck, L.* AU - Rombauts, S.* AU - Sato, S.* AU - Cheung, F.* AU - Gouzy, J.* AU - Wang, X.* AU - Mudge, J.* AU - Vasdewani, J.* AU - Schiex, T.* AU - Spannagl, M. AU - Monaghan, E.* AU - Nicholson, C.* AU - Humphray, S.J.* AU - Schoof, H.* AU - Mayer, K.F.X. AU - Rogers, J.* AU - Quétier, F.* AU - Oldroyd, G.E.* AU - Debellé, F.* AU - Cook, D.R.* AU - Retzel, E.F.* AU - Roe, B.A.* AU - Town, C.D.* AU - Tabata, S.* AU - van de Peer, Y.* AU - Young, N.D.* C1 - 2631 C2 - 23886 SP - 14959-14964 TI - Legume genome evolution viewed through the Medicago truncatula and Lotus japonicus genomes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 40 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AU - Lee, S.* AU - Chen, J.* AU - Zhou, G.* AU - Shi, R.Z.* AU - Bouffard, G.G.* AU - Kocherginsky, M.* AU - Ge, X.* AU - Sun, M.* AU - Jayathilaka, N.* AU - Kim, Y.C.* AU - Emmanuel, N.* AU - Bohlander, S.K. AU - Minden, M. AU - Kline, J.* AU - Ozer, O.* AU - Larson, R.A.* AU - LeBeau, M.M.* AU - Green, E.D.* AU - Trent, J.* AU - Karrison, T.* AU - Liu, P.P.* AU - Wang, S.M.* AU - Rowley, J.D.* C1 - 3014 C2 - 23986 SP - 1030-1035 TI - Gene expression profiles in acute myeloid leukemia with common translocations using SAGE. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 4 PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - Differences in the cleavage specificities of constitutive proteasomes and immunoproteasomes significantly affect the generation of MHC class I ligands and therefore the activation of CD8-positive T cells. Based on these findings, we investigated whether proteasomal specificity also influences CD8-positive T cells during thymic selection by peptides derived from self proteins. We find that one of the self peptides responsible for positive selection of ovalbumin-specific OT-1 T cells, which is derived from the f-actin capping protein (Cpalpha1), is efficiently generated only by immunoproteasomes. Furthermore, OT-1 mice backcrossed onto low molecular mass protein 7 (LMP7)-deficient mice show a 50% reduction of OT-1 cells. This deficiency is also observed after transfer of BM from OT-1 mice in LMP7-deficient mice and can be corrected by the injection of the Cpalpha1 peptide. Interestingly, WT and LMP7-deficient mice mount comparable immune responses to the ovalbumin-derived epitope SIINFEKL. However, their cytotoxic T lymphocytes (CTL) differ in the use of T cell receptor Vbeta genes. CTL derived from WT mice use Vbeta8 or Vbeta5 (the latter is also used by OT-1 cells), whereas SIINFEKL-specific CTL from LMP7-deficient mice are exclusively Vbeta8-positive. Taken together, our experiments provide strong evidence that proteasomal specificity shapes the repertoire of T cells participating in antigen-specific immune responses. AU - Osterloh, P.* AU - Linkemann, K. AU - Tenzer, S.* AU - Rammensee, H.-G.* AU - Radsak, M.P.* AU - Busch, D.H. AU - Schild, H.* C1 - 2439 C2 - 24022 SP - 5042-5047 TI - Proteasomes shape the repertoire of T cells participating in antigen-specific immune responses. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 13 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - MENX is a recessive multiple endocrine neoplasia-like syndrome in the rat. The tumor spectrum in MENX overlaps those of human multiple endocrine neoplasia (MEN) types 1 and 2. We mapped the MenX locus to the distal part of rat chromosome 4, excluding the homologs of the genes responsible for the MEN syndromes (RET and MEN1) and syndromes with an endocrine tumor component (VHL and NF1). We report the fine mapping of the disease locus and the identification of a homozygous frameshift mutation in Cdkn1b, encoding the cyclin-dependent kinase inhibitor p27(Kip1). As a consequence of the mutation, MENX-affected rats show dramatic reduction in p27(Kip1) protein. We have identified a germ-line nonsense mutation in the human CDKN1B gene in a MEN1 mutation-negative patient presenting with pituitary and parathyroid tumors. Expanded pedigree analysis shows that the mutation is associated with the development of an MEN1-like phenotype in multiple generations. Our findings demonstrate that germ-line mutations in p27(Kip1) can predispose to the development of multiple endocrine tumors in both rats and humans. AU - Pellegata, N.S. AU - Quintanilla-Martinez, L. AU - Siggelkow, H.* AU - Samson, E. AU - Bink, K. AU - Höfler, H. AU - Fend, F.* AU - Graw, J. AU - Atkinson, M.J.* C1 - 5327 C2 - 23939 SP - 15558-15563 TI - Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 42 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease. AU - Poët, M.* AU - Kornak, U.* AU - Schweizer, M.* AU - Zdebik, A.A.* AU - Scheel, O.* AU - Hölter, S.M. AU - Wurst, W. AU - Schmitt, A.* AU - Fuhrmann, J.C.* AU - Planells-Cases, R.* AU - Mole, S.E.* AU - Hübner, C.A.* AU - Jentsch, T.J.* C1 - 1815 C2 - 23952 SP - 13854-13859 TI - Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 103 IS - 37 PB - National Academy of Sciences PY - 2006 SN - 0027-8424 ER - TY - JOUR AB - IFN-gamma secretion by natural killer (NK) cells is pivotal to several tumor and viral immune responses, during which NK and dendritic cells cooperation is required. We show here that macrophages are mandatory for NK cell IFN-gamma secretion in response to erythrocytes infected with Plasmodium falciparum (Pf), a causative agent of human malaria. In addition, direct sensing of Pf infection by NK cells induces their production of the proinflammatory chemokine CXCL8, without triggering their granule-mediated cytolytic programs. Despite their reported role in Pf recognition, Toll-like receptor (TLR) 2, TLR9, and TLR11 are individually dispensable for NK cell activation induced by Pf-infected erythrocytes. However, IL-18R expression on NK cells, IL-18 production by macrophages, and MyD88 on both cell types are essential components of this previously undescribed pathway of NK cell activation in response to a parasite infection. AU - Baratin, M.* AU - Roetynck, S.* AU - Lépolard, C.* AU - Falk, C. AU - Sawadogo, S.* AU - Uematsu, S.* AU - Akira, S.* AU - Ryffel, B.* AU - Tiraby, J.-G.* AU - Alexopoulou, L.* AU - Kirschning, C.J.* C1 - 2435 C2 - 23069 SP - 14747-14752 TI - Natural killer cell and macrophage cooperation in MyD88-dependent innate responses to Plasmodium falciparum. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 102 IS - 41 PB - National Academy of Sciences PY - 2005 SN - 0027-8424 ER - TY - JOUR AB - Despite the presence of neural stem cells and ongoing neurogenesis in some regions of the adult mammalian brain, neurons are not replaced in most brain regions after injury. With the aim to unravel factors contributing to the failure of neurogenesis in the injured cerebral cortex, we examined the expression of cell fate determinants after acute brain injuries, such as stab wound or focal ischemia, and in a model of chronic amyloid deposition. Although none of the neurogenic factors, such as Pax6, Mash1, Ngn2, was detected in the injured parenchyma, we observed a strong up-regulation of the bHLH transcription factor Olig2, but not Olig1, upon acute and chronic injury. To examine the function of Olig2 in brain lesion, we injected retroviral vectors containing a dominant negative form of Olig2 into the lesioned cortex 2 days after a stab wound. Antagonizing Olig2 function resulted in a significant number of infected cells generating immature neurons that were not observed after injection of the control virus. These data, therefore, imply Olig2 as a repressor of neurogenesis in cells reacting to brain injury and open innovative perspectives toward evoking endogenous neuronal repair. AU - Buffo, A. AU - Vosko, O.* AU - Ertürk, D. AU - Hamann, G.F.* AU - Jucker, M.* AU - Rowitch, D.* AU - Götz, M. C1 - 3702 C2 - 23216 SP - 18183-18188 TI - Expression pattern of the transcription factor Olig2 in response to brain injuries: Implications for neuronal repair. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 102 IS - 50 PB - National Academy of Sciences PY - 2005 SN - 0027-8424 ER - TY - JOUR AB - The activating receptor NKG2D recognizes a wide range of different ligands, some of which are primarily expressed in "stressed" tissues or on tumor cells. Until now, similar stimulatory effects on natural killer and CD8+ T cells have been described for all NKG2D ligands, and the NKG2D receptor/ligand system has therefore been interpreted as a sensor system involved in tumor immune surveillance and activation of immune responses. We show here that the NKG2D ligands H60 and MIC class 1 chain-related protein A (MICA) can also mediate strong suppressive effects on T cell proliferation. Responsiveness to H60- and MICA-mediated suppression requires IL-10 and involves a receptor other than NKG2D. These findings might provide explanations for the observation that strong in vivo NKG2D ligand expression, such as that on tumor cells, sometimes fails to support effective immune responses and links this observation to a distinct subgroup of NKG2D ligands. AU - Kriegeskorte, A.K.* AU - Gebhardt, F.E.* AU - Porcellini, S.* AU - Schiemann, M. AU - Stemberger, C.* AU - Franz, TJ.* AU - Huster, K.M. AU - Carayannopoulos, L.N.* AU - Yokoyama, W.M.* AU - Colonna, M.* AU - Siccardi, A.G.* AU - Bauer, S.* AU - Busch, D.H. C1 - 2273 C2 - 23311 SP - 11805-11810 TI - NKG2D-independent suppression of T cell proliferation by H60 and MICA. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 102 IS - 33 PB - National Academy of Sciences PY - 2005 SN - 0027-8424 ER - TY - JOUR AB - A major advantage of the mouse model lies in the increasing information on its genome, transcriptome, and proteome, as well as in the availability of a fast growing number of targeted and induced mutant alleles. However, data from comparative transcriptome and proteome analyses in this model organism are very limited. We use DNA chip-based RNA expression profiling and 2D gel electrophoresis, combined with peptide mass fingerprinting of liver and kidney, to explore the feasibility of such comprehensive gene expression analyses. Although protein analyses mostly identify known metabolic enzymes and structural proteins, transcriptome analyses reveal the differential expression of functionally diverse and not yet described genes. The comparative analysis suggests correlation between transcriptional and translational expression for the majority of genes. Significant exceptions from this correlation confirm the complementarities of both approaches. Based on RNA expression data from the 200 most differentially expressed genes, we identify chromosomal colocalization of known, as well as not yet described, gene clusters. The determination of 29 such clusters may suggest that coexpression of colocalizing genes is probably rather common. AU - Mijalski, T. AU - Harder, A.* AU - Kersten, M.* AU - Horsch, M. AU - Strom, T.M. AU - Liebscher, H.V. AU - Lottspeich, F.* AU - Hrabě de Angelis, M. AU - Beckers, J. C1 - 2326 C2 - 22734 SP - 8621-8626 TI - Identification of coexpressed gene clusters in a comparative analysis of transcriptome and proteome in mouse tissues. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 102 IS - 24 PB - National Academy of Sciences PY - 2005 SN - 0027-8424 ER - TY - JOUR AB - A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community. AU - Schnütgen, F.* AU - De-Zolt, S.* AU - van Sloun, P.* AU - Hollatz, M. AU - Floß, T. AU - Hansen, J. AU - Altschmied, J.* AU - Seisenberger, C. AU - Ghyselinck, N.B.* AU - Ruiz, P.* AU - Chambon, P.* AU - Wurst, W. AU - von Melchner, H.* C1 - 1116 C2 - 22742 SP - 7221-7226 TI - Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 102 IS - 20 PB - National Academy of Sciences PY - 2005 SN - 0027-8424 ER - TY - JOUR AB - Can CD4(+) and CD8(+) "memory" T cells that are generated and maintained in the context of low-level virus persistence protect, in the absence of antibody, against a repeat challenge with the same pathogen? Although immune T cells exert effective, long-term control of a persistent gamma-herpesvirus (gammaHV68) in Ig(-/-) microMT mice, subsequent exposure to a high dose of the same virus leads to further low-level replication in the lung. This lytic phase in the respiratory tract is dealt with effectively by the recall of memory T cells induced by a gammaHV68 recombinant (M3LacZ) that does not express the viral M3 chemokine binding protein. At least for the CD8(+) response, greater numbers of memory T cells confer enhanced protection in the M3LacZ-immune mice. However, neither WT gammaHV68 nor the minimally persistent M3LacZ primes the T cell response to the extent that a WT gammaHV68 challenge fails to establish latency in the microMT mice. Memory CD4(+) and CD8(+) T cells thus act together to limit gammaHV68 infection but are unable to provide absolute protection against a high-dose, homologous challenge. AU - Andreansky, S.* AU - Liu, H.* AU - Adler, H. AU - Koszinowski, U.H.* AU - Efstathiou, S.* AU - Doherty, P.C.* C1 - 3034 C2 - 21677 SP - 2017-2022 TI - The limits of protection by "memory" T cells in lg -/-mice persistently infected with a γ-herpesvirus. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 7 PB - National Academy of Science PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - The interplay of environmental and genetic factors in the developmental organization of the hippocampus has not been fully elucidated. The neuropeptide corticotropin-releasing factor (CRF) is released from hippocampal interneurons by environmental signals, including stress, to increase synaptic efficacy. In the early postnatal hippocampus, we have previously characterized a transient population of CRF-expressing Cajal-Retzius-like cells. Here we queried whether this stress-activated neuromodulator influences connectivity in the developing hippocampal network. Using mice deficient in the principal hippocampal CRF receptor [CRF(1)(-/-)] and organotypic cultures grown in the presence of synthetic CRF, or CRF receptor antagonists, we found robust effects of CRF on dendritic differentiation in hippocampal neurons. In CRF(1)(-/-) mice, the dendritic trees of hippocampal principal cells were exuberant, an effect that was induced in normal hippocampi in vitro by the presence of CRF(1) antagonists. In both cases, total dendritic length and dendritic branching were significantly increased. In contrast, exogenous synthetic CRF blunted the dendritic growth in hippocampal organotypic cultures. Taken together, these findings suggest that endogenous CRF, if released excessively by previous early postnatal stress, might influence neuronal connectivity and thus function of the immature hippocampus. AU - Chen, Y.* AU - Bender, R.A.* AU - Brunson, K.L.* AU - Pomper, J.K.* AU - Grigoriadis, D.E.* AU - Wurst, W. AU - Baram, T.Z.* C1 - 3392 C2 - 22338 SP - 15782-15787 TI - Modulation of dendritic differentiation by corticotropin-releasing factor in the developing hippocampus. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 44 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - The extracellular calcium-sensing receptor (CaSR) plays a pivotal role in the regulation of extracellular calcium such that abnormalities, which result in a loss or gain of function, lead to hypercalcemia or hypocalcemia, respectively, in patients. Mice carrying CaSR knockout alleles develop hypercalcemia that mimics the disorders observed in humans. To date, there is no mouse model for an activating CaSR mutation. Here, we describe such a mouse model, named Nuf, originally identified for having opaque flecks in the nucleus of the lens in a screen for eye mutants. Nuf mice also display ectopic calcification, hypocalcemia, hyperphosphatemia, and inappropriately reduced levels of plasma parathyroid hormone. These features are similar to those observed in patients with autosomal dominant hypocalcemia. Inheritance studies of Nuf mice revealed that the trait was transmitted in an autosomal-dominant manner, and mapping studies located the locus to chromosome 16, in the vicinity of the CaSR gene (Mouse Genome Database symbol Gprc2a). DNA sequence analysis revealed the presence of a Gprc2a missense mutation, Leu723Gln. Transient expression of wild-type and mutant CaSRs in human embryonic kidney 293 cells demonstrated that the mutation resulted in a gain of function of the CaSR, which had a significantly lower EC(50). Thus, our results have identified a mouse model for an activating CaSR mutation, and the development of ectopic calcification and cataract formation, which tended to be milder in the heterozygote Nuf mice, indicates that an evaluation for such abnormalities in autosomal dominant hypocalcemia patients who have activating CaSR mutations is required. AU - Hough, T.A.* AU - Bogani, D.* AU - Cheeseman, M.T.* AU - Favor, J. AU - Nesbit, M.A.* AU - Thakker, R.V.* AU - Lyon, M.F.* C1 - 2316 C2 - 22164 SP - 13566-13571 TI - Activating calcium-sensing receptor mutation in the mouse is associated with cataracts and ectopic calcification. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 37 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - Several recent studies have demonstrated that T-helper cell-dependent events during the initial priming period are required for the generation of CD8(+) T cell-mediated protective immunity. The underlying mechanisms of this phenomenon have not yet been determined, mostly because of difficulties in studying memory T cells or their precursor populations at early stages during immune responses. We identified IL-7 receptor (CD127) surface expression as a marker for long-living memory T cells, most importantly allowing the distinction between memory and effector T cells early after in vivo priming. The combination of surface staining for CD127 and CD62L further separates between two functionally distinct memory cell subsets, which are similar (if not identical) to cell subsets recently described as central memory T cells (CD127(high) and CD62L(high)) and peripheral effector memory T cells (CD127(high) and CD62L(low)). Using this new tool of memory T cell analysis, we demonstrate that CD8(+) T cell priming in the absence of T cell help or CD40L specifically alters the generation of the effector memory T cell subset, which appears to be crucial for immediate memory responses and long-term maintenance of effective protective immunity. Our data reveal a unique strategy to obtain information about the quality of long-term protective immunity early during an immune response, a finding that may be applied in a variety of clinical settings, including the rapid monitoring of vaccination success. AU - Huster, K.M.* AU - Busch, V.* AU - Schiemann, M.* AU - Linkemann, K.* AU - Kerksiek, K.M.* AU - Wagner, H.* AU - Busch, D. C1 - 3709 C2 - 22053 SP - 5610-5615 TI - Selective expression of IL-7 receptor on memory T cells identifies early CD40L-dependent generation of distinct CD8+ memory T cell subsets. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 15 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - The metabolism of poly(ADP-ribose) (PAR) is critical for genomic stability in multicellular eukaryotes. Here, we show that the failure to degrade PAR by means of disruption of the murine poly(ADP-ribose) glycohydrolase (PARG) gene unexpectedly causes early embryonic lethality and enhanced sensitivity to genotoxic stress. This lethality results from the failure to hydrolyze PAR, because PARG null embryonic day (E) 3.5 blastocysts accumulate PAR and concurrently undergo apoptosis. Moreover, embryonic trophoblast stem cell lines established from early PARG null embryos are viable only when cultured in medium containing the poly(ADP-ribose) polymerase inhibitor benzamide. Cells lacking PARG also show reduced growth, accumulation of PAR, and increased sensitivity to cytotoxicity induced by N-methyl-N′-nitro-N-nitrosoguanidine and menadione after benzamide withdrawal. These results provide compelling evidence that the failure to degrade PAR has deleterious consequences. Further, they define a role for PARG in embryonic development and a protective role in the response to genotoxic stress. AU - Koh, D.W.* AU - Lawler, A.M.* AU - Poitras, M.F.* AU - Sasaki, M.* AU - Wattler, S.* AU - Nehls, M.C.* AU - Stöger, T. AU - Poirier, G.G.* AU - Dawson, V.L.* AU - Dawson, T.M.* C1 - 1805 C2 - 22325 SP - 17699-17704 TI - Failure to degrade poly(ADP-ribose) causes increased sensitivity to cytotoxicity and early embryonic lethality. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 51 PB - PNAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - Zea mays L. ssp. mays, or corn, one of the most important crops and a model for plant genetics, has a genome approximately 80% the size of the human genome. To gain global insight into the organization of its genome, we have sequenced the ends of large insert clones, yielding a cumulative length of one-eighth of the genome with a DNA sequence read every 6.2 kb, thereby describing a large percentage of the genes and transposable elements of maize in an unbiased approach. Based on the accumulative 307 Mb of sequence, repeat sequences occupy 58% and genic regions occupy 7.5%. A conservative estimate predicts approximately 59,000 genes, which is higher than in any other organism sequenced so far. Because the sequences are derived from bacterial artificial chromosome clones, which are ordered in overlapping bins, tagged genes are also ordered along continuous chromosomal segments. Based on this positional information, roughly one-third of the genes appear to consist of tandemly arrayed gene families. Although the ancestor of maize arose by tetraploidization, fewer than half of the genes appear to be present in two orthologous copies, indicating that the maize genome has undergone significant gene loss since the duplication event. AU - Messing, J.* AU - Bharti, A.K.* AU - Karlowski, W.M. AU - Gundlach, H. AU - Kim, H.R.* AU - Yu, Y.* AU - Wei, F.* AU - Fuks, G.* AU - Soderlund, C.A.* AU - Mayer, K.F.X. AU - Wing, R.A.* C1 - 3375 C2 - 22225 SP - 14349-14354 TI - Sequence composition and genome organization of maize. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 40 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - Creation of fusion genes by balanced chromosomal translocations is one of the hallmarks of acute myeloid leukemia (AML) and is considered one of the key leukemogenic events in this disease. in t(12;13)(p13;q12) AML, ectopic expression of the homeobox gene CDX2 was detected in addition to expression of the ETV6-CDX2 fusion gene, generated by the chromosomal translocation. Here we show in a murine model of t(12;13)(p13;q12) AML that myeloid leukemogenesis is induced by the ectopic expression of CDX2 and not by the ETV6-CDX2 chimeric gene. Mice transplanted with bone marrow cells retrovirally engineered to express Cdx2 rapidly succumbed to fatal and transplantable AML. The transforming capacity of Cdx2 depended on an intact homeodomain and the N-terminal transactivation domain. Transplantation of bone marrow cells expressing ETV6-CDX2 failed to induce leukemia. Furthermore, coexpression of ETV6-CDX2 and Cdx2 in bone marrow cells did not accelerate the course of disease in transplanted mice compared to Cdx2 alone. These data demonstrate that activation of a protooncogene by a balanced chromosomal translocation can be the pivotal leukemogenic event in AML, characterized by the expression of a leukemia-specific fusion gene. Furthermore, these findings link protooncogene activation to myeloid leukemogenesis, an oncogenic mechanism so far associated mainly with lymphoid leukemias and lymphomas. AU - Rawat, V.P.S. AU - Cusan, M.* AU - Deshpande, A. AU - Hiddemann, W. AU - Quintanilla-Martinez, L. AU - Humphries, R.K.* AU - Bohlander, S.K. AU - Feuring-Buske, M. AU - Buske, C. C1 - 4965 C2 - 21687 SP - 817-822 TI - Ectopic expression of the homeobox gene Cdx2 is the transforming event in a mouse model of t(12;13)(p13;q12) acute myeloid leukemia. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 3 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - O-mannosylation is an important protein modification in eukaryotes that is initiated by an evolutionarily conserved family of protein O-mannosyltransferases. The first mammalian protein O-mannosyltransferase gene described was the human POMT1. Mutations in the hPOMT1 gene are responsible for Walker-Warburg syndrome (WWS), a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities. During embryogenesis, the murine Pomt1 gene is prominently expressed in the neural tube, the developing eye, and the mesenchyme. These sites of expression correlate with those in which the main tissue alterations are observed in WWS patients. We have inactivated a Pomt1 allele by gene targeting in embryonic stem cells and produced chimeras transmitting the defect allele to offspring. Although heterozygous mice were viable and fertile, the total absence of Pomt1(-/-) pups in the progeny of heterozygous intercrosses indicated that this genotype is embryonic lethal. An analysis of the mutant phenotype revealed that homozygous Pomt1(-/-) mice suffer developmental arrest around embryonic day (E) 7.5 and die between E7.5 and E9.5. The Pomt1(-/-) embryos present defects in the formation of Reichert's membrane, the first basement membrane to form in the embryo. The failure of this membrane to form appears to be the result of abnormal glycosylation and maturation of dystroglycan that may impair recruitment of laminin, a structural component required for the formation of Reichert's membrane in rodents. The targeted disruption of mPomt1 represents an example of an engineered deletion of a known glycosyltransferase involved in O-mannosyl glycan synthesis. AU - Willer, T.* AU - Prados, B.* AU - Falcon-Perez, J.* AU - Renner-Müller, I.* AU - Przemeck, G.K.H. AU - Lommel, M.* AU - Coloma, A.* AU - Valero, M.C.* AU - Hrabě de Angelis, M. AU - Tanner, W.* AU - Wolf, E.* C1 - 4597 C2 - 21950 SP - 14126-14131 TI - Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 39 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AB - Lipopolysaccharides (LPS) are cell-surface components of Gram-negative bacteria and are microbe-/pathogen-associated molecular patterns in animal pathosystems. As for plants, the molecular mechanisms of signal transduction in response to LPS are not known. Here, we show that Arabidopsis thaliana reacts to LPS with a rapid burst of NO, a hallmark of innate immunity in animals. Fifteen LPS preparations (among them Burkholderia cepacia, Pseudomonas aeruginosa, and Erwinia carotovora) as well as lipoteichoic acid from Gram-positive Staphylococcus aureus were found to trigger NO production in suspension-cultured Arabidopsis cells as well as in leaves. NO was detected by confocal laser-scanning microscopy in conjunction with the fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, by electron paramagnetic resonance, and by a NO synthase (NOS) assay. The source of NO was addressed by using T-DNA insertion lines. Interestingly, LPS did not activate the pathogen-inducible varP NOS, but AtNOS1, a distinct NOS previously associated with hormonal signaling in plants. A prominent feature of LPS treatment was activation of defense genes, which proved to be mediated by NO. Northern analyses and transcription profiling by using DNA microarrays revealed induction of defense-associated genes both locally and systemically. Finally, AtNOS1 mutants showed dramatic susceptibility to the pathogen Pseudomonas syringae pv. tomato DC3000. In sum, perception of LPS and induction of NOS contribute toward the activation of plant defense responses. AU - Zeidler, D. AU - Zähringer, U.* AU - Gerber, I.* AU - Dubery, I.* AU - Hartung, T.* AU - Bors, W. AU - Hutzler, P. AU - Durner, J. C1 - 4429 C2 - 22126 SP - 15811-15816 TI - Innate immunity in Arabidopsis thaliana: Lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 101 IS - 44 PB - NAS PY - 2004 SN - 0027-8424 ER - TY - JOUR AU - Drexler, I. AU - Staib, C. AU - Kastenmüller, W. AU - Stevanovic, St.* AU - Schmidt, B.* AU - Lemonnier, F.A.* AU - Rammensee, H.-G.* AU - Busch, D.H.* AU - Bernhard, H.* AU - Erfle, V. AU - Sutter, G. C1 - 22261 C2 - 21028 SP - 217-222 TI - Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 100 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Hansen, J. AU - Floß, T. AU - van Sloun, P.* AU - Füchtbauer, E.-M.* AU - Vauti, F.* AU - Arnold, H.-H.* AU - Schnütgen, F.* AU - Wurst, W.* AU - von Melchner, H.* AU - Ruiz, P.* C1 - 9928 C2 - 21177 SP - 9918-9922 TI - A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 100 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Humme, S. AU - Reisbach, G. AU - Feederle, R.* AU - Delecluse, H.-J.* AU - Bousset, K.* AU - Hammerschmidt, W. AU - Schepers, A. C1 - 22336 C2 - 21193 SP - 10989-10994 TI - The EBV nuclear antigen 1 (EBNA1) enhances B cell immortalization several thousandfold. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 100 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Schroeder, T. AU - Fraser, S.T.* AU - Ogawa, M.* AU - Nishikawa, S.* AU - Oka, C.* AU - Bornkamm, G.W. AU - Nishikawa, S.-I.* AU - Honjo, T.* AU - Just, U. C1 - 22224 C2 - 20941 SP - 4018-4023 TI - Recombination signal sequence-binding protein Jk alters mesodermal cell fate decisions by suppressing cardiomyogenesis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 100 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Staege, M.S.* AU - Lee, S.P.* AU - Frisan, T.* AU - Mautner, J. AU - Scholz, S.* AU - Pajic, A. AU - Rickinson, A.B.* AU - Masucci, M.G.* AU - Polack, A. AU - Bornkamm, G.W. C1 - 22246 C2 - 21003 SP - 4550-4555 TI - MYC overexpression imposes a nonimmunogenic phenotype on Epstein-Barr virus-infected B cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Stieber, J.* AU - Herrmann, S.* AU - Feil, S.* AU - Löster, J. AU - Feil, R.* AU - Biel, M.* AU - Hofmann, F.* AU - Ludwig, A.* C1 - 9929 C2 - 21473 SP - 15235-15240 TI - The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 100 PY - 2003 SN - 0027-8424 ER - TY - JOUR AU - Adler, B. AU - Schaadt, E. AU - Kempkes, B. AU - Zimber-Strobl, U. AU - Baier, B. AU - Bornkamm, G.W. C1 - 21963 C2 - 20487 SP - 437-442 TI - Control of Epstein-Barr virus reactivation by activated CD40 and viral latent membrane protein 1. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PY - 2002 SN - 0027-8424 ER - TY - JOUR AB - Pax6 is a key regulator of eye development in vertebrates and invertebrates, and heterozygous loss-of-function mutations of the mouse Pax6 gene result in the Small eye phenotype, in which a small lens is a constant feature. To provide an understanding of the mechanisms underlying this haploinsufficient phenotype, we evaluated in Pax6 heterozygous mice the effects of reduced Pax6 gene dosage on the activity of other transcription factors regulating eye formation. We found that Six3 expression was specifically reduced in lenses of Pax6 heterozygous mouse embryos. Interactions between orthologous genes from the Pax and Six families have been identified in Drosophila and vertebrate species, and we examined the control of Pax6 and Six3 gene expression in the developing mouse lens. Using in vitro and transgenic approaches, we found that either transcription factor binds regulatory sequences from the counterpart gene and that both genes mutually activate their expression. These studies define a functional relationship in the lens in which Six3 expression is dosage-dependent on Pax6 and where, conversely, Six3 activates Pax6. Accordingly, we show a rescue of the Pax6 haploinsufficient lens phenotype after lens-specific expression of Six3 in transgenic mice. This phenotypic rescue was accompanied by cell proliferation and activation of the platelet-derived growth factor alpha-R/cyclin D1 signaling pathway. Our findings thus provide a mechanism implicating gene regulatory interactions between Pax6 and Six3 in the tissue-specific defects found in Pax6 heterozygous mice. AU - Goudreau, G.* AU - Petrou, P.* AU - Reneker, L.W.* AU - Graw, J. AU - Löster, J. AU - Gruss, P.* C1 - 9925 C2 - 20239 SP - 8719-8724 TI - Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PB - NAS PY - 2002 SN - 0027-8424 ER - TY - JOUR AU - Neuhierl, B. AU - Feederle, R.* AU - Hammerschmidt, W. AU - Delecluse, H.J.* C1 - 21961 C2 - 20483 SP - 1-6 TI - Glycoprotein gp110 of Epstein-Barr virus determines viral tropism and efficiency of infection. JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2002 SN - 0027-8424 ER - TY - JOUR AU - Solakoglu, O.* AU - Maierhofer, C. AU - Lahr, G.* AU - Breit, E.* AU - Scheunemann, P.* AU - Heumos, I.* AU - Pichlmeier, U.* AU - Schlimok, G.* AU - Oberneder, R.* AU - Köllermann, M.W.* AU - Köllermann, J.* AU - Speicher, M.R. AU - Pantel, K.* C1 - 9926 C2 - 20541 SP - 2246-2251 TI - Heterogenous proliferative potential of occult metastatic cells in bone marrow of patients with solid epithelial tumors. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PY - 2002 SN - 0027-8424 ER - TY - JOUR AU - Staege, M.S. AU - Lee, S.P.* AU - Frisan, T.* AU - Mautner, J. AU - Scholz, S.* AU - Pajic, A. AU - Rickinson, A.B.* AU - Masucci, M.G.* AU - Polack, A. AU - Bornkamm, G.W. C1 - 21974 C2 - 20498 SP - 4550-4555 TI - MYC overexpression imposes a nonimmunogenic phenotype on Epstein-Barr virus-infected B cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PY - 2002 SN - 0027-8424 ER - TY - JOUR AU - Wang, T.-L.* AU - Maierhofer, C. AU - Speicher, M.R. AU - Lengauer, C.* AU - Vogelstein, B.* AU - Kinzler, K.W.* AU - Velculescu, V.E.* C1 - 9927 C2 - 20621 SP - 16156-16161 TI - Digital karyotyping. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 99 PY - 2002 SN - 0027-8424 ER - TY - JOUR AB - Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalomocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex. AU - Cahana, A.* AU - Escamez, T.* AU - Nowakowski, R.S.* AU - Hayes, N.L.* AU - Giacobini, M.* AU - von Holst, A.* AU - Shmueli, O.* AU - Sapir, T.* AU - McConnell, S.K.* AU - Wurst, W. AU - Martinez, S.* C1 - 3350 C2 - 22824 SP - 6429-6434 TI - Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 98 IS - 11 PB - National Academy of Sciences PY - 2001 SN - 0027-8424 ER - TY - JOUR AU - Kiernan, A.E.* AU - Ahituv, N.* AU - Fuchs, H. AU - Balling, R. AU - Avraham, K.B.* AU - Steel, K.P.* AU - Hrabě de Angelis, M. C1 - 22313 C2 - 21121 SP - 3873-3878 TI - The Notch ligand Jagged1 is required for inner ear sensory development. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 98 PY - 2001 SN - 0027-8424 ER - TY - JOUR AU - Klessig, D.F.* AU - Durner, J. AU - Noad, R.* AU - Navarre, D.A.* AU - Wendehenne, D.* AU - Kumar, D.* AU - Zhou, J.M.* AU - Shah, J.* AU - Zhang, S.* AU - Kachroo, P.* C1 - 21839 C2 - 20054 SP - 8849-8855 TI - Nitric oxide and salicylic acid signaling in plant defense. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 97 PY - 2000 SN - 0027-8424 ER - TY - JOUR AU - Lonjou, C.* AU - Barnes, K.* AU - Chen, H.* AU - Cookson, W.O.C.M.* AU - Deichmann, K.A.* AU - Hall, I.P.* AU - Holloway, J.W.* AU - Laitinen, T.* AU - Palmer, L.J.* AU - Wjst, M. AU - Morton, N.E.* C1 - 21583 C2 - 19709 SP - 10942-10947 TI - A first trial of retrospective collaboration for positional cloning in complex inheritance : Assay of the cytokine region on chromosome 5 by the Consortium on Asthma Genetics (COAG). JO - Proc. Natl. Acad. Sci. U.S.A. VL - 97 PY - 2000 SN - 0027-8424 ER - TY - JOUR AU - Meyer, M.R.* AU - Angele, A.* AU - Kremmer, E. AU - Kaupp, U.B.* AU - Müller, F.* C1 - 21602 C2 - 19733 SP - 10595-10600 TI - A cGMP-signaling pathway in a subset of olfactory sensory neurons. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 97 PY - 2000 SN - 0027-8424 ER - TY - JOUR AU - Caloca, M.J.* AU - Garcia-Bermejo, M.L.* AU - Blumberg, P.M.* AU - Lewin, N.E.* AU - Kremmer, E. AU - Mischak, H.* AU - Wang, S.* AU - Nacro, K.* AU - Bienfait, B.* AU - Marquez, V.* AU - Kazanietz, M.G.* C1 - 21062 C2 - 19092 SP - 11854-11859 TI - ß2-Chimaerin is a novel target for diacylglycerol: Binding properties and changes in subcellular localization mediated by lignad binding to its C1 domain. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 96 PY - 1999 SN - 0027-8424 ER - TY - JOUR AB - On the basis of the B lymphotropic Epstein-Barr virus (EBV), we have constructed a virus-free packaging cell line that allows encapsidation of plasmids into herpesvirus particles. This cell line harbors an EBV mutant whose packaging signals have been deleted. The gene vectors, which can encompass very large, contiguous pieces of foreign DNA, carry all cis-acting elements involved in amplification and encapsidation into virus-like particles as well as those essential for extrachromosomal maintenance in the recipient cell. Although this first-generation packaging cell line suffers from unwanted recombination between the helper virus genome and gene vector DNAs, this approach opens the way to delivery and stable maintenance of any transgene in human B cells. AU - Delecluse, H.J. AU - Pich, D. AU - Hilsendegen, T. AU - Baum, C.* AU - Hammerschmidt, W. C1 - 33207 C2 - 35620 SP - 5188-5193 TI - A first-generation packaging cell line for Epstein-Barr virus-derived vectors. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 96 IS - 9 PY - 1999 SN - 0027-8424 ER - TY - JOUR AU - Durner, J. AU - Gow, A.J.* AU - Stamler, J.S.* AU - Glazebrock, J.* C1 - 21809 C2 - 20011 SP - 14206-14207 TI - Ancient origins of nitric oxide signaling in biological systems. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 96 PY - 1999 SN - 0027-8424 ER - TY - JOUR AB - With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes. To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid. Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV. Because any genetic modification is possible in E. coli, this experimental approach opens the way to the genetic analysis of all EBV functions. Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed. Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E. coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed. AU - Delecluse, H.-J. AU - Hilsendegen, T. AU - Pich, D. AU - Zeidler, R.* AU - Hammerschmidt, W. C1 - 27630 C2 - 32775 SP - 8245-8250 TI - Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 95 IS - 14 PY - 1998 SN - 0027-8424 ER - TY - JOUR AB - We describe a new mouse frameshift mutation (Pax2(1Neu)) with a 1-bp insertion in the Pax2 gene. This mutation is identical to a previously described mutation in a human family with renal-coloboma syndrome [Sanyanusin, P., McNoe, L. A., Sullivan, M. J., Weaver, R. G. & Eccles, M. R. (1995) Hum. Mol. Genet. 4, 2183-2184]. Heterozygous mutant mice exhibit defects in the kidney, the optic nerve, and retinal layer of the eye, and in homozygous mutant embryos, development of the optic nerve, metanephric kidney, and ventral regions of the inner ear is severely affected. In addition, we observe a deletion of the cerebellum and the posterior mesencephalon in homozygous mutant embryos demonstrating that, in contrast to mutations in Pax5, which is also expressed early in the mid-hindbrain region, loss of Pax2 gene function alone results in the early loss of the mid-hindbrain region. The mid-hindbrain phenotype is similar to Wnt1 and En1 mutant phenotypes, suggesting the conservation of gene regulatory networks between vertebrates and Drosophila. AU - Favor, J. AU - Sandulache, R. AU - Neuhäuser-Klaus, A. AU - Pretsch, W. AU - Chatterjee, B. AU - Senft, E. AU - Wurst, W. AU - Blanquet, V. AU - Grimes, P.* AU - Spörle, R. AU - Schughart, K. C1 - 22870 C2 - 31198 SP - 13870-13875 TI - The mouse Pax21Neu mutation is identical to a human PAX2 mutation in a family with renal-coloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 93 IS - 24 PB - National Academy of Sciences PY - 1996 SN - 0027-8424 ER - TY - JOUR AB - Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy. AU - Kempkes, B. AU - Pich, D. AU - Zeidler, R. AU - Hammerschmidt, W. C1 - 27616 C2 - 32768 SP - 5875-5879 TI - Immortalization of human primary B lymphocytes in vitro with DNA. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 92 IS - 13 PY - 1995 SN - 0027-8424 ER - TY - JOUR AU - Schartl, M. AU - Erbelding-Denk, C. AU - Hölter, S.* AU - Nanda, I. AU - Schmid, M. AU - Schröder, J.H. AU - Epplen, J.T. C1 - 19773 C2 - 12922 SP - 7064-7068 TI - Reproductive fallure of the dominant males in a poecillid fish species, Limia perugiae. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 90 PY - 1993 SN - 0027-8424 ER - TY - JOUR AB - Hierarchical structures among male individuals in a population are frequently reflected in differences in aggressive and reproductive behavior and access to the females. In general, social dominance requires large investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesis has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Burke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poeciliid fish Limia perugiae, in small mating groups the dominant male has a mating success of 100%, whereas in larger groups its contribution to the offspring unexpectedly drops to zero. AU - Schartl, M.* AU - Erbelding-Denk, C. AU - Hölter, S.* AU - Nanda, I.* AU - Schmid, M.W.* AU - Schröder, J.H. AU - Epplen, J.T.* C1 - 40416 C2 - 37987 SP - 7064-7068 TI - Reproductive failure of dominant males in the poeciliid fish Limia perugiae determined by DNA fingerprinting. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 90 IS - 15 PY - 1993 SN - 0027-8424 ER - TY - JOUR AB - The frequency and pattern of mutations at codon 61 of the c-Ha-ras gene have been analyzed in 195 liver tumors and 132 precancerous liver lesions from various rodent strains with differing susceptibility to hepatocarcinogenesis. By using the polymerase chain reaction and allele-specific oligonucleotide hybridization, C → A transversions at the first base and A → T transversions or A → G transitions at the second base of c-Ha-ras codon 61 were detected in 20-60% of spontaneous or carcinogen-induced liver tumors of the C3H/He, CBA, CF1, and B6C3F1 mouse strains, which are highly susceptible to hepatocarcinogenesis. No such mutations, however, could be found in any of the 31 liver tumors of the insensitive C57BL/6J and BALB/c mouse strains or in any of the 35 liver tumors of the comparatively resistant Wistar rat. Further analyses of c-Ha-ras codon 12 mutations in liver tumors from the three insensitive rodent strains also failed to give any positive results. In early precancerous liver lesions, C-Ha-ras codon 61 mutations were found in 13-14% of lesions of the sensitive C3H/He and B6C3F1 mouse strains but not in any of the 34 lesions of the insensitive C57BL/6J mouse. Taken together, our results indicate a close correlation between the mutational activation of the c-Ha-ras gene in liver tumors of the different rodent strains and their susceptibility to hepatocarcinogenesis, whereby the mutations appear to provide a selective growth advantage, leading to a clonal expansion of the mutated liver cell population, only in livers of sensitive but not of insensitive strains. AU - Buchmann, A.* AU - Bauer-Hofmann, R.* AU - Mahr, J.* AU - Drinkwater, N.R.* AU - Luz, A. AU - Schwarz, M.A.* C1 - 34132 C2 - 40218 SP - 911-915 TI - Mutational activation of the c-Ha-ras gene in liver tumors of different rodent strains: Correlation with susceptibility to hepatocarcinogenesis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 88 IS - 3 PY - 1991 SN - 0027-8424 ER - TY - JOUR AU - Maier-Greiner, U.H. AU - Obermaier-Skrobranek, B.M.M. AU - Estermaier, L.M. AU - Kammerloher, W. AU - Freund, C. AU - Wülfing, C. AU - Burkert, U.I. AU - Matern, D.H. AU - Breuer, M. AU - Eulitz, M. AU - Küfrevioglu, Ö.I. AU - Hartmann, G.R. C1 - 19239 C2 - 12310 SP - 4260-4264 TI - Isolation and Properties of a Nitrile Hydratase from the Soil Fungus Myrothecium Verrucaria that is Highly Specific for the Fertilizer Cyanamide and Cloning of its Gene. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 88 PY - 1991 SN - 0027-8424 ER - TY - JOUR AU - Eulitz, M. AU - Weiss, D.T. AU - Solomon, A. C1 - 18334 C2 - 11524 SP - 6542-6546 TI - Immunoglobulin Heavy-chain-associated Amyloidosis. JO - Proc. Natl. Acad. Sci. U.S.A. VL - 87 PY - 1990 SN - 0027-8424 ER -