Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions

We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional ‘slow freezing’ methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS + 0.5 M sucrose at 37 °C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions

Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis

Domains in Arabidopsis proteins NRPE1 and SPT5-like, composed almost exclusively of repeated motifs in which only WG or GW sequences and an overall amino-acid preference are conserved, have been experimentally shown to bind multiple molecules of Argonaute (AGO) protein(s). Domain swapping between the WG/GW domains of NRPE1 and the human protein GW182 showed a conserved function. As classical sequence alignment methods are poorly-adapted to detect such weakly-conserved motifs, we have developed a tool to carry out a systematic analysis to identify genes potentially encoding AGO-binding GW/WG proteins. Here, we describe exhaustive analysis of the Arabidopsis genome for all regions potentially encoding proteins bearing WG/GW motifs and consider the possible role of some of them in AGO-dependent mechanisms. We identified 20 different candidate WG/GW genes, encoding proteins in which the predicted domains range from 92aa to 654aa. These mostly correspond to a limited number of families: RNA-binding proteins, transcription factors, glycine-rich proteins, translation initiation factors and known silencing-associated proteins such as SDE3. Recent studies have argued that the interaction between WG/GW-rich domains and AGO proteins is evolutionarily conserved. Here, we demonstrate by an in silico domain-swapping simulation between plant and mammalian WG/GW proteins that the biased amino-acid composition of the AGO-binding sites is conserved

The De Novo Cytosine Methyltransferase DRM2 Requires Intact UBA Domains and a Catalytically Mutated Paralog DRM3 during RNA–Directed DNA Methylation in Arabidopsis thaliana

Eukaryotic DNA cytosine methylation can be used to transcriptionally silence repetitive sequences, including transposons and retroviruses. This silencing is stable between cell generations as cytosine methylation is maintained epigenetically through DNA replication. The Arabidopsis thaliana Dnmt3 cytosine methyltransferase ortholog DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is required for establishment of small interfering RNA (siRNA) directed DNA methylation. In mammals PIWI proteins and piRNA act in a convergently evolved RNA–directed DNA methylation system that is required to repress transposon expression in the germ line. De novo methylation may also be independent of RNA interference and small RNAs, as in Neurospora crassa. Here we identify a clade of catalytically mutated DRM2 paralogs in flowering plant genomes, which in A.thaliana we term DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3). Despite being catalytically mutated, DRM3 is required for normal maintenance of non-CG DNA methylation, establishment of RNA–directed DNA methylation triggered by repeat sequences and accumulation of repeat-associated small RNAs. Although the mammalian catalytically inactive Dnmt3L paralogs act in an analogous manner, phylogenetic analysis indicates that the DRM and Dnmt3 protein families diverged independently in plants and animals. We also show by site-directed mutagenesis that both the DRM2 N-terminal UBA domains and C-terminal methyltransferase domain are required for normal RNA–directed DNA methylation, supporting an essential targeting function for the UBA domains. These results suggest that plant and mammalian RNA–directed DNA methylation systems consist of a combination of ancestral and convergent features

NOF1 Encodes an Arabidopsis Protein Involved in the Control of rRNA Expression

The control of ribosomal RNA biogenesis is essential for the regulation of protein synthesis in eukaryotic cells. Here, we report the characterization of NOF1 that encodes a putative nucleolar protein involved in the control of rRNA expression in Arabidopsis. The gene has been isolated by T-DNA tagging and its function verified by the characterization of a second allele and genetic complementation of the mutants. The nof1 mutants are affected in female gametogenesis and embryo development. This result is consistent with the detection of NOF1 mRNA in all tissues throughout plant life's cycle, and preferentially in differentiating cells. Interestingly, the closely related proteins from zebra fish and yeast are also necessary for cell division and differentiation. We showed that the nof1-1 mutant displays higher rRNA expression and hypomethylation of rRNA promoter. Taken together, the results presented here demonstrated that NOF1 is an Arabidopsis gene involved in the control of rRNA expression, and suggested that it encodes a putative nucleolar protein, the function of which may be conserved in eukaryotes

Plant ARGONAUTEs: Features, Functions and Unknowns

ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA(sRNA)– based gene silencing pathways controlling gene expression and transposon activity. In plants, AGOs regulate key biological processes such as development, response to stress, genome structure and integrity, and pathogen defense. Canonical functions of plant AGO–sRNA complexes include the endonucleolytic cleavage or translational inhibition of target RNAs, and the methylation of target DNAs. Here, I provide a brief update on the major features, molecular functions and biological roles of plant AGOs. A special focus is given to the more recent discoveries related to emerging molecular or biological functions of plant AGOs, as well as to the major unknowns in the plant AGO field.This work was supported by an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 655841 to A.C.Carbonell Olivares, A. (2017). Plant ARGONAUTEs: Features, Functions and Unknowns. En Plant Argonaute Proteins: Methods and Protocols. Springer Link. 1-21. https://doi.org/10.1007/978-1-4939-7165-7_1121Meister G (2013) Argonaute proteins: functional insights and emerging roles. Nat Rev Genet 14(7):447–459. doi: 10.1038/nrg3462Huntzinger E, Izaurralde E (2011) Gene silencing by microRNAs: contributions of translational repression and mRNA decay. Nat Rev Genet 12(2):99–110. doi: 10.1038/nrg2936Cerutti H, Casas-Mollano JA (2006) On the origin and functions of RNA-mediated silencing: from protists to man. Curr Genet 50(2):81–99. doi: 10.1007/s00294-006-0078-xFang X, Qi Y (2016) RNAi in plants: an argonaute-centered view. Plant Cell 28(2):272–285. doi: 10.1105/tpc1500920Kapoor M, Arora R, Lama T, Nijhawan A, Khurana JP, Tyagi AK, Kapoor S (2008) Genome-wide identification, organization and phylogenetic analysis of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analysis during reproductive development and stress in rice. BMC Genomics 9:451. doi: 10.1186/1471-2164-9-451Morel JB, Godon C, Mourrain P, Beclin C, Boutet S, Feuerbach F, Proux F, Vaucheret H (2002) Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14(3):629–639. doi: 10.1105/tpc010358Yamasaki T, Kim EJ, Cerutti H, Ohama T (2016) Argonaute3 is a key player in miRNA-mediated target cleavage and translational repression in Chlamydomonas. Plant J 85(2):258–268. doi: 10.1111/tpj13107Schroda M (2006) RNA silencing in Chlamydomonas: mechanisms and tools. Curr Genet 49(2):69–84. doi: 10.1007/s00294-005-0042-1Arif MA, Frank W, Khraiwesh B (2013) Role of RNA interference (RNAi) in the moss Physcomitrella patens. Int J Mol Sci 14(1):1516–1540. doi: 10.3390/ijms14011516Zhang H, Xia R, Meyers BC, Walbot V (2015) Evolution, functions, and mysteries of plant ARGONAUTE proteins. Curr Opin Plant Biol 27:84–90. doi: 10.1016/jpbi201506011Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8(11):884–896. doi: 10.1038/nrg2179Tolia NH, Joshua-Tor L (2007) Slicer and the argonautes. Nat Chem Biol 3(1):36–43. doi: 10.1038/nchembio848Song JJ, Smith SK, Hannon GJ, Joshua-Tor L (2004) Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305(5689):1434–1437. doi: 10.1126/science1102514Nakanishi K, Weinberg DE, Bartel DP, Patel DJ (2012) Structure of yeast Argonaute with guide RNA. Nature 486(7403):368–374. doi: 10.1038/nature11211Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133(1):128–141. doi: 10.1016/jcell200802033Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, Wu L, Li S, Zhou H, Long C, Chen S, Hannon GJ, Qi Y (2008) Sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133(1):116–127. doi: 10.1016/jcell200802034Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y (2008) The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol 49(4):493–500. doi: 10.1093/pcp/pcn043Zhu H, Hu F, Wang R, Zhou X, Sze SH, Liou LW, Barefoot A, Dickman M, Zhang X (2011) Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development. Cell 145(2):242–256. doi: 10.1016/jcell201103024Zhang X, Niu D, Carbonell A, Wang A, Lee A, Tun V, Wang Z, Carrington JC, Chang CE, Jin H (2014) ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis. Nat Commun 5:5468. doi: 10.1038/ncomms6468Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science 305(5689):1437–1441. doi: 10.1126/science1102513Sheng G, Zhao H, Wang J, Rao Y, Tian W, Swarts DC, van der Oost J, Patel DJ, Wang Y (2014) Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage. Proc Natl Acad Sci U S A 111(2):652–657. doi: 10.1073/pnas1321032111Baumberger N, Baulcombe DC (2005) Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A 102(33):11928–11933. doi: 10.1073/pnas0505461102Qi Y, Denli AM, Hannon GJ (2005) Biochemical specialization within Arabidopsis RNA silencing pathways. Mol Cell 19(3):421–428. doi: 10.1016/jmolcel200506014Carbonell A, Fahlgren N, Garcia-Ruiz H, Gilbert KB, Montgomery TA, Nguyen T, Cuperus JT, Carrington JC (2012) Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24(9):3613–3629. doi: 10.1105/tpc112099945Qi Y, He X, Wang XJ, Kohany O, Jurka J, Hannon GJ (2006) Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443(7114):1008–1012. doi: 10.1038/nature05198Ji L, Liu X, Yan J, Wang W, Yumul RE, Kim YJ, Dinh TT, Liu J, Cui X, Zheng B, Agarwal M, Liu C, Cao X, Tang G, Chen X (2011) ARGONAUTE10 and ARGONAUTE1 regulate the termination of floral stem cells through two microRNAs in Arabidopsis. PLoS Genet 7(3):e1001358. doi: 10.1371/journalpgen1001358Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297(5589):2053–2056. doi: 10.1126/science1076311Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP (2002) Prediction of plant microRNA targets. Cell 110(4):513–520. doi: 10.1016/S0092-8674(02)00863-2Mallory AC, Reinhart BJ, Jones-Rhoades MW, Tang G, Zamore PD, Barton MK, Bartel DP (2004) MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5′ region. EMBO J 23(16):3356–3364. doi: 10.1038/sjemboj7600340German MA, Pillay M, Jeong DH, Hetawal A, Luo S, Janardhanan P, Kannan V, Rymarquis LA, Nobuta K, German R, De Paoli E, Lu C, Schroth G, Meyers BC, Green PJ (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26(8):941–946. doi: 10.1038/nbt1417Addo-Quaye C, Eshoo TW, Bartel DP, Axtell MJ (2008) Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol 18(10):758–762. doi: 10.1016/jcub200804042Arribas-Hernandez L, Kielpinski LJ, Brodersen P (2016) mRNA decay of most Arabidopsis miRNA targets requires slicer activity of AGO1. Plant Physiol 171(4):2620–2632. doi: 10.1104/pp.16.00231Cuperus JT, Carbonell A, Fahlgren N, Garcia-Ruiz H, Burke RT, Takeda A, Sullivan CM, Gilbert SD, Montgomery TA, Carrington JC (2010) Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis. Nat Struct Mol Biol 17(8):997–1003. doi: 10.1038/nsmb1866Montgomery TA, Yoo SJ, Fahlgren N, Gilbert SD, Howell MD, Sullivan CM, Alexander A, Nguyen G, Allen E, Ahn JH, Carrington JC (2008) AGO1-miR173 complex initiates phased siRNA formation in plants. Proc Natl Acad Sci U S A 105(51):20055–20062. doi: 10.1073/pnas0810241105Allen E, Xie Z, Gustafson AM, Carrington JC (2005) microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121(2):207–221. doi: 10.1016/jcell200504004Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19(18):2164–2175. doi: 10.1101/gad1352605Rajagopalan R, Vaucheret H, Trejo J, Bartel DP (2006) A diverse and evolutionarily fluid set of microRNAs in Arabidopsis thaliana. Genes Dev 20(24):3407–3425. doi: 10.1101/gad1476406Arribas-Hernandez L, Marchais A, Poulsen C, Haase B, Hauptmann J, Benes V, Meister G, Brodersen P (2016) The slicer activity of ARGONAUTE1 Is required specifically for the phasing, not production, of trans-acting short interfering RNAs in Arabidopsis. Plant Cell 28(7):1563–1580. doi: 10.1105/tpc1600121Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P, Yamamoto YY, Sieburth L, Voinnet O (2008) Widespread translational inhibition by plant miRNAs and siRNAs. Science 320(5880):1185–1190. doi: 10.1126/science1159151Li S, Le B, Ma X, Li S, You C, Yu Y, Zhang B, Liu L, Gao L, Shi T, Zhao Y, Mo B, Cao X, Chen X (2016) Biogenesis of phased siRNAs on membrane-bound polysomes in Arabidopsis. Elife 5:e22750. doi: 10.7554/eLife22750Zeng Y, Yi R, Cullen BR (2003) MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci U S A 100(17):9779–9784. doi: 10.1073/pnas1630797100Iwakawa HO, Tomari Y (2013) Molecular insights into microRNA-mediated translational repression in plants. Mol Cell 52(4):591–601. doi: 10.1016/jmolcel201310033Li S, Liu L, Zhuang X, Yu Y, Liu X, Cui X, Ji L, Pan Z, Cao X, Mo B, Zhang F, Raikhel N, Jiang L, Chen X (2013) MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell 153(3):562–574. doi: 10.1016/jcell201304005Li JF, Chung HS, Niu Y, Bush J, McCormack M, Sheen J (2013) Comprehensive protein-based artificial microRNA screens for effective gene silencing in plants. Plant Cell 25(5):1507–1522. doi: 10.1105/tpc113112235Liu MJ, SH W, JF W, Lin WD, YC W, Tsai TY, Tsai HL, SH W (2013) Translational landscape of photomorphogenic Arabidopsis. Plant Cell 25(10):3699–3710. doi: 10.1105/tpc113114769Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15(11):2730–2741. doi: 10.1105/tpc016238Chen X (2004) A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303(5666):2022–2025. doi: 10.1126/science1088060Gandikota M, Birkenbihl RP, Hohmann S, Cardon GH, Saedler H, Huijser P (2007) The miRNA156/157 recognition element in the 3′ UTR of the Arabidopsis SBP box gene SPL3 prevents early flowering by translational inhibition in seedlings. Plant J 49(4):683–693. doi: 10.1111/j1365-313X200602983xYang L, Wu G, Poethig RS (2012) Mutations in the GW-repeat protein SUO reveal a developmental function for microRNA-mediated translational repression in Arabidopsis. Proc Natl Acad Sci U S A 109(1):315–320. doi: 10.1073/pnas1114673109Mallory AC, Hinze A, Tucker MR, Bouche N, Gasciolli V, Elmayan T, Lauressergues D, Jauvion V, Vaucheret H, Laux T (2009) Redundant and specific roles of the ARGONAUTE proteins AGO1 and ZLL in development and small RNA-directed gene silencing. PLoS Genet 5(9):e1000646. doi: 10.1371/journalpgen1000646Hou CY, Lee WC, Chou HC, Chen AP, Chou SJ, Chen HM (2016) Global analysis of truncated RNA ends reveals new insights into ribosome stalling in plants. Plant Cell 28(10):2398–2416. doi: 10.1105/tpc1600295Rogers K, Chen X (2013) Biogenesis, turnover, and mode of action of plant microRNAs. Plant Cell 25(7):2383–2399. doi: 10.1105/tpc113113159Behm-Ansmant I, Rehwinkel J, Doerks T, Stark A, Bork P, Izaurralde E (2006) mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20(14):1885–1898. doi: 10.1101/gad1424106Wu L, Fan J, Belasco JG (2006) MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci U S A 103(11):4034–4039. doi: 10.1073/pnas0510928103Schirle NT, MacRae IJ (2012) The crystal structure of human Argonaute2. Science 336(6084):1037–1040. doi: 10.1126/science1221551Pfaff J, Hennig J, Herzog F, Aebersold R, Sattler M, Niessing D, Meister G (2013) Structural features of Argonaute-GW182 protein interactions. Proc Natl Acad Sci U S A 110(40):E3770–E3779. doi: 10.1073/pnas1308510110Ma X, Kim EJ, Kook I, Ma F, Voshall A, Moriyama E, Cerutti H (2013) Small interfering RNA-mediated translation repression alters ribosome sensitivity to inhibition by cycloheximide in Chlamydomonas reinhardtii. Plant Cell 25(3):985–998. doi: 10.1105/tpc113109256Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11(3):204–220. doi: 10.1038/nrg2719Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2(5):E104. doi: 10.1371/journalpbio0020104Herr AJ, Jensen MB, Dalmay T, Baulcombe DC (2005) RNA polymerase IV directs silencing of endogenous DNA. Science 308(5718):118–120. doi: 10.1126/science1106910Kanno T, Huettel B, Mette MF, Aufsatz W, Jaligot E, Daxinger L, Kreil DP, Matzke M, Matzke AJ (2005) Atypical RNA polymerase subunits required for RNA-directed DNA methylation. Nat Genet 37(7):761–765. doi: 10.1038/ng1580Onodera Y, Haag JR, Ream T, Costa Nunes P, Pontes O, Pikaard CS (2005) Plant nuclear RNA polymerase IV mediates siRNA and DNA methylation-dependent heterochromatin formation. Cell 120(5):613–622. doi: 10.1016/jcell200502007Haag JR, Ream TS, Marasco M, Nicora CD, Norbeck AD, Pasa-Tolic L, Pikaard CS (2012) In vitro transcription activities of Pol IV, Pol V, and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing. Mol Cell 48(5):811–818. doi: 10.1016/jmolcel201209027Pontes O, Li CF, Costa Nunes P, Haag J, Ream T, Vitins A, Jacobsen SE, Pikaard CS (2006) The Arabidopsis chromatin-modifying nuclear siRNA pathway involves a nucleolar RNA processing center. Cell 126(1):79–92. doi: 10.1016/jcell200605031Li CF, Pontes O, El-Shami M, Henderson IR, Bernatavichute YV, Chan SW, Lagrange T, Pikaard CS, Jacobsen SE (2006) An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana. Cell 126(1):93–106. doi: 10.1016/jcell200605032El-Shami M, Pontier D, Lahmy S, Braun L, Picart C, Vega D, Hakimi MA, Jacobsen SE, Cooke R, Lagrange T (2007) Reiterated WG/GW motifs form functionally and evolutionarily conserved ARGONAUTE-binding platforms in RNAi-related components. Genes Dev 21(20):2539–2544. doi: 10.1101/gad451207Li CF, Henderson IR, Song L, Fedoroff N, Lagrange T, Jacobsen SE (2008) Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana. PLoS Genet 4(2):e27. doi: 10.1371/journalpgen0040027Bies-Etheve N, Pontier D, Lahmy S, Picart C, Vega D, Cooke R, Lagrange T (2009) RNA-directed DNA methylation requires an AGO4-interacting member of the SPT5 elongation factor family. EMBO Rep 10(6):649–654. doi: 10.1038/embor200931He XJ, Hsu YF, Zhu S, Wierzbicki AT, Pontes O, Pikaard CS, Liu HL, Wang CS, Jin H, Zhu JK (2009) An effector of RNA-directed DNA methylation in Arabidopsis is an ARGONAUTE 4- and RNA-binding protein. Cell 137(3):498–508. doi: 10.1016/jcell200904028Zhong X, Du J, Hale CJ, Gallego-Bartolome J, Feng S, Vashisht AA, Chory J, Wohlschlegel JA, Patel DJ, Jacobsen SE (2014) Molecular mechanism of action of plant DRM de novo DNA methyltransferases. Cell 157(5):1050–1060. doi: 10.1016/jcell201403056Cao X, Jacobsen SE (2002) Locus-specific control of asymmetric and CpNpG methylation by the DRM and CMT3 methyltransferase genes. Proc Natl Acad Sci U S A 99(Suppl 4):16491–16498. doi: 10.1073/pnas162371599Lahmy S, Pontier D, Bies-Etheve N, Laudie M, Feng S, Jobet E, Hale CJ, Cooke R, Hakimi MA, Angelov D, Jacobsen SE, Lagrange T (2016) Evidence for ARGONAUTE4-DNA interactions in RNA-directed DNA methylation in plants. Genes Dev 30(23):2565–2570. doi: 10.1101/gad289553116Zheng X, Zhu J, Kapoor A, Zhu JK (2007) Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing. EMBO J 26(6):1691–1701. doi: 10.1038/sjemboj7601603Havecker ER, Wallbridge LM, Hardcastle TJ, Bush MS, Kelly KA, Dunn RM, Schwach F, Doonan JH, Baulcombe DC (2010) The Arabidopsis RNA-directed DNA methylation Argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22(2):321–334. doi: 10.1105/tpc109072199Eun C, Lorkovic ZJ, Naumann U, Long Q, Havecker ER, Simon SA, Meyers BC, Matzke AJ, Matzke M (2011) AGO6 functions in RNA-mediated transcriptional gene silencing in shoot and root meristems in Arabidopsis thaliana. PLoS One 6(10):e25730. doi: 10.1371/journalpone0025730Duan CG, Zhang H, Tang K, Zhu X, Qian W, Hou YJ, Wang B, Lang Z, Zhao Y, Wang X, Wang P, Zhou J, Liang G, Liu N, Wang C, Zhu JK (2015) Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA-directed DNA methylation. EMBO J 34(5):581–592. doi: 10.15252/embj201489453McCue AD, Panda K, Nuthikattu S, Choudury SG, Thomas EN, Slotkin RK (2015) ARGONAUTE 6 bridges transposable element mRNA-derived siRNAs to the establishment of DNA methylation. EMBO J 34(1):20–35. doi: 10.15252/embj201489499Zhang Z, Liu X, Guo X, Wang XJ, Zhang X (2016) Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing. Nat Plants 2(5):16049. doi: 10.1038/nplants201649Wu J, Yang Z, Wang Y, Zheng L, Ye R, Ji Y, Zhao S, Ji S, Liu R, Xu L, Zheng H, Zhou Y, Zhang X, Cao X, Xie L, Wu Z, Qi Y, Li Y (2015) Viral-inducible Argonaute18 confers broad-spectrum virus resistance in rice by sequestering a host microRNA. Elife 4:05733. doi: 10.7554/eLife05733Wu J, Yang R, Yang Z, Yao S, Zhao S, Wang Y, Li P, Song X, Jin L, Zhou T, Lan Y, Xie L, Zhou X, Chu C, Qi Y, Cao X, Li Y (2017) ROS accumulation and antiviral defence control by microRNA528 in rice. Nat Plants 3:16203. doi: 10.1038/nplants2016203Wei W, Ba Z, Gao M, Wu Y, Ma Y, Amiard S, White CI, Rendtlew Danielsen JM, Yang YG, Qi Y (2012) A role for small RNAs in DNA double-strand break repair. Cell 149(1):101–112. doi: 10.1016/jcell201203002Oliver C, Santos JL, Pradillo M (2014) On the role of some ARGONAUTE proteins in meiosis and DNA repair in Arabidopsis thaliana. Front Plant Sci 5:177. doi: 10.3389/fpls201400177Ye R, Chen Z, Lian B, Rowley MJ, Xia N, Chai J, Li Y, He XJ, Wierzbicki AT, Qi Y (2016) A Dicer-independent route for biogenesis of siRNAs that direct DNA methylation in Arabidopsis. Mol Cell 61(2):222–235. doi: 10.1016/jmolcel201511015Dolata J, Bajczyk M, Bielewicz D, Niedojadlo K, Niedojadlo J, Pietrykowska H, Walczak W, Szweykowska-Kulinska Z, Jarmolowski A (2016) Salt stress reveals a new role for ARGONAUTE1 in miRNA biogenesis at the transcriptional and posttranscriptional levels. Plant Physiol 172(1):297–312. doi: 10.1104/pp1600830Singh RK, Gase K, Baldwin IT, Pandey SP (2015) Molecular evolution and diversification of the Argonaute family of proteins in plants. BMC Plant Biol 15(1):23. doi: 10.1186/s12870-014-0364-6Singh RK, Pandey SP (2015) Evolution of structural and functional diversification among plant Argonautes. Plant Signal Behav 10(10):e1069455. doi: 10.1080/1559232420151069455Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C (1998) AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J 17(1):170–180. doi: 10.1093/emboj/171170Kidner CA, Martienssen RA (2004) Spatially restricted microRNA directs leaf polarity through ARGONAUTE1. Nature 428(6978):81–84. doi: 10.1038/nature02366Sorin C, Bussell JD, Camus I, Ljung K, Kowalczyk M, Geiss G, McKhann H, Garcion C, Vaucheret H, Sandberg G, Bellini C (2005) Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17(5):1343–1359. doi: 10.1105/tpc105031625Yang L, Huang W, Wang H, Cai R, Xu Y, Huang H (2006) Characterizations of a hypomorphic argonaute1 mutant reveal novel AGO1 functions in Arabidopsis lateral organ development. Plant Mol Biol 61(1-2):63–78. doi: 10.1007/s11103-005-5992-7Kidner CA, Martienssen RA (2005) The developmental role of microRNA in plants. Curr Opin Plant Biol 8(1):38–44. doi: 10.1016/jpbi200411008Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y (2009) Rice microRNA effector complexes and targets. Plant Cell 21(11):3421–3435. doi: 10.1105/tpc109070938Vaucheret H (2008) Plant ARGONAUTES. Trends Plant Sci 13(7):350–358. doi: 10.1016/jtplants200804007Hunter C, Sun H, Poethig RS (2003) The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr Biol 13(19):1734–1739Adenot X, Elmayan T, Lauressergues D, Boutet S, Bouche N, Gasciolli V, Vaucheret H (2006) DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr Biol 16(9):927–932. doi: 10.1016/jcub200603035Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, Alexander AL, Carrington JC (2006) Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr Biol 16(9):939–944. doi: 10.1016/jcub200603065Axtell MJ, Jan C, Rajagopalan R, Bartel DP (2006) A two-hit trigger for siRNA biogenesis in plants. Cell 127(3):565–577. doi: 10.1016/jcell200609032Hunter C, Willmann MR, Wu G, Yoshikawa M, de la Luz G-NM, Poethig SR (2006) Trans-acting siRNA-mediated repre

Effects of SR 49059, a new orally active and specific vasopressin V1 receptor antagonist, on vasopressin-induced vasoconstriction in humans

We have evaluated the efficacy of SR 49059, a new orally active and specific vasopressin V1 receptor antagonist (arginine-vasopressin [AVP]), in the blockade of the vascular effects of exogenous AVP in healthy subjects. In preliminary experiments, two procedures to measure the V1 vascular effects of AVP were assessed. First, the AVP-induced changes in skin blood flow were investigated by the injection of increasing doses of AVP intradermally, with or without a previous local vasodilation with calcitonin gene-related peptide (CGRP). In a second protocol, AVP was infused intra-arterially, and the changes in radial artery diameter and blood flow were measured. The intradermal injection of AVP caused significant decreases in skin blood flow, and the use of CGRP increased the sensitivity of the method by a factor of 10(2) to 10(3). AVP infused intra-arterially caused dose-dependent decreases in the radial artery diameter and blood flow. In the main study, the potency and efficacy of SR 49059 to block the AVP-induced changes in skin blood flow were assessed in 12 healthy men with a double-blind, triple crossover study design. The subjects were randomized to receive a placebo orally and 30 mg and 300 mg of the antagonist at a 1-week interval. The subjects were then further randomized to evaluate the efficacy of the same doses of the antagonist to block the vasoconstriction of the radial artery induced by an intra-arterial infusion of AVP. SR 49059 inhibits, dose-dependently and significantly, the AVP-induced changes in skin blood flow, with a peak effect occurring between 2 and 6 hours after injection. In addition, the 300-mg dose of SR 49059 completely blocked the vasoconstriction of the radial artery induced by AVP. In conclusion, skin blood-flow measurement, after intradermal injection of AVP on a skin area vasodilated with CGRP, is an effective method to investigate the V1 vascular effect of AVP in humans. SR 49059 is a potent and specific antagonist of V1 receptors, which blocks the AVP-induced vasoconstriction

From combinations to multitarget-directed ligands: A continuum in Alzheimer's disease polypharmacology

The continued drug discovery failures in complex neurodegenerative diseases, including Alzheimer's disease (AD), has raised questions about the classical paradigm \u201cone-drug, one-target, one-disease.\u201d In parallel, the ever-increasing awareness of the multiplicity of the underlying pathways has led to the affirmation of polypharmacological approaches. Polypharmacology, which broadly embodies the use of pharmaceutical agents acting on multiple targets, seems to be the best way to restore the complex diseased network and to provide disease-modifying effects in AD. In this review, our aim is to provide a roadmap into a world that is still only partly explored and that should be seen as a continuum of pharmacological opportunities, from drug combinations to multitarget-directed ligands (both codrugs and hybrids). Each modality has unique features that can be effectively exploited by medicinal chemists. We argue that understanding their advantages and drawbacks is very helpful in choosing a proper approach and developing successful AD multitarget drug-discovery endeavors. We also briefly dwell on (co)target validation, an aspect that is quite often neglected, but critical for an efficient clinical translation. We substantiate our discussion with instructive examples taken from the recent literature. Our wish is that, in spite of the specter of the high attrition rates, best researchers preferring to enter, stay, and progress in the field would help grow the sector and develop AD polypharmacology to full potential