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Supplementary Table 1: Significantly upregulated genes in the “malignant” cluster of PNETs (p<0.05) Fold change Gene (WDEC/WDET) FEV protein 11.61 adenylate cyclase 2 (brain) 4.47 nuclear receptor subfamily 4, group A, member 2 4.45 growth arrest and DNA-damage-inducible, beta 3.28 plexin B1 3.10 neuronal PAS domain protein 2 2.92 caldesmon 1 2.81 drebrin 1 2.43 hypothetical protein BC013764 2.41 TGFB-induced factor (TALE family homeobox) 2.26 Homo sapiens cDNA FLJ12843 fis, clone NT2RP2003293, mRNA sequence 2.17 nuclear prelamin A recognition factor 2.15 chromobox homolog 4 (Pc class homolog, Drosophila) 2.14 RuvB (E coli homolog)-like 1 [Homo sapiens], mRNA sequence 2.03 latent transforming growth factor beta binding protein 4 2.02 putative glialblastoma cell differentiation-related 1.92 hypothetical protein FLJ10298 1.92 protein tyrosine phosphatase type IVA, member 1 1.90 ADP-ribosylation factor-like 4 1.88 NIMA (never in mitosis gene a)-related kinase 7 1.87 RAB11B, member RAS oncogene family 1.87 myelin transcription factor 1 1.86 centaurin, beta 2 1.83 Homo sapiens cDNA FLJ38575 fis, clone HCHON2007046, mRNA sequence 1.83 SH3 domain binding glutamic acid-rich protein like 3 1.81 immunoglobulin heavy constant gamma 3 (G3m marker) 1.77 chromobox homolog 3 (HP1 gamma homolog, Drosophila) 1.74 basic transcription element binding protein 1 1.73 H1 histone family, member X 1.73 protein phosphatase 2A, regulatory subunit B' (PR 53) 1.73 Homo sapiens cDNA FLJ30096 fis, clone BNGH41000045, mRNA sequence 1.73 KIAA0397 gene product 1.71 OK/SW-CL.4 [Homo sapiens], mRNA sequence 1.69 high-mobility group 20B 1.69 phosphoserine phosphatase 1.68 Homo sapiens mRNA; cDNA DKFZp434B142 (from clone DKFZp434B142), mRNA sequence 1.67 nuclear mitotic apparatus protein 1 1.67 hypothetical protein 628 1.66 PRKC, apoptosis, WT1, regulator 1.64 E3 ubiquitin ligase SMURF1 1.64 chromodomain protein, Y chromosome-like 1.63 hypothetical protein FLJ21839 1.62 hypothetical protein MGC2550 1.62 Suppl. Table 1 (continued) Fold Change Gene (WDEC/WDET) DAZ associated protein 2 1.61 replication factor C (activator 1) 2, 40kDa 1.60 likely ortholog of mouse tubulin, delta 1 1.59 FUS interacting protein (serine-arginine rich) 1 1.58 fetal Alzheimer antigen 1.58 hypothetical protein FLJ13910 1.58 Homo sapiens clone 23718 mRNA sequence 1.58 plectin 1, intermediate filament binding protein 500kDa 1.58 hypothetical protein MGC3260 1.58 inhibitor of growth family, member 4 1.57 transducin-like enhancer of split 3 (E(sp1) homolog, Drosophila) 1.57 high mobility group nucleosomal binding domain 3 1.57 Homo sapiens full length insert cDNA clone YY61D04, mRNA sequence 1.57 nucleoporin 62kDa 1.56 phosphoribosyl pyrophosphate synthetase-associated protein 2 1.56 PRO0974 [Homo sapiens], mRNA sequence 1.56 small nuclear RNA activating complex, polypeptide 3, 50kDa 1.56 actin, gamma 1 1.55 supervillin 1.55 glucosamine (N-acetyl)-6-sulfatase (Sanfilippo disease IIID) 1.55 polypyrimidine tract binding protein 1 1.54 calcium binding protein Cab45 precursor 1.54 hypothetical protein FLJ20294 1.53 intersectin 1 (SH3 domain protein) 1.52 Sp3 transcription factor 1.52 vacuolar protein sorting 41 (yeast) 1.51 Homo sapiens cDNA FLJ38058 fis, clone CTONG2014898, mRNA sequence 1.51 transmembrane 9 superfamily member 1 1.50 Supplementary Table 2: Significantly downregulated genes in the “malignant” cluster of PNETs (p<0.05) Fold Change Gene ((WDEC/WDET) ATP-binding cassette, sub-family C (CFTR/MRP), member 8 0.29 SH3-domain GRB2-like endophilin B1 0.41 Rag D protein 0.42 protocadherin 17 0.42 glucokinase (hexokinase 4, maturity onset diabetes of the young 2) 0.43 neuropeptide Y 0.46 hypothetical protein FLJ20559 0.46 sortilin-related receptor, L(DLR class) A repeats-containing 0.46 Homo sapiens, clone IMAGE:4133122, mRNA, mRNA sequence 0.47 pentaxin-related gene, rapidly induced by IL-1 beta 0.47 AD037 protein 0.47 Consensus includes gb:AF059180 /DEF=Homo sapiens mutant beta-globin (HBB) gene, complete cds /FEA=mRNA /DB_XREF=gi:4837722 /UG=Hs.155376 hemoglobin, beta 0.48 Homo sapiens cDNA: FLJ21930 fis, clone HEP04301, highly similar to HSU90916 Human clone 23815 mRNA sequence 0.49 growth factor receptor-bound protein 10 0.49 tyrosinase-related protein 1 0.51 alpha-methylacyl-CoA racemase 0.53 inositol 1,4,5-triphosphate receptor, type 3 0.54 FN5 protein 0.55 Hr44 antigen 0.55 phosphodiesterase 1C, calmodulin-dependent 70kDa 0.55 hemoglobin, beta 0.56 hemoglobin, delta 0.56 glutamate receptor, metabotropic 1 0.56 Hemoglobin gamma-G [Homo sapiens], mRNA sequence 0.56 Alpha one globin [Homo sapiens], mRNA sequence 0.57 hemoglobin, gamma G 0.58 hemoglobin, alpha 1 0.59 carbonic anhydrase IV 0.59 Endomucin-1 [Homo sapiens], mRNA sequence 0.60 carboxypeptidase A4 0.60 Rho guanine nucleotide exchange factor (GEF) 10 0.60 ESTs, Moderately similar to hypothetical protein FLJ20489 [Homo sapiens] [H.sapiens] 0.60 ADP-ribosylation factor 4-like 0.63 hemoglobin, alpha 2 0.63 adrenergic, alpha-2A-, receptor 0.63 Consensus includes gb:V00489 /DEF=Human alpha-globin gene with flanks /FEA=mRNA /DB_XREF=gi:28548 /UG=Hs.272572 hemoglobin, alpha 2 0.64 membrane protein, palmitoylated 1, 55kDa 0.64 lipase, hormone-sensitive 0.65 neuromedin B 0.65 a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 2 0.66 neurofibromin 2 (bilateral acoustic neuroma) 0.66 Supplementary Table 3: Significantly upregulated genes in GI carcinoids when compared to PNETs (p<0.05) Fold Change GI- Gene carcinoids/PNET extracellular matrix protein 1 28.08 solute carrier family 18 (vesicular monoamine), member 1 25.32 lectin, galactoside-binding, soluble, 4 (galectin 4) 24.04 cytochrome P450, subfamily IIIA, polypeptide 7 23.91 epidermal growth factor receptor pathway substrate 8 related protein 3 21.13 homeo box C6 20.79 forkhead box A1 14 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 (mitochondrial) 12.53 glucosidase, beta, acid 3 (cytosolic) 12.04 immunoglobulin kappa constant 11.1 cadherin 17, LI cadherin (liver-intestine) 10.6 apolipoprotein A-I 10.21 neuropeptide Y receptor Y1 9.99 Papillary thyroid carcinoma-encoded protein, mRNA sequence 9.47 hypothetical protein BC013764 9.02 anterior gradient 2 homolog (Xenepus laevis) 8.81 hephaestin 8.67 flavin containing monooxygenase 5 8.67 cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 4 8.52 neurogranin (protein kinase C substrate, RC3) 8.42 HRAS-like suppressor 7.68 apolipoprotein D 7.67 Consensus includes gb:BF195104 /FEA=EST /DB_XREF=gi:11081627 /DB_XREF=est:7n15a07.x1 /CLONE=IMAGE:3564565 /UG=Hs.56729 lymphocyte-specific protein 1 7.63 tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin) 7.6 serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 7.31 glycoprotein M6A 7.3 B-factor, properdin 7.14 myosin IA 6.98 immunoglobulin heavy constant mu 6.67 interferon consensus sequence binding protein 1 6.39 retinoic acid receptor responder (tazarotene induced) 3 6.38 cytochrome P450, subfamily IIIA (niphedipine oxidase), polypeptide 5 5.73 KIAA1054 protein 5.7 breast carcinoma amplified sequence 1 5.68 breast cancer anti-estrogen resistance 3 5.56 homeodomain only protein 5.56 T cell receptor beta locus 5.48 carboxylesterase 1 (monocyte/macrophage serine esterase 1) 5.41 KIAA0233 gene product 5.36 lymphocyte antigen 75 5.33 retinoic acid receptor responder (tazarotene induced) 1 5.3 Suppl. Table 3 (continued) Fold Change GI- Gene carcinoids/PNET Kruppel-like factor 5 (intestinal) 5.23 dopa decarboxylase (aromatic L-amino acid decarboxylase) 5.13 FEV protein 5 coagulation factor V (proaccelerin, labile factor) 4.73 sodium channel, voltage-gated, type III, alpha polypeptide 4.65 hypothetical protein DKFZp434B0417 4.62 phospholipase A2, group X 4.59 heparan sulfate (glucosamine) 3-O-sulfotransferase 1 4.42 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 10 (GalNAc-T10) 4.4 Homo sapiens mRNA; cDNA DKFZp564N1116 (from clone DKFZp564N1116), mRNA sequence 4.34 Mst3 and SOK1-related kinase 4.33 KIAA0830 protein 4.3 protocadherin LKC 4.27 absent in melanoma 1 4.07 ESTs, Moderately similar to T00636 hypothetical protein F21856_2 - human [H.sapiens] 4.05 KIAA0703 gene product 3.96 SMA5 3.92 ribonuclease, RNase A family, 4 3.91 annexin A13 3.82 fibroblast growth factor 13 3.79 hypothetical protein MGC2376 3.78 chemokine-like factor 1 3.66 hypothetical protein MGC2742 3.65 ret proto-oncogene (multiple endocrine neoplasia and medullary thyroid carcinoma 1, Hirschsprung disease) 3.62 DKFZP586C1619 protein 3.58 fatty-acid-Coenzyme A ligase, long-chain 5 3.58 zinc finger protein 36, C3H type-like 2 3.52 KIAA1013 protein 3.44 latexin protein 3.44 KIAA0648 protein 3.4 kinesin family member 13B 3.37 potassium voltage-gated channel, delayed-rectifier, subfamily S, member 3 3.37 SMA3 3.35 hypothetical protein FLJ14639 3.31 S100 calcium binding protein A6 (calcyclin) 3.31 homolog of rat orphan transporter v7-3 3.29 hypothetical protein MGC4309 3.29 tumor necrosis factor, alpha-induced protein 3 3.28 inositol 1,4,5-triphosphate receptor, type 1 3.28 Homo sapiens, clone IMAGE:3659798, mRNA, mRNA sequence 3.26 Homo sapiens, clone IMAGE:3659798, mRNA, mRNA sequence 3.26 plexin C1 3.24 cytochrome P450, subfamily XXVIIA (steroid 27-hydroxylase, cerebrotendinous xanthomatosis), polypeptide 1 3.16 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) 3.16 Suppl. Table 3 (continued) Fold Change GI- Gene carcinoids/PNET
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  • WO 2013/064736 Al 10 May 2013 (10.05.2013) P O P CT

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    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization I International Bureau (10) International Publication Number (43) International Publication Date WO 2013/064736 Al 10 May 2013 (10.05.2013) P O P CT (51) International Patent Classification: BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, C12N 9/10 (2006.01) DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (21) International Application Number: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, PCT/FI2012/05 1048 ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (22) International Filing Date: NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, 3 1 October 2012 (3 1.10.2012) RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (25) Filing Language: English ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 201 16074 1 November 201 1 (01. 11.201 1) FI GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (71) Applicant: VALIO LTD [FI/FI]; Meijeritie 6, FI-00370 TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Helsinki (FI). EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, (72) Inventors: RAJAKARI, Kirsi; c/o Valio Ltd, Meijeritie 6, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, FI-00370 Helsinki (FI).
  • Four-Dimensional Live Imaging of Apical Biosynthetic Trafficking Reveals a Post-Golgi Sorting Role of Apical Endosomal Intermediates

    Four-Dimensional Live Imaging of Apical Biosynthetic Trafficking Reveals a Post-Golgi Sorting Role of Apical Endosomal Intermediates

    Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates Roland Thuenauera,b,1,2, Ya-Chu Hsua, Jose Maria Carvajal-Gonzaleza,3, Sylvie Debordea,4, Jen-Zen Chuanga, Winfried Römerc,d, Alois Sonnleitnerb, Enrique Rodriguez-Boulana,5, and Ching-Hwa Sunga,5 aMargaret M. Dyson Vision Research Institute, Weill Medical College of Cornell University, New York, NY 10065; bCenter for Advanced Bioanalysis Linz, 4020 Linz, Austria; and cInstitute of Biology II, and dBIOSS Centre for Biological Signalling Studies, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany Edited by Keith E. Mostov, University of California School of Medicine, San Francisco, CA, and accepted by the Editorial Board January 17, 2014 (received for review March 11, 2013) Emerging data suggest that in polarized epithelial cells newly is an important regulator of biological processes that require synthesized apical and basolateral plasma membrane proteins apical trafficking, e.g., lumen formation during epithelial tubu- traffic through different endosomal compartments en route to the logenesis (11), apical secretion of discoidal/fusiform vesicles in respective cell surface. However, direct evidence for trans-endo- bladder umbrella cells (12), and apical microvillus morphogenesis somal pathways of plasma membrane proteins is still missing and and rhodopsin localization in fly photoreceptors (13). However, the mechanisms involved are poorly understood. Here, we imaged despite the physiological importance of trans-endosomal traf- the entire biosynthetic route of rhodopsin-GFP, an apical marker in ficking, the underlying mechanisms remain largely unclear. epithelial cells, synchronized through recombinant conditional ag- Previous studies on trans-endosomal trafficking in polarized gregation domains, in live Madin-Darby canine kidney cells using epithelial cells have relied on pulse chase/cell fractionation pro- spinning disk confocal microscopy.
  • Biosynthetic Investigation of Phomopsins Reveals a Widespread Pathway for Ribosomal Natural Products in Ascomycetes

    Biosynthetic Investigation of Phomopsins Reveals a Widespread Pathway for Ribosomal Natural Products in Ascomycetes

    Biosynthetic investigation of phomopsins reveals a widespread pathway for ribosomal natural products in Ascomycetes Wei Dinga, Wan-Qiu Liua, Youli Jiaa, Yongzhen Lia, Wilfred A. van der Donkb,c,1, and Qi Zhanga,b,1 aDepartment of Chemistry, Fudan University, Shanghai 200433, China; bDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and cHoward Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 23, 2016 (received for review November 24, 2015) Production of ribosomally synthesized and posttranslationally mod- dehydroaspartic acid, 3,4-dehydroproline, and 3,4-dehydrovaline ified peptides (RiPPs) has rarely been reported in fungi, even (Fig. 1A). though organisms of this kingdom have a long history as a prolific Early isotopic labeling studies showed that Ile, Pro, and Phe were source of natural products. Here we report an investigation of the all incorporated into the phomopsin scaffold (24). Because a Phe phomopsins, antimitotic mycotoxins. We show that phomopsin is a hydroxylase that can convert Phe to Tyr has not been found in fungal RiPP and demonstrate the widespread presence of a path- Ascomycetes, the labeling study seems incongruent with a ribosomal way for the biosynthesis of a family of fungal cyclic RiPPs, which we route to phomopsins, in which Tyr, not Phe, would need to be in- term dikaritins. We characterize PhomM as an S-adenosylmethionine– corporated into the phomopsin structure via a tRNA-dependent dependent α-N-methyltransferase that converts phomopsin A to pathway. Here we report our investigation of the biosynthetic path- an N,N-dimethylated congener (phomopsin E), and show that the way of phomopsins, which explains the paradoxical observations in methyltransferases involved in dikaritin biosynthesis have evolved the early labeling studies and unequivocally demonstrates that pho- differently and likely have broad substrate specificities.