chap 21 bacteriophages

8/3/2019 Chap 21 Bacteriophages

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The Best for Last: Bacteriophages

Chapter 21

Bacteriophages attached to a bacterium.


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21.1 Bacteriophage Research History

Bacteriophagesviruses that infect bacteria Discovered after recognition of bacterial

hosts in the 1880s (the golden age of

microbiology) Frederick W. Twort observed glassy

transformation

Used a Chamberland filterto discover thefilterable bacteriophages that lysed cultures ofmicrococci

His work was not acknowledged for 5 years


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Felix dHerelle (1873-1849)

Credited as the sole-discoverer of bacteriophages

1917 report by dHerelledescribes the lysis ofdysentery-causing bacteria

grown in liquid medium

Tworts paper was not cited indHerelles publication

SPL/Photo Researchers, Inc. .

Figure 21.01: Felix d'Herelle (1873-1949)


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Felix dHerelles Research

2 directionsDetermining the biological nature of

bacteriophages

Exploring the use of bacteriophagesas therapy to treat bacterial infections

in a preantibiotic era


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dHerelle and Bacteriophage Therapy

1919 field trials to control an epidemic ofchicken typhoid ( ) caused by the

bacterium Salmonella gallinarum

Inoculated chickens either orally or by injectionwith bacteriophages

Flocks treated with bacteriophages suffered fewerdeaths and shorter epidemics that did not reoccur

Positive results motivated dHerelle to conducthuman trials in the 1920s


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dHerelles Human Trials

To prove that the bacteriophagepreparations were safe:

dHerelle injectedhimselfFamily membersCoworkers

No harmful effects observed


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dHerelles Bacteriophage Experiments, cont.

1925 report by dHerelle regarding experiments at theLeague of Nations quarantine station in Alexandria,Egypt

Injected 4 patients suffering from laboratory confirmedbubonic ( ) plague with bacteriophages into the

bubos present in their lymph nodes

All 4 patients recovered rapidly

After this success, dHerrelle traveled the worldcontinuing bacteriophage therapy as a means ofcontrolling cholera outbreaks


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dHerelles Career

Accepted an appointment as a professor ofprotobiology at Yale University in 1928

Played a role in establishing a bacteriophageinstitute in Tbilisi, Soviet Georgia in 1934 This institute exists today as the Georgia Eliava

Institute of Bacteriophage, Microbiology and Virology


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Bacteriophage Therapy Abandoned in the 1930s

Published reports were not consistent The therapy appeared to be hit or miss The main reason for the abandonment of

bacteriophage therapy was the development of

antibiotics

Today the Western world has a renewed interest inbacteriophage therapy to combat antibiotic-resistant

strains of pathogenic bacteria


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The History of the Georgia Eliava Institute of

Bacteriophage, Microbiology and Virology

Founded in 1923 by Professor Giorgyi Eliava Felix dHerelle visited Eliava at the Institute during

1934-1935 via invites by Josef Stalin

Stalin was interested in bacteriophage therapy for military use

Eliava was executed in 1937 dHerelle did not return to the Institute after his death


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Activities of the Georgia Eliava Institute

Continued after Eliavas death Research focus centered around bacteriological

and bacteriophage research

The Institute change names in 1952 and wasreorganized in 1988

The Institute manufactured bacteriophage sprays,salves , ointments and pills

The Institute was damaged during the Georgian civilwar.

Thousands of bacteriophage samples were lost The Institute was revitalized by energetic

entrepreneurs after a 1997 BBC Broadcast entitled TheVirus That Cures


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The American Phage Group

Formed in the 1940s Consisted of scientists from universities

throughout the U.S. that were studying

bacteria and bacteriophages

Max DelbruckSalvador LuriaAlfred Hershey

The Phage Group spent summers doingresearch experiments at Cold Spring

Harbor (Long Island, NY)


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The American Phage Group, cont.

Optimized experiments to study the biology ofbacteriophages such as:One-step growth experimentsPlaque assaysFocused on a selected group of

authorized bacteriophages such as the

Type (T) bacteriophages


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Bacteriophage Lysis and Plaque Assays

Figure 21.2a: Bacterial cells growing in

log phase (left) and being lysed bybacteriophages (right).

David B. Fankhauser, PhD, University of Cincinnati Clermont College

Figure 21.2b: Bacteriophage plaque

assays from one-step growthexperiments.


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21.2 Bacteriophage Ecology

Scientific community estimates that aquaticcommunities contain 4-6 X 1030 bacteria and 1X 1031 bacteriophages

Bacteriophages recycle bacterial carbon in themarine environment

Marine microbial ecology is a rapidlydeveloping field


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21.3 The Biology of Bacteriophages

Composition and Structure

Over 5,100 bacteriophages have beenanalyzed using the transmission electron

microscope

ICTV recognizes 1 order 13 families 31 genera


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Bacteriophage Structure

4 basic shapes or symmetries Binary (head and tail structure) Icosahedral (also called cubic) Helical (filamentous) Pleomorphic ( )

The majority of bacteriophages contain a headand tail structure


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Representatives of the 13 Bacteriophage Families

Grouped by their types of nucleic acid genomes.

Figure 21-3

Adapted from H. -W. Ackermann, Arch. Virol. 146 (2001): 843-857.


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Electron Micrographs of Tailed Bacteriophages

Figure 21-4

Courtesy of Maria Schnos and used with permission of Ross Inman, Department ofMolecular Virology, Bock Laboratories, University of Wisconsin-Madisonx

Photos courtesy of ICTVdB Picture Gallery. Used with permission from Hans-W. Ackermann, Department of Medical Biology, LavalUniversity.

Bacteriophage 3/K26Bacillus licheniformis virusEnterobactervirus P2


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Bacteriophage Structure and Genomes

3 families of bacteriophages are enveloped 96% of all bacteriophages contain dsDNA genomes The remaining 4% contain ssDNA, ssRNA or dsRNA

genomes

Genomes may code for as few as 3-5 genes to asmany as 100 The genomes of bacteriophages contain unusual or

modified bases that protect them from degradation byhost nucleases during phage infection

Most famous group of bacteriophages T-Type


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Only Two Forms of DNA Genomes in

Prokaryotes

Genome

Virion

1. Lineard.s.

25~120(x106

daltons)

Complexmorphology

2. Circulars.s. 1.7~2.7(x106

daltons)

Simpleicosahedral or

helical capsids


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21.4 Overview of Bacteriophage Infection

Bacteriophages Possess Alternative Lifestyles: Lytic

vs Temperate Phages

Bacteriophages adsorb to the surface receptormolecules of bacteria during the first step of infection

Receptors may be: Pili Proteins Oligosaccharides Lipopolysaccharides (LPS)


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Adsorption and Penetration

T4 bacteriophages anchors its tail fibers in the LPSlayer of its bacterial host

This adsorption step causes a conformational change,resulting in contraction of the tail sheath and

penetration of the cell membrane and the

bacteriophage

The bacteriophage DNA genome is subsequentlyinjected into the host cell through the tail tube


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Bacteriophage T4 Adsorption and Penetration

Figure 21.5 Bacteriophage T4 penetrating through the E. coliK-12

Omp C outer membrane protein receptor.


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Other Bacteriophages

May bind to other receptors Bacteriophages that do not have tails penetrate the

host by producing polysaccharide-degrading enzymes

that digest components of the bacterial envelope or

cell wall


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Resistance to Bacteriophage Infection

Bacteria become resistant to phage infection whentheir host cell receptors are altered by mutation

There is interest in engineering new receptor-recognition elements into the tail fibers of well-

characterized bacteriophages so they can infect

genetically distant hosts


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Genome Penetration

Not an injection process Bacteriophages may package lysozyme in the base of

its tail and uses the enzyme to degrade a portion of

the peptidoglycan of the bacterial cell wall

The DNA is drawn into the cell by a process that is notwell understood for most bacteriophages

After the DNA enters the cell, it circularizes rapidly bysticky ends or termini or the linear ends are modified

and protected from bacterial nucleases


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Transcription and Translation are Coupled

Bacterial host RNA polymerase recognizes theviral DNA promoters and begins transcriptionofearly genes

Translation of the early genes is coupled withtranscription

Late genes are transcribed and translated


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Temporal Gene Expression

Class I: 0-6 min, transcribed byE. coliRNA polymerase.

Class II: 6-12 min, involvingDNA synthesis and nuclease.

Class III: 8-20 min, for viralstructural proteins.

Transcribed by T7

RNA polymerase


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Assembly and Release Process

Lytic Infection

After all bacteriophage parts are produced, newbacteriophages are assembled

A copy of the genomic DNA is reeled into apreassembled icosahedral head

A few molecules of lysozyme are packaged into thetail plate

Phage lysin, endolysins, muramidases or virolysinshydrolyze bonds in the murein or peptidoglycan of the

cell wall, allowing the viruses to escape or be released

Holin is used to create pores in the inner membrane ofthe host, allowing the lysin or other enzymes to

facilitate bacteriophage release


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Lytic Infection

Figure 21.06: E. colibacteriophage T4 lytic infection cycle.


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T7 bacteriophageDNA


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Lysogenic (Temperate) Infections

These bacteriophages infect their hosts but do not killthem.

Instead their genome becomes integrated into aspecific region of the host chromosome

The integrated bacteriophage genome is called aprophage

The viral DNA replicates every time the cell copies itschromosomal DNA during cell division


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Lysogenic (Temperate) Infections, cont

Some temperate phages encode transposase whichallows the bacteriophage to insert randomly into the

chromosome

Other bacteriophages integrate into site specificlocations within the chromosome

All bacteriophage gene expression is repressed by arepressor protein


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Infection Cycle of a Temperate Bacteriophage

Figure 21.07: Infection cycle of a temperate bacteriophage.


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Derepression or Induction

If the repressorloses function (becomesinactivated), viral DNA is excised from thebacterial chromosome and the excised DNA

acts like a lytic virus

Spontaneous derepression orinductionhappens about 1 in every 10,000 cell divisions

Temperate bacteriophages can carry hostgenes from one bacterial cell to another in aprocess called transduction


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Phage

Icosahedral head (55 nm in diameter) Tail 15x135 nm long Linear double-stranded DNA About 50 kb with 12 unpaired bases at both

ends called cohesive ends.

Biological active form is circular rather thanlinear.

Lytic and lysogenic life cycle. Rolling circle replication mechanism.


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Lytic cycle and lysogenic cycle


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Regulatory elements that control

lysogenic and lytic development


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Repression of the lytic genes in a lysogenic cells

CI: OR1 > OR2 > OR3


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repressor

1. The CI gene of phage encodes a repressor.2. CI: 27 kD and form dimmer.3. Binds to OL (for transcription in a left direction)4. Binds to OR (for transcription in a right direction)


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Autogenous regulation of phage repressor synthesis

1. CIO

L1,O

R1

(1>2>3)

2. OR1PR, OL1PL, OR3PRM

3. PR:cro

4. PL :N5. CIbind

OL1,L2,R1, R2,

6.RNA polymerasebind PRPL(cro, N)


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Autogenous regulation of phage repressor synthesis

7. CI Bind OR2PRM,CI

8. PRM: Repressor Maintenance9. Repressor,bind OR1,R210.Repressor,bind

OR1,R2,R311. Repressor bind OR3 Block

PRM (self regulation)

12.OR2, CI repressor

13.U.V.activate proteaseCI repressor


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Lytic Cycle (45 mins)

1. Immediately early genes:Cro: control of repressor and other thingsN: antiterminator, prevent termination at TL1

and TR1.

2. Delayed-early genes:O and P proteins: for circular DNA replication.Q: 2nd antiterminator, prevent termination at TR4.

3. Late genes:Block early genes, rolling-circle replication.


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Genes and recognition sites involved in the

lytic regulatory cascade

1. Cro, N, Q: regulatory proteins required for lytic development.2. O

R

PR

, OL

PL

: leftward and rightward transcription.

3. OR1, OR2, OR3: three repressor binding sites.4. nutL, nutR: N protein binding site prevent transcription

termination at tL1 and tR1.

Express delayedearly genes

Express

late genes


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Functions ofcro at early and late stages of lytic cycle

Cro: OR3 > OR2 > OR1

CI: OR1 > OR2 > OR3


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N protein functions as an antiterminator at tR1 and tL1

N, RNA polymerasetR1

N: antiterminator

1. N, RNA polymerasereadthrough stop site (tR1)

2. nut: N utilization3. Nus: N utilization substance4. RNA polymeraseN,

NusB/S10, NusA,pass

tR1,


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lysogenic or lytic cycle?

1. [cro] [CI]OR, OL2. CIICIII3. PRE: promoter for repressor establishment,CI croantisense mRNA


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lysogenic or lytic cycle?

Poor growth condition CIIICII lysogenyGood growth condition CIII (less active)CII lytic

RNA PolCII

bind PRE,CII,CIII


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Regulatory elements that control lysogenic and lytic

development


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Phage

rolling circlereplication


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21.5 Bacteriophages Create

Pathogenic Bacteria in Nature

Lysogenic conversiona prophage may carrygenes that alter the phenotype of a lysogenicbacterium.

e.g. Corynebacterium diphtheriae ( )produces a toxin responsible for diphtheria only ifit carries the temperate bacteriophage calledphage.

The toxgene is located near one end of the phagegenome

1883 Klebsdiphtheriae 1884 Loeffer


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Other Examples of Lysogenic Conversion

IfStreptococcus pyogenes( contains atemperate bacteriophage T2,the bacterium changes to apyrogenic orerythrogenicexotoxinproducing bacterium

This exotoxin causes the rashofscarlet fever ( )

Figure 21.08: This child has the Scarlet Fever rash caused

by a lysogenic Streptococcus pyogenes bacterium.


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( )

( )

( )( )

( )


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21.6 Control of Bacteriophages in

Industrial Fermentations

Bacteria are used in a variety of foodfermentation processes:

Yogurt Cheese Sauerkraut Soy sauce


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Pharmaceutical and biotechnology industriesuse bacteria on a large scale to produce

products such as: Alcohols Vitamins Amino acids Enzymes Hormones Biopolymers Antibiotics Other therapeutics


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Bacteriophages are a Threat to Fermentation

and Pharmaceutical Industries

Attacks by bacteriophages can results in considerableeconomic losses

About 1-10% of dairy product fermentation batches arelost to bacteriophage infection

Often happens because the raw starting materials arecontaminated with undetectable numbers of bacteriophages

Laboratory staff are trained to expect and respond tobacteriophage outbreaks

Bacteriophages are ubiquitous Bacteriophage seasons are January to March and October to

November


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If a bacteriophage were the size of a

cockroach- these cockroaches would

cover the surface of the earth in a layerthat is 31,071 miles or 50,000 km deep!

Bacteriophages are ubiquitous

- Bacteriophage researcher Roger Hendrix


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Microbe Fermentation Vessel Used to Make

Enzymes for Medical and Industrial Use

Figure 21-9Courtesy of Shawn Walshaw-Wertz, Cherokee Pharmaceuticals LLC


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21.7 Biofilms and Bacteriophages

Bacteria form complex communities called biofilms onsolid wet surfaces in natures such as

River rocks Walls of limestone caves Pipes Industrial equipment Hulls of ships Teeth Lining of the colon Sutures Lungs of Cystic Fibrosis Patients Medical devices such as catheters ( ), heart valves Surrounding tissues of the heart Orthopedic devices Facial implants


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Biofilms, cont.

Biofilms may consist of a single bacterial species tohundreds or thousands of bacterial species

The bacteria produce extracellular polysaccharidepolymers that surround and encase the microbes,

facilitating their adhesion to surfaces

Biofilms can cause environmental problems


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Biofilms Can Cause Chronic Bacterial

Infections in Humans

Biofilms are a challenge to medical care Bacteria commonly isolated from medical devices:

Enterococcus faecalis

S. aureus

S. epidermidis Streptococcus viridans E. coli Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa


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Biofilms Can Cause Chronic Bacterial

Infections in Humans, cont.

These bacteria originate from the skin of the patient orthe healthcare workers, entry ports of catheters etc.

Preventative strategies to treat biofilms on medicaldevices:

Application of silver-impregnated catheters Coating devices with antibiotics Disinfection at the surface Adding inline filters into the ports of catheters

Bacteriophages studies to pretreat catheter surfacesto control biofilms are promising


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EM ofStaphylococcus aureus Bacteria Found

on a Patients Catheter

Figure 21.10: EM ofStaphylococcus aureus bacteria found on the surface

of a patient's catheter. Magnified 2363X.


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21.8 FDA-Approved Listeria-Specific

Bacteriophage Preparation on Ready-to-Eat Meats

Listeriosis ( ) is a foodborne illnesscaused by the bacterium Listeriamonocytogenes

Listeriosis occurs in pregnant women (cancause a miscarriage, stillbirth or prematuredelivery), newborns and people with weakened

immune systems

2006FDA approved LISTEX P100which is acocktail of 6 bacteriophages in the form of an

application spray


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LISTEX P100

LISTEX P100 will be used in clean rooms ofcheese, meat, and poultry processing plantsto inhibit the growth of Listeria on products

such as lunch meat and hot dogs

LISTEX P100 will not be declared as aningredient on the label of a treated product

p 592Vi Fil 21 1


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Bacteriophage Therapy

Makes a Comeback

3 Georgia woodsman suffered from burnscaused by Soviet-era makeshift heaters

packed with radiation (Strontium 90 )

Their burns became infected withStaphylococcus aureus

Antibiotics failed to prevent the bacteria frominvading deeply into the wounds

p.592Virus File 21-1


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Bacteriophage Therapy

Makes a Comeback, cont.

The men were rushed to a hospital in Tbilisiwhere doctors placed bacteriophage patches

on the wounds

The infections cleared in a few weeks The men were stable enough to undergo skin

grafting abroad


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Figure 21.VF01: These wounds were treated

with the bacteriophage powder PhagoBioDerm.


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Bacteriophage Therapy Hurdles Being Addressed

Contamination of bacteriophage stocks withbacterial toxins and debris that causes sideeffects in patients

Narrow host range of bacteriophages The elimination of bacteriophages by the

hosts immune system (antibodies)

Bacterial host resistance to phage infection Lytic bacteriophages are ideal for therapy

50% of all bacteriophages are temperate

chap 21 bacteriophages

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