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A Mouse in the 12R-, Alox12b, Disrupts Formation of the Epidermal Permeability Barrier Jennifer L. Moran1, Haiyan Qiu1, Annick Turbe-Doan1, Yujuan Yun1, William E. Boeglin2, Alan R. Brash2 and David R. Beier1

Nonbullous congenital ichthyosiform erythroderma (NCIE) is a nonsyndromic form of autosomal recessive congenital characterized by hyperkeratosis and a disruption in the epidermal permeability barrier. Identification of in two (LOXs), ALOX12B (12R-LOX) and ALOXE3 (eLOX3), and further functional studies implicate ALOX12B and ALOXE3 in the etiology of NCIE. Here, we report a mutation in Alox12b in the recessive ethylnitrosurea-induced mouse mutant, mummy (Alox12bmmy-Bei). mummy mutants have red, scaly skin and die perinatally. Histologically, mummy mutants display defects in the . We mapped mummy to a 1.9 Mb interval on Chr. 11 containing Alox12b (12R-LOX), Aloxe3 (eLOX3) and Alox15b (8-LOX). Sequencing of all three identified a nonsense mutation in the catalytic domain of Alox12b.We demonstrate that mummy mutants have a disrupted epidermal permeability barrier and that the nonsense mutation in mummy abolishes the activity of 12R-LOX. The mummy mutant provides a mouse model for LOX-mediated NCIE and is the first described mouse mutant affecting epidermal barrier formation identified by forward genetics. Journal of Investigative Dermatology (2007) 127, 1893–1897; doi:10.1038/sj.jid.5700825; published online 12 April 2007

INTRODUCTION of two types: (LI) and nonbullous The epidermal permeability barrier is a specialized epidermal congenital ichthyosiform erythroderma (NCIE, CIE, or NBCIE) structure that protects the skin from dehydration and the entry (reviewed in Akiyama et al., 2003). The genetic basis of of chemical or infectious agents and is essential for terrestrial several ARCI loci have been identified, and mutations in life. Barrier function is provided by the stratum corneum, the some genes can result in multiple forms of ARCI (reviewed in outer layer of the epidermis composed of terminally Akiyama et al., 2003; Richard, 2004; Akiyama, 2006). Many differentiated keratin-filled cells with crosslinked cornified ARCI disease genes encode proteins involved in the synthesis envelopes (CEs) sealed by lipid bilayers. Incomplete barrier of the CE or epidermal lipid metabolism, such as TGM1,a function in humans is observed in premature infants, transglutaminase that crosslinks the CE; ABCA12, an ATP- inflammatory skin diseases such as , and in binding cassette transporter involved in lipid trafficking, and congenital ichthyoses (reviewed in Richard, 2004; Segre, ALOX12B and ALOXE3, two lipoxygenases (LOXs) involved 2006). in fatty acid metabolism. Nonsyndromic autosomal recessive congenital ichthyosis In the mouse, several genes required for epidermal barrier (ARCI) is a genetically and clinically heterogeneous class of function have been identified. These include CE and skin diseases marked by scaly skin and impaired epidermal intercellular junction structural proteins, proteins involved barrier function. The most common clinical cases of ARCI are in CE protein crosslinking and lipid synthesis, and transcrip- tion factors (reviewed in Segre, 2003, 2006). Two such genes 1Genetics Division, Brigham and Women’s Hospital, Harvard Medical are known human congenital ichthyoses disease genes: Tgm1 School, Boston, Massachusetts 02115, USA and 2Department of (autosomal recessive LI and NCIE) and loricrin (autosomal Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee, dominant NCIE) (reviewed in Richard, 2004). Here, we USA describe the first mouse mutation in the 12R-LOX, Alox12b, Correspondence: Dr Jennifer L. Moran, Genetics Division, Brigham and Women’s Hospital, Harvard Medical School, 77 Avenue Louis Pasteur, which is mutated in some human NCIE-affected individuals. Boston, Massachusetts 02115, USA. E-mail: [email protected] Abbreviations: EPB, epidermal permeability barrier; HETE, RESULTS hydroperoxyeicosatetraenoic acid; LOX, lipoxygenase; SNP, single The recessive mummy mutant was identified in an ethyl- nucleotide polymorphism; NCIE, nonbullous congenital ichthyosiform erythroderma; ARCI, autosomal recessive congenital ichthyosis nitrosurea mutagenesis screen for recessive neonatal develop- Received 21 September 2006; revised 9 February 2007; accepted 15 February mental defects (Herron et al., 2002). Affected mice are born 2007; published online 12 April 2007 with red, shiny skin that rapidly desiccates and appears scaly

& 2007 The Society for Investigative Dermatology www.jidonline.org 1893 JL Moran et al. Mouse 12R-LOX Mutation Disrupts Skin Barrier

Control mummy a ab A/J B6 5,712 5,716 6,436 6,525 6,526 6,523 6,522 6,344 6,437 6,524 6,435 6,527

M BPP2222222222 Position UUUUAAAAAAAAAA (Mb) Marker

26.7 rs3090752 30.7 rs4228643 55.2 rs4228731 58.1 rs4228770 cd 69.6 D11Mit29 73.2 D11Mit194 79.0 rs13481117

b MUM_Alox12b_14AJ.F Fragment base #267. C T CCTGG A TC T C T CCTTGGA C T Figure 1. Gross phenotype and histology of newborn mummy mutants. (a–b) mummy mutants are distinguishable from unaffected littermates at birth by their red skin that rapidly desiccates and appears scaly. (c–d) Newborn A/J mummy dorsal skin shows a thickened stratum corneum by hematoxylin and eosin staining of sections (n ¼ 9 affected mice; n ¼ 10 unaffected mice, evaluated at E17.5, E18.5, and newborn). Bar ¼ 150 mm. MUM_Alox12b_14A.F Fragment base #304. Base 304 of 485 (Figure 1a and b). The mutation causes lethality within CTCC TGA A TC T 24 hours of birth. Histological analysis of newborn epidermis C TTCCG AA TC T demonstrates a thickened, compressed stratum corneum in the affected mice in contrast to the normal basket-weave mummy corneum layers in unaffected littermates (Figure 1c and d). The initial map position of the mummy mutation was determined by genotyping 10 affected intercross progeny and two parentals with a fixed whole genome single nucleotide polymorphism (SNP) panel of 394 SNPs (Moran et al., 2006). Figure 2. Genetic mapping and identification of the mummy mutation. Retention of homozygous alleles for the mutagenized strain (a)Themummy mutation was mapped to a 21 Mb interval on Chr 11 between (A/J) and positive logarithm of odds (LOD) association at a SNPs rs4228770 (58.1 Mb) and rs13481117 (79.0 Mb) by performing a whole genome scan of 394 SNPs on two parental (A/J Â C57BL/6J) F1 and 10 affected single locus demonstrates linkage (LOD 2.81) to a 21 Mb F2 progeny. Alleles for the mutated A/J strain (M) are red; C57BL/6J interval on (Chr) 11 (rs4228770 (58.1 Mb) – background strain (B), blue; heterozygotes, yellow; and no calls, white. P for rs13481117 (79.0 Mb) (Figure 2a)). The genetic interval was parental; U for unaffected and A for affected mice. Genotypes from two further refined to 1.9 Mb (D11Mit115 (67.4 Mb) – D11Mit298 nonrecombinant simple-sequence length polymorphism markers, D11Mit29 (69.3 Mb)) by genotyping recombinant affected mice and an (69.6 Mb) and D11Mit194 (73.2 Mb), are also shown. (b) Chromatograms of additional 15 affected progeny with simple-sequence length Alox12b genomic sequence in wild-type A/J (top) and mummy-affected (middle, bottom) mice showing a G to A mutation, which introduces a polymorphism markers (data not shown). The refined 1.9 Mb premature stop codon in mummy mutants. genetic interval contains 41 RefSeq genes. Three genes in the 1.9 Mb genetic interval, Alox12b, Aloxe3, and Alox15b, were evaluated for candidates for the The mutation introduces a nonsense codon into the mummy mutation based on several criteria. Firstly, these Alox C-terminal LOX catalytic domain (W633X), which results in genes comprise a family of epithelial-specific LOXs (hepoxy- truncation of the protein by 68 amino acids. Mutated lins) expressed in the mouse epidermis (Sun et al., 1998; transcript is present in mummy homozygote skin, indicating Heidt et al., 2000). Secondly, Alox15b (Alox8; 8-LOX) the absence of nonsense-mediated mRNA decay (data not expression is upregulated in a mouse model of severe shown). dermatitis (Schneider et al., 2004). Thirdly, mutations in LOXs are lipid-peroxidizing that oxygenate ALOX12B (12R-LOX) and ALOXE3 (eLOX3) have been polyunsaturated fatty acids to hydroperoxide derivatives identified in individuals with ARCI, specifically the ARCI (Samuelsson et al., 1987). Mouse Alox12b protein (12R- subtype NCIE, an ichthyosis characterized by hyperkeratosis LOX) oxygenates arachidonate methyl rather than and a disruption of the epidermal permeability barrier (Jobard to 12R-hydroperoxyeicosatetraenoic acid et al., 2002; Eckl et al., 2005). Sequencing of all exons and (HETE) (Krieg et al., 1999). To assess the affect of the mutation intron–exon boundaries from all three genes was performed on the enzymatic activity of 12R-LOX, we overexpressed on genomic DNA from mummy-affected mutant mice. histidine (His)-tagged mutant W633X and wild-type Alox12b We identified a G to A single change in exon 14 proteins in COS7 cells and assayed for 12R-LOX activity by of the Alox12b (Figure 2b). The Alox12b gene encodes incubating cell extracts with [14C]-labeled arachidonic acid one transcript with 15 exons and a 701 amino-acid protein. methyl ester substrate. Analysis using reversed-phase high-

1894 Journal of Investigative Dermatology (2007), Volume 127 JL Moran et al. Mouse 12R-LOX Mutation Disrupts Skin Barrier

12 Control mummy 11 a Standards 89 5 12-HETE 15 Me ester UV 235 nm

E17.5

CPM 12-HETE 400 WT Alox12b 12-HETE Me ester 200

0 Mutant Alox12b 200

0 0 51015 20 Retention time (minutes) E18.5 Figure 3. Reversed-phase HPLC analysis of catalytic activities of wild-type and mutant Alox12b. The His-tagged enzymes expressed in COS7 cells were incubated with [14C]arachidonate methyl ester as substrate and the products analyzed by reversed-phase HPLC using a Waters Symmetry C18 column (25 Â 0.46 cm) with a solvent system of methanol/water/acetic acid (85:15:0.01, by volume) run at a flow rate of 1 ml/minute on an Agilent 2100 series HPLC system equipped with an on-line Flo-One Beta radioactive 105 detector. Arachidonate methyl ester is used as the test substrate because b 100 +/− mouse 12R-LOX does not metabolize free arachidonic acid (Krieg et al., +/+ 1999). Top trace: profile of unlabeled standards added to the sample before 95 injection and detected with UV recording at 235 nm. Middle trace: 90 14C-labeled products formed by wild-type Alox12b – note that the [14C]12- 85 HETE methyl ester product formed from the [14C]arachidonate methyl ester is 80 mainly hydrolyzed to the free acid by esterase activity in the COS cell extract body weight Percent 14 −/− (cf. ref (Krieg et al., 1999)). Lower trace: C elution profile from mutant 75 Alox12b shows no detectable products. 70 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Time (hours) performance liquid chromatography (HPLC) revealed that Figure 4. Analysis of epidermal permeability barrier function in mummy wild-type Alox12b converted the substrate to 12R-HETE and mice. (a) Widespread permeability of X-gal substrate in affected mice at E17.5 12R-HETE methyl ester, whereas mutant Alox12b-W633X (n ¼ 5À/À; n ¼ 13 þ /À; n ¼ 7À/À) and E18.5 (n ¼ 2À/À; n ¼ 3 þ /À; produced no detectable products (Figure 3). Expression levels n ¼ 4 þ / þ ) compared with littermate controls. (b) The weight of intercross of wild-type and mutant Alox12b proteins, as detected by progeny was measured every hour for the first 6 hours after birth. Dramatic Western blotting with an antibody against the His tag, were weight loss of mummy homozygotes at E17.5 and E18.5 (n ¼ 5À/À), 21–27% equivalent (data not shown). Thus, the mummy mutation weight loss compared with no more than 3% for control littermates (n ¼ 16 þ /À; n ¼ 7 þ / þ ), shows a lack of barrier function. W633X completely eliminates Alox12b enzymatic activity. Identification of a loss-of-function mutation in mouse Alox12b, association of mutations in human ALOX12B in An additional test of skin barrier function is to monitor NCIE, and the neonatal mummy phenotype all suggest that mice for dehydration. E17.5 and E18.5 mice were dissected epidermal permeability barrier function may be compro- and revived, and weights were measured every hour for 6 h. mised in mummy mice. Formation of the permeability barrier mummy mutants lost between 21 and 27% of their body is a highly patterned process that begins at E16 in the mouse weight within 6 hours of birth indicating rapid dehydration, and is completed by birth (Hardman et al., 1998). Disruption the likely cause of perinatal lethality (Figure 4b). of the skin barrier is lethal in mice, as demonstrated by the study of several perinatal lethal gene-targeted mutations DISCUSSION affecting the skin barrier (reviewed in Segre, 2003; Leyvraz Here we describe the first mouse mutation in the 12R-LOX, et al., 2005; Herrmann et al., 2005). To test whether mummy Alox12b, which was identified in an ethylnitrosurea muta- mutants have a defective skin barrier, a dye permeability genesis screen for recessive perinatal developmental defects. assay was performed on untreated E17.5 and E18.5 fetuses. In mummy (Alox12bmmy-Bei) mice, a nonsense mutation in X-gal substrate does not penetrate skin with a functional Alox12b leads to premature truncation of the protein and loss barrier and accessibility of X-gal to the skin produces a of LOX catalytic activity. Alox12b deficiency in mice leads to colored precipitate through endogenous b-galactosidase a lack of epidermal permeability barrier function and activity (Hardman et al., 1998). mummy mutants do not perinatal lethality. In humans, mutations in ALOX12B show evidence of barrier acquisition as demonstrated by blue underlie the genetic basis for some cases of NCIE, one of staining across the entire body at E17.5 and E18.5 (Figure 4a). the main clinical forms of ARCI. Like the mouse nonsense

www.jidonline.org 1895 JL Moran et al. Mouse 12R-LOX Mutation Disrupts Skin Barrier

mutation, all human point mutations tested result in abolish- Genetic mapping ment or significant impairment of 12R-LOX activity (Eckl mummy was identified in an ethylnitrosurea mutagenesis screen for et al., 2005; Yu et al., 2005). autosomal recessive developmental phenotypes (Herron et al., The mummy mutant provides a mouse model for human 2002). The mutation was generated on the A/J strain and maintained NCIE, as mummy skin shares some of the features of NCIE- by serial backcross to FVB/NJ. The mutation was outcrossed to affected skin. NCIE-affected individuals display erythroder- C57BL/6J for mapping and phenotypic analysis. Ten affected mic, scaly skin, an increase in stratum corneum thickness F2-affected progeny and two F1 male parents were genotyped with (hyperkeratosis), inflammation and impaired epidermal bar- a whole genome SNP panel containing 394 SNPs and simple- rier function, with a range in phenotype severity (reviewed in sequence length polymorphisms (Moran et al., 2006). Recombinant Akiyama et al., 2003). mummy mutants are born with shiny, affected mice and an additional 15 affected mice were genotyped red skin that rapidly desiccates because of a defective with simple-sequence length polymorphism markers to narrow the epidermal barrier. Histologically, the stratum corneum of genetic interval to 1.9 Mb (D11Mit115 (67.4 Mb) – D11Mit298 late gestational and neonatal skin is thickened. Although (69.3 Mb); Genome Build 36). NCIE is not typically lethal, mummy mice die within 24 hours of birth, presumably because of rapid dehydration. Thus, we DNA sequencing are not able to observe the effects of Alox12b deficiency in High-throughput sequencing of exons and exon/intron boundaries of the adult mouse. Lethality in mice and not humans has also the Alox12b, Aloxe3, and Alox15b genes was performed as been observed in transglutaminase-mediated barrier defects described previously (Moran et al., 2006). Genomic DNA from the and ARCI, and may be owing to the lack of medical care for wild-type A/J strain and two mummy-affected mice were used as newborn mice or the larger surface area-to-volume ratio of sequencing templates. Alox12b Vega Gene ID:OTTMUSG000000- mice compared with humans (Matsuki et al., 1998; Kuramoto 005964 (www.ensembl.org/Mus_musculus). et al., 2002; Segre 2006). Alox12b is a member of a family of epithelial-specific LOXs expressed in neonatal and adult mouse epidermis (Sun Skin permeability assay et al., 1998; Heidt et al., 2000). Alox12b mRNA is first E17.5-E18.5 untreated fetuses were stained with X-gal solution at detected in the skin and other epithelial tissues at E15.5 and is low pH to assay endogenous epidermal b-galactosidase activity as highly expressed in newborn mouse epidermis (Sun et al., described by Hardman et al. (1998). 1998). Alox12b’s expression in late granular and squamous keratinocytes led to the proposal that Alox12b plays a role in skin barrier formation and terminal differentiation (Heidt Wild-type and mutant Alox12b activity assay et al., 2000). The skin barrier begins to form at E16 when a cDNA amplification and cloning. Total RNA was extracted from precursor of the stratum corneum is first detected (Hardman wild-type and mutant E18.5 skin with a Qiagen RNeasy Midi Kit et al., 1998). We observed a defect in epidermal permeability (Qiagen, Valencia, CA). First strand cDNA was synthesized using barrier (EPB) formation in mummy mice at E17.5. Given the Superscript III First Strand cDNA Synthesis Kit (oligo-dT primer; expression of Alox12b and both the barrier and stratum Invitrogen, Carlsbad, CA) and subsequently used as a template for corneum defects in mummy mice, we speculate that Alox12b polymerase chain reaction (PCR) amplification of wild-type Alox12b activity is required for terminal differentiation of keratino- cDNA with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) cytes and formation of a functional stratum corneum. To and cDNA-specific primers (forward primer 50-GTGGTTCCCGCTG elucidate potentially a mechanism for the epidermal defects, ACTGC-30; reverse primer 50-TTACAGTGATTTTATTGGA-30). PCR examination of the morphology and integrity of CEs, lipid product was cloned into pCR2.1TOPO and the cDNA sequence was synthesis and processing, and ultrastructural cellular archi- verified by full-length sequencing. tecture will need to be performed. Wild-type and mutant cDNAs were cloned into the Gateway The identification of several human ARCI disease genes, system for mammalian expression (Invitrogen). Briefly, the wild-type ALOX12B, ALOXE3, ichthyin, and FLJ39501, has led to the Alox12b cDNA pCR2.1TOPO clone was used as a template for PCR elucidation of an arachidonic acid metabolism LOX-depen- amplification of wild-type and mutant cDNA with Pfx50 DNA dent pathway in ARCI pathogenesis (Jobard et al., 2002; Yu Polymerase (Invitrogen). PCR were performed with a common et al., 2003; Lefevre et al., 2004; Lefevre et al., 2006). It is still forward and distinct reverse primers; the mutant cDNA primer unclear whether the LOX-dependent pathway plays a contained the point mutation (forward 50-CACCATGGCCACCTA structural or signaling role in epidermal differentation. The TAAAGTC-30; wild-type reverse primer 30-CAGAAGGGTGGACTA mouse mutant mummy provides a model that will facilitate GATGGA-30; mutant reverse primer 30-GCTCCCGACAGAGAGTT further exploration of the role of LOXs in epidermal barrier CAG-30). Products were cloned into the pENTR/SD/D-TOPO vector formation and terminal differentiation of the skin. using the pENTR Directional TOPO Cloning Kit (Invitrogen) according to manufacturer’s protocol. After isolation of plasmid DNA with the Purelink HQ Mini Plasmid Kit (Invitrogen), wild-type MATERIALS AND METHODS and mutant Alox12b cDNAs were cloned into the Gateway Histology destination vector pDEST26 (N-terminal fusion vector with in-frame Newborn heads were fixed in Bouin’s, embedded in paraffin, 6 Â His-tag) by LR recombination using Gateway LR Clonase sectioned and stained with hematoxylin and eosin. enzyme mix according to manufacturer’s protocol.

1896 Journal of Investigative Dermatology (2007), Volume 127 JL Moran et al. Mouse 12R-LOX Mutation Disrupts Skin Barrier

Transfection and expression of wild-type and mutant proteins Heidt M, Furstenberger G, Vogel S, Marks F, Krieg P (2000) Diversity of Subconfluent COS7 cells were transiently transfected using a 1:4 mouse lipoxygenases: Identification of a subfamily of epidermal ratio of plasmid to FuGENE 6 Reagent (Roche Applied Science, isozymes exhibiting a differentiation-dependent mRNA expression pattern. Lipids 35:701–7 Indianapolis, IN) according to the manufacturer’s instructions. After Herrmann T, Grone H-J, Langbein L, Kaiser I, Gosch I, Bennemann U et al. 48 hours, cells were rinsed and harvested in phosphate-buffered (2005) Disturbed epidermal structure in mice with temporally controlled saline, pelleted, and then sonicated in 200 ul buffer (50 mM Tris (pH Fatp4 deficiency. J Invest Dermatol 125:1228–35 7.5) and 150 mM NaCl) on ice using a Virsonic 100 (Virtis) at setting Herron BJ, Lu W, Rao C, Liu S, Peters H, Bronson RT et al. (2002) Efficient 6 for 10 seconds. generation and mapping of recessive developmental mutations using ENU mutagenesis. Nat Genet 30:185–9 Jobard F, Lefevre C, Karaduman A, Blanchet-Bardon C, Emre S, Weissenbach J 12R-LOX activity assay et al. (2002) Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase Reversed phase-HPLC analyses of wild-type and mutant cell lysates (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1. Hum Mol Genet were performed essentially as described (Chen et al., 2006). Briefly, 11:107–13 100 ml of sonicated cell lysates were incubated with freshly purified 14 Krieg P, Siebert M, Kinzig A, Bettenhausen R, Marks F, Furstenberger G (1999) [1- C]arachidonic acid methyl ester (500,000 d.p.m., added in 1 ul Murine 12(R)-lipoxygenase: Functional expression, genomic structure of ethanol, 50 mM final concentration) at 371C for 30 minutes. Lipids and chromosomal localization. FEBS Lett 446:142–8 were extracted using a Bligh and Dyer protocol, evaporated to Kuramoto N, Takizawa T, Takizawa T, Matsuki M, Morioka H, Robinson JM dryness under nitrogen and stored in methanol at À201C. Extracts et al. (2002) Development of ichthyosiform skin compensates for defective permeability barrier functio in mice lacking transglutaminase were analyzed by reversed-phase HPLC using a Waters Symmetry 1. J Clin Invest 109:243–50 C18 column (25 Â 0.46 cm) with a solvent system of methanol/water/ Lefevre C, Bouadjar B, Ferrnad V, Tadini G, Megarbane A, Lathrop M et al. acetic acid (80:20:0.01, by volume) run at a flow rate of 1 ml/minute (2006) Mutations in a new cytochrome p450 gene in lamellar ichthyosis on an Agilent 2100 series HPLC system with Diode Array Detector type 3. Hum Mol Genet 15:767–76 and on-line Flo-One Beta radioactive detector. Before injection, a Lefevre C, Bouadjar B, Karaduman A, Jobard F, Saker S, Ozguc M et al. (2004) mixture of unlabeled HETE standards and 12-HETE methyl ester was Mutations in ichthyin a new gene on chromosome 5q33 in a new form of autosomal recessive congenital ichthyosis. Hum Mol Genet 13:2473–82 added to each sample to help establish the precise retention times for comparison with 14C-labeled products. Leyvraz C, Charles R-P, Rubera I, Guitard M, Rotman S, Breiden B et al. (2005) The epidermal barrier function is dependent on the serine The authors’ institutions approved all described studies. protease CAP1/Prss8. J Cell Biol 170:487–96 Matsuki M, Yamashita F, Ishida-Yamamoto A, Yamada K, Kinoshita C, Fushiki CONFLICT OF INTEREST S et al. (1998) Defective stratum corneum and early neonatal death in The authors state no conflict of interest. mice lacking the gene for transglutaminase 1 (keratinocyte transgluta- minase). Proc Natl Acad Sci USA 95:1044–9 ACKNOWLEDGMENTS Moran JL, Bolton AD, Tran PV, Brown A, Dwyer ND, Manning DK et al. 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