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Nucleotide metabolism – Part 1 (purine biosynthesis). By Henry Wormser, Ph.D Professor of Medicinal Chemistry. Biological significance of nucleotide metabolism. Nucleotides make up nucleic acids (DNA and RNA) Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP)

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Nucleotide metabolism part 1 purine biosynthesis l.jpg

Nucleotide metabolism – Part 1(purine biosynthesis)


Henry Wormser, Ph.D

Professor of Medicinal Chemistry

Biological significance of nucleotide metabolism l.jpg

Biological significance of nucleotide metabolism

  • Nucleotides make up nucleic acids (DNA and RNA)

  • Nucleotide triphosphates are the “energy carriers” in cells (primarily ATP)

  • Many metabolic pathways are regulated by the level of the individual nucleotides

    • Example: cAMP regulation of glucose release

  • Adenine nucleotides are components of many of the coenzymes

    • Examples: NAD+, NADP+, FAD, FMN, coenzyme A

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Dietary nucleotides

  • do not contribute energy as do carbs, proteins and fats

  • are not incorporated into RNA or DNA unless given I.V.

  • normally metabolized to individual components (bases, sugar and phosphate)

  • purines are converted to uric acid which is then excreted

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Medical significance of nucleotide metabolism

  • Anticancer agents:

    • Rapidly dividing cells biosynthesize lots of purines and pyrimidines, but other cells reuse them. Cancer cells are rapidly dividing, so inhibitor of nucleotide metabolism kill them

  • Antiviral agents

    • Zidovudine (Retrovir)

    • Lamivudine (Epivir)

    • Valacyclovir (Valtrex)

  • Structures of nucleotide building blocks and nucleotides l.jpg

    Structures of nucleotide building blocks and nucleotides

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    Structures of nucleotide buildingblocks and nucleotides

    guanine: comes from guano; thymine –thymus gland

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    Ribonucleotide – phosphate = ribonucleoside

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    Biosynthesis of the purine nucleotide system

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    Synthesis of Inosine Monophosphate

    • Basic pathway for biosynthesis of purine ribonucleotides

    • Starts from ribose-5-phosphate which is derived from the pentose phosphate pathway

    • Requires 11 steps overall

    • occurs primarily in the liver

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    The big picture

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    Steps 1 thru 3

    • Step 1:Activation of ribose-5-phosphate

      • enzyme: ribose phosphate pyrophosphokinase

      • product: 5-phosphoribosyl-a-pyrophosphate (PRPP)

      • PRPP is also a precursor in the biosynthesis of pyrimidine nucleotides and the amino acids histidine and tryptophan

    Step 1 purine synthesis l.jpg

    Step 1: purine synthesis

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    Steps 1 thru 3

    • Step 2: acquisition of purine atom 9

      • enzyme: amidophosphoribosyl transferase

      • displacement of pyrophosphate group by glutamine amide nitrogen (inversion of configuration – a to b

      • product: b-5-phosphoribosylamine

    Steps 1 and 2 are tightly regulated by feedback inhibition

    Step 2 purine synthesis commited step l.jpg

    Step 2: purine synthesis:commited step

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    Steps 1 thru 3

    • Step 3: acquisition of purine atoms C4, C5, and N7

      • enzyme: glycinamide synthetase

      • b-phosphoribosylamine reacts with ATP and glycine

      • product: glycinamide ribotide (GAR)

    Step 3 purine synthesis l.jpg

    Step 3 : purine synthesis

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    Steps 4 thru 6

    • Step 4: acquisition of purine atom C8

      • formylation of free a-amino group of GAR

      • enzyme: GAR transformylase

      • co-factor of enzyme is N10-formyl THF

    • Step 5: acquisition of purine atom N3

      • The amide amino group of a second glutamine is transferred to form formylglycinamidine ribotide (FGAM)

    • Step 6: closing of the imidazole ring or formation of 5-aminoimidazole ribotide

    Step 6 purine synthesis l.jpg

    Step 6: purine synthesis

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    Step 7

    • Step 7: acquisition of C6

      • C6 is introduced as HCO3-

      • enzyme: AIR carboxylase (aminoimidazole ribotide carboxylase)

      • product: CAIR (carboxyaminoimidazole ribotide)

      • enzyme composed of 2 proteins: PurE and PurK (synergistic proteins)

    Step 7 purine synthesis l.jpg

    Step 7: purine synthesis

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    Steps 8 thru 11

    • Step 8: acquisition of N1

      • N1 is acquired from aspartate in an amide condensation reaction

      • enzyme: SAICAR synthetase

      • product: 5-aminoimidazole-4-(N-succinylocarboxamide)ribotide (SAICAR)

      • reaction is driven by hydrolysis of ATP

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    Step 8: purine synthesis

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    Steps 8 thru 11

    • Step 9: elimination of fumarate

      • Enzyme: adenylosuccinate lyase

      • Product: 5-aminoimidazole-4-carboxamide ribotide (AICAR)

    • Step 10: acquisition of C2

      • Another formylation reaction catalyzed by AICAR transformylase

      • Product: 5-formaminoimidazole-4-carboxamide ribotide (FAICAR)

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    Step 9: purine synthesis

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    Step 10: purine synthesis

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    Step 11

    • cyclization or ring closure to form IMP

    • water is eliminated

    • in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis

    • once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells

    Step 11 purine synthesis l.jpg

    Step 11: purine synthesis

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    Synthesis of adenine

    and guanine nucleotides

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    Purine nucleoside diphosphates and triphosphates:

    - to be incorporated into DNA and RNA, nucleoside

    monophosphates (NMP’s) must be converted into

    nucleoside triphosphates (NTP’s)

    - nucleoside monophosphate kinases (adenylate & guanylate kinases)

    - nucleoside diphosphate kinase

    Regulation of purine nucleotide biosynthesis l.jpg

    Regulation of purine nucleotide biosynthesis

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    The purine salvage pathway

    • Purine bases created by degradation of RNA or DNA and intermediate of purine synthesis were costly for the cell to make, so there are pathways to recover these bases in the form of nucleotides

    • Two phosphoribosyl transferases are involved:

      • APRT (adenine phosphoribosyl transferase) for adenine

      • HGPRT (hypoxanthine guanine phosphoribosyl transferase) for guanine or hypoxanthine

    Salvage of purines l.jpg

    Salvage of purines

    Adenine phosphoribosyltransferase (APRT)

    Salvage of purines37 l.jpg

    Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue)

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

    Hypoxanthine + PRPP IMP + Ppi

    Guanine + PRPPGMP + Ppi

    Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells

    Salvage of purines

    Lesch nyhan syndrome l.jpg

    Lesch-Nyhan syndrome

    • there is a defect or lack in the HGPRT enzyme

    • the rate of purine synthesis is increased about 200X

    • uric acid level rises and there is gout

    • in addition there are mental aberrations

    • patients will self-mutilate by biting lips and fingers off

    Lesch nyhan syndrome39 l.jpg

    Lesch-Nyhan syndrome

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    Salvage of purine bases

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    Next: Part 2 - biosynthesis of pyrimidine


    Nucleotide metabolism part 2 pyrimidine biosynthesis l.jpg

    Nucleotide metabolism – Part 2(pyrimidine biosynthesis)


    Henry Wormser, Ph.D

    Professor of Medicinal Chemistry

    Synthesis of pyrimidine ribonucleotides l.jpg

    Synthesis of pyrimidine ribonucleotides

    • shorter pathway than for purines

    • base is made first, then attached to ribose-P (unlike purine biosynthesis)

    • only 2 precursors (aspartate and glutamine, plus HCO3-) contribute to the 6-membered ring

    • requires 6 steps (instead of 11 for purine)

    • the product is UMP (uridine monophosphate)

    Origin of atoms in pyrimidine ring l.jpg

    Origin of atoms in pyrimidine ring

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    The big picture

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    Step 1: synthesis of carbamoyl phosphate

    • Condensation of glutamine, bicarbonate in the presence of ATP

    • Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme used here

    Step 1 pyrimidine synthesis l.jpg

    Step 1: pyrimidine synthesis

    CPS-II is the major site of regulation in animals: UDP and

    UTP inhibit the enzyme and ATP and PRPP activate it

    It is the committed step in animals

    Step 2 synthesis of carbamoyl aspartate l.jpg

    Step 2: synthesis of carbamoyl aspartate

    • enzyme is aspartate transcarbamoylase (ATCase)

    • catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Pi

    • ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTP

    • carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step

    Step 2 pyrimidine synthesis l.jpg

    Step 2: pyrimidine synthesis

    Step 3 ring closure to form dihydroorotate l.jpg

    Step 3: ring closure to form dihydroorotate

    • enzyme: dihydroorotase

    • forms a pyrimidine from carbamoyl aspartate

    • water is released in this process

    Step 3 pyrimidine synthesis l.jpg

    Step 3: pyrimidine synthesis

    Slide52 l.jpg

    • the first 3 enzymatic reactions are catalyzed by 3 separate proteins/enzymes in E. coli

    • in animals, all 3 steps are found in a multifunctional enzyme (210 kD). This allows “channeling” of the substrates and products between active sites without releasing them to the medium where they could be degraded.

    • The acronym CAD is used as a name for the multienzyme: carbamoyl phosphate synthetase, aspartate transcarbamoylase and dihydroorotase

    • channeling also increases the overall rate of multistep processes

    Step 4 oxidation of dihydroorotate to orotate l.jpg

    Step 4: oxidation of dihydroorotate to orotate

    • an irreversible reaction

    • enzyme: dihydroorotate dehydrogenase

    • oxidizing power is derived from quinones (thru coenzyme Q)

    Step 4 pyrimidine synthesis l.jpg

    Step 4: pyrimidine synthesis

    Step 5 acquisition of ribose phosphate moiety l.jpg

    Step 5: acquisition of ribose phosphate moiety

    • enzyme: orotate phosphoribosyl transferase

    • ribose phosphate originates from PRPP

    • product is orotidine-5’-monophosphate (OMP)

    • orotate phosphoribosyl transferase is also used in salvage of uracil and cytosine to their corresponding nucleotide

    Step 5 pyrimidine synthesis l.jpg

    Step 5: pyrimidine synthesis

    Step 6 decarboxylation of omp l.jpg

    Step 6: decarboxylation of OMP

    • enzyme: OMP decarboxylase

    • product: uridine monophosphate (UMP)

    • in animals, steps 5 and 6 are catalyzed by a single polypeptide with 2 active sites

    Step 6 pyrimidine synthesis l.jpg

    Step 6: pyrimidine synthesis

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    The big picture again

    Orotic aciduria l.jpg

    Orotic aciduria

    • an inherited human disease caused by a deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine synthesis

    • large amounts of orotic acid in urine

    • retarded growth and severe anemia

    • treat by administration (injection) of uridine and/or cytidine

    Leflunomide arava l.jpg

    Leflunomide (Arava)

    • Leflunomide is an isoxazole immunomodulatory agent which inhibits dihydroorotate dehydrogenase) and has antiproliferative activity. Several in vivo and in vitro experimental models have demonstrated an anti-inflammatory effect.

    • It is currently used as a DMARD in patients with serious rheumatoid arthritis

    Leflunomide arava63 l.jpg

    Leflunomide (Arava)

    Activation of leflunomide l.jpg

    Activation of leflunomide

    Opening of the isoxazole yields a reactive compound which

    can then inhibit the enzyme dihydroorotate dehydrogenase

    Synthesis of uridine and cytidine triphosphate l.jpg

    Synthesis of uridine and cytidine triphosphate

    (in bacteria, ammonia donates the amino group)

    Regulation of pyrimidine nucleotide biosynthesis l.jpg

    Regulation of pyrimidine nucleotide biosynthesis

    UTP and CTP are feeback inhibitors of CPS II

    Formation of deoxyribonucleotides l.jpg

    Formation of deoxyribonucleotides

    All pathways shown previously led to synthesis of ribonucleotides

    dADP, dGDP, dUDP and dCDP are all synthesized by the same enzyme

    Synthesized from nucleoside diphosphate (not mono or triphosphate) by

    ribonucleotide reductase

    Synthesis of dtmp l.jpg

    Synthesis of dTMP

    • Methylation of d-UMP via N5,N10-methylene THF

    • Reaction inhibited by 5-fluorouracil (Efudex)

    Slide72 l.jpg

    Activation of 5-fluorouracil

    Dihydrofolate reductase l.jpg

    Dihydrofolate reductase

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    Next - Part 3: catabolism

    Nucleotide metabolism part 3 nucleotide degradation l.jpg

    Nucleotide metabolism – Part 3(nucleotide degradation)


    Henry Wormser, Ph.D

    Professor of Medicinal Chemistry

    Nucleotide degradation l.jpg

    Nucleotide degradation

    Slide79 l.jpg


    of AMP

    Pentostatin l.jpg


    previously called deoxycoformycin (DCF)

    a purine analog with a 7-membered-ring

    potent inhibitor of adenosine deaminase

    ADA is a key enzyme which regulates

    adenosine levels in cells

    indicated for refractory hairy cell leukemia

    other uses: chronic lymphocytic leukemia

    and lymphomas

    Adenosine deaminase l.jpg

    Adenosine deaminase

    Ada deficiency l.jpg

    ADA deficiency

    • In the absence of ADA lymphocytes are destroyed

    • deoxyadenosine is not destroyed, is converted to dAMP and then into dATP

    • dATP is a potent feedback inhibitor of deoxynucleotide biosynthesis

    • this leads to SCID (severe combined immunodeficiency disease)

    • Infants with this deficiency have a high fatality rate due to infections

    Ada deficiency84 l.jpg

    ADA deficiency

    • treatment consists of administering pegylated ADA which can remain in the blood for 1 – 2 weeks

    • more efficient is gene therapy: replacing the gene that is missing or defective

    • gene therapy has been performed on selected patients

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    Degradation of

    GMP and XMP

    Catabolism of purines l.jpg


    Slide89 l.jpg


    • a disorder associated with abnormal amounts of urates in the body

    • early stage: recurring acute non-articular arthritis

    • late stage: chronic deforming polyarthritis and eventual renal complication

    • disease with rich history dating back to ancient Greece

    Slide90 l.jpg


    • once fashionable to associate gout with intelligence

    • people with gout:

      • Isaac Newton

      • Benjamin Frankin

      • Martin Luther

      • Charles Darwin

      • Samuel Johnson

    Slide91 l.jpg


    • prevails mainly in adult males

    • rarely encountered in premenopausal women

    • symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be seen under polarized light)

    • usually affect joints in the lower extremities (the big toe is the classic site)

    Slide92 l.jpg


    Four stages of gout l.jpg

    Four Stages of Gout

    1. asymptomatic hyperuricemia

    2. acute gouty arthritic attacks

    3. asymptomatic intercritical period

    4. tophaceous gout (characterized by the formation of tophi in joints)

    • podagra (big toe)

    • cheiagra (wrist) according to Hippocrates

    • gonadra (knee)

    Diagnostic features l.jpg

    Diagnostic features

    • usually affect joints in the lower extremities ( 95%)

    • onset is fast and sudden

    • pain is usually severe; joint may be swollen, red and hot

    • attack may be accompanied by fever, leukocytosis and an elevated ESR

    Drugs which may induce hyperuricemia l.jpg

    Drugs which may induce hyperuricemia

    • niacin

    • thiazides and other diuretics

    • low dose aspirin

    • pyrazinamide

    • ethambutol

    • cyclosporine

    • cytotoxic drugs

    Non pharmacological approaches l.jpg

    Non-pharmacological approaches

    • Avoid purine rich foods:

      • red meat and organ meat (liver, kidneys)

      • shellfish, anchovies, mackerel, herring

      • meat extracts and gravies

      • peas and beans, aspargus, lentils

      • beer, lager, other alcoholic beverages

    • Weight loss

    • Control alcohol (binge drinking)

    Pharmacological management of gout l.jpg

    Pharmacological management of gout

    • based on the premise that the hyperuricemia is due to both overproduction and underexcretion of uric acid

    • symptomatic relief of pain is also achieved with analgesics (i.e. indomethacin)

    • drugs used:

      • analgesics (NSAIDs)

      • uricosuric agents

      • xanthine oxidase inhibitors

    Therapy of acute gout l.jpg

    Therapy of acute gout

    • treat with colchicine or NSAIDs

    • avoid aspirin

    • do not treat with allopurinol or uricosuric drugs

    • uric acid lowering agents should never be started or stopped during acute attack

    • pain resolution occurs within 48-72 hrs

    Colchicine l.jpg


    a non-basic alkaloid from the seeds and corms of Colchicum autumnale

    (Meadow Safron)

    Colchicine100 l.jpg


    • used in the symptomatic treatment of acute attacks of gout

    • decreases leukocyte motility, decreases phagocytosis and lactic acid production

    • not used in other forms of arthritis

    • a very potent drug

    • can cause severe GI distress and abdominal pain

    Probenecid benemid l.jpg

    Probenecid (Benemid)

    A uricosuric agent

    Probenecid benemid102 l.jpg

    Probenecid (Benemid)

    • inhibits the tubular reabsorption of uric acid

    • it can also inhibit the tubular excretion of certain organic acid via the transporter

    • used in gout to promote the elimination of uric acid (not effective in acute attack)

    • also used to enhance plasma concentration of certain antiinfectives (beta lactams)

    Allopurinol zyloprim l.jpg

    ALLOPURINOL (Zyloprim)

    • prevention of attacks of gouty arthitis and nephropathy

    • also used during chemotherapy of cancer and to prevent recurrent calcium oxalate calculi

    • metabolized to oxypurinol (also an inhibitor of xanthine oxidase)

    • inhibits the metabolism of certain anticancer drugs (6-MP, azathioprine)

    Allopurinol zyloprim104 l.jpg

    Allopurinol (Zyloprim)

    An inhibitor of xanthine oxidase; prevents the formation of uric acid from

    precursorial purines

    Fate of uric acid l.jpg

    Fate of uric acid

    • in human and other primates uric acid is the final product of purine degradation and is excreted in the urine

    • the same is true in bird, reptiles and many insects

    • in other mammals uric acid is oxidized to allantoin (urate oxidase)

    • teleost (bony) fish convert allantoin to allantoic acid

    • cartilaginous fish and amphibian further degrade allantoic acid to urea

    • and finally marine invertebrates decompose urea to ammonia

    Rasburicase elitek l.jpg

    Rasburicase (Elitek)

    A recombinant form of uric

    acid oxidase. Used for initial management of plasma uric acid levels in pediatric patients with leukemia, lymphoma, and solid tumor malignancies who are receiving anticancer therapy expected to result in tumor lysis and subsequent elevation of plasma uric acid.

    Slide109 l.jpg

    Catabolism of a pyrimidine

    Formation of deoxyribonucleotides112 l.jpg

    Formation of deoxyribonucleotides

    • ribonucleotide reductase studied by JoAnne Stubbe (Wisconsin, then MIT)

    • very complex enzyme; contains:

      • Tyrosine radical

      • 2 non-heme irons

      • Two catalytically active cysteine residues

      • Cys are reduced by other proteins – thioredoxin

      • Ribo. Reductase is the therapeutic target of the anticancer drug hydroxyurea

    Mechanism of ribonucleotide reductase l.jpg

    Mechanism of ribonucleotide reductase

    • Free radical mechanism involving tyrosyl residues and cysteine residues on the enzyme

    • The enzyme is a dimer of dimers:

      • R1 – a dimer of identical a subunits (85 kD each)

      • R2 – a dimer of identical b subunits (45 kD each)

    Reduction of the disulfide bond in ribonucleotide reductase l.jpg

    Reduction of the disulfide bond in ribonucleotide reductase

    • 2 proteins can perform this reductive reaction:

      • Thioredoxin (ubiquitous 12 kD monomer)

      • Glutaredoxin which functions similarly to thioredoxin. Oxidized glutredoxin is reduced by glutathione (g-glutamylcysteinylglycine)

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    Regeneration of thioredoxin and ribonucleotide reductase

    Hydroxyurea hydrea l.jpg

    HYDROXYUREA (Hydrea)

    • inhibits the enzyme ribonucleotide reductase

      • this enzyme causes ribonucleotides to be converted to deoxyribonucleotides

      • DNA synthesis cannot occur

      • cell are killed in the S phase

      • drug holds other cells in the G1 phase

    • primarily used to treat chronic myelogenous leukemia

    • cancer cell develop resistance by:

      • increasing quantity of inhibited enzyme

      • decreasing sensitivity of enzyme for inhibitor

    • used orally

    • major side effect is leukopenia

    Gemcitabine gemzar l.jpg

    GEMCITABINE (Gemzar)

    Another inhibitor of ribonucleotide reductase:indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer

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    The purine nucleotide cycle for anaplerotic replenishment of citric acid cycle intermediates in skeletal muscle

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    The end

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