protects

uncapped

budding

yeast

telomeres

from

exonuclease

-LRB-

Mediator

of

Replication

Checkpoint

-RRB-

is

a

component

of

the

DNA

replication

fork

machinery

and

is

necessary

for

checkpoint

activation

after

replication

stress

In

this

study

we

addressed

the

role

of

at

uncapped

telomeres

Our

experiments

show

that

contributes

to

the

vitality

of

both

and

telomere

capping

mutants

Cells

with

telomere

capping

defects

containing

or

a

checkpoint

defective

allele

exhibit

similar

growth

suggesting

growth

defects

of

are

not

due

to

checkpoint

defects

This

is

in

accordance

with

activation

after

telomere

uncapping

Poor

growth

of

mutants

in

the

absence

of

is

a

result

of

enhanced

single

stranded

DNA

accumulation

at

uncapped

telomeres

Consistent

with

this

deletion

of

encoding

a

nuclease

that

contributes

to

single

stranded

DNA

accumulation

after

telomere

uncapping

improves

growth

of

strains

and

decreases

ssDNA

production

Our

observations

show

that

a

core

component

of

the

replication

fork

plays

an

important

role

in

telomere

capping

protecting

from

nucleases

and

checkpoint

pathways

Introduction

Telomeres

are

specialized

DNA

protein

complexes

at

the

end

of

eukaryotic

chromosomes

Proper

telomere

structure

is

essential

for

chromosome

integrity

and

genome

stability

because

telomeres

protect

natural

chromosome

ends

from

degradation

and

end-to-end

fusion

and

because

they

ensure

complete

genome

replication

Telomeres

differ

from

Double

Strand

Breaks

-LRB-

DSBs

-RRB-

in

that

they

normally

fail

to

activate

DNA

repair

or

DNA

damage

checkpoint

pathways

If

that

was

the

case

then

they

would

undergo

recombination

and

chromosomal

fusions

and

dicentric

chromosomes

would

be

generated

-LSB-

-RSB-

Many

proteins

associate

with

telomeric

DNA

These

proteins

ensure

that

telomeres

behave

differently

from

DSB

ends

and

help

maintain

chromosomal

stability

Some

telomeric

proteins

bind

specifically

to

dsDNA

and

others

show

higher

affinity

to

ssDNA

In

budding

yeast

there

are

numerous

proteins

with

affinity

for

telomeric

dsDNA

such

as

and

-LSB-

-RSB-

The

budding

yeast

telomeric

ssDNA

ends

are

thought

to

be

protected

by

three

essential

proteins

and

-LSB-

-RSB-

If

telomeres

become

uncapped

they

activate

a

DNA

damage

response

pathway

leading

to

cell

cycle

arrest

-LSB-

-RSB-

Moreover

recently

it

has

been

suggested

that

telomeres

trigger

a

transient

DNA

damage

response

in

each

S

phase

in

order

to

complete

DNA

replication

and

cap

chromosome

ends

-LSB-

-RSB-

DNA

damage

response

pathways

are

complex

networks

which

include

among

other

components

mediators

Mediators

facilitate

the

transmission

of

the

DNA

damage

signal

from

sensors

to

downstream

effectors

activation

of

the

latter

affecting

cell

cycle

progression

-LSB-

-RSB-

-LRB-

Mediator

of

Replication

Checkpoint

-RRB-

appears

to

take

the

mediator

role

in

activation

under

replication

stress

-LSB-

-RSB-

However

parallel

pathways

exist

because

in

mutants

activation

occurs

through

-LRB-

another

mediator

protein

-RRB-

presumably

because

the

accumulation

of

DNA

damage

rather

than

replication

defects

in

mutants

leads

to

activation

of

and

thereby

activation

of

-LSB-

-RSB-

also

appears

to

be

directly

involved

in

DNA

replication

and

because

of

this

cells

display

a

slow

S

phase

-LSB-

-RSB-

is

an

active

component

of

the

replication

machinery

loaded

onto

DNA

shortly

after

replication

initiates

and

moving

with

other

components

of

replication

forks

-LSB-

-RSB-

In

the

presence

of

hydroxyurea

a

type

of

replication

stress

appears

to

form

a

stable

replication-pausing

complex

preventing

the

uncoupling

of

the

replication

machinery

from

DNA

synthesis

-LSB-

-RSB-

According

to

this

model

mediates

activation

of

under

conditions

of

replication

stress

so

that

subsequent

DNA

repair

events

occur

and

cell

replication

resumes

normal

function

-LSB-

-RSB-

However

recent

experiments

suggest

that

the

role

of

at

stalled

replication

forks

is

more

than

activating

since

cells

defective

in

activation

are

not

defective

in

replication

fork

initiation

or

progression

-LSB-

-RSB-

is

a

mutant

allele

in

which

SQ/TQ

residues

have

been

substituted

with

AQ

resulting

in

its

inability

to

mediate

phosphorylation

and

activation

of

-LSB-

-RSB-

Although

is

involved

in

the

DNA

replication

checkpoint

it

has

been

shown

that

it

is

not

required

for

the

DNA

damage

checkpoint

since

double

mutants

arrest

in

at

non-permissive

temperatures

-LSB-

-RSB-

It

has

been

reported

that

activation

of

in

response

to

telomere

shortening

still

occurs

in

the

absence

of

and

that

is

responsible

for

this

activation

in

telomerase-deficient

cells

in

which

telomeres

continually

shorten

until

they

activate

a

checkpoint

-LSB-

-RSB-

Surprisingly

though

double

mutants

arrest

cell

division

suggesting

that

is

not

required

for

cell

cycle

arrest

in

telomerase

negative

cells

In

contrast

after

induced

telomere

uncapping

activation

is

entirely

and

-LSB-

-RSB-

Here

we

investigated

the

role

of

at

uncapped

telomeres

using

the

temperature

sensitive

and

mutations

to

uncap

telomeres

Our

experiments

indicate

that

protects

telomeres

from

the

DNA

damage

response

and

that

the

role

of

in

DNA

replication

forks

rather

than

in

checkpoint

activation

is

important

for

protection

of

telomeres

Materials

and

methods

Yeast

strains

and

plasmids

All

strains

in

the

background

are

and

they

contain

an

mutation

-LRB-

Supplemental

Table

-RRB-

therefore

yeast

extract/peptone/dextrose

-LRB-

YEPD

-RRB-

was

supplemented

with

adenine

at

mg/l

Strains

are

in

the

background

-LRB-

Supplemental

Table

-RRB-

and

they

were

generated

by

mating

a

single

gene

deletion

mutant

array

-LSB-

-RSB-

with

a

query

strain

-LSB-

-RSB-

To

construct

strains

standard

genetic

procedures

of

transformation

and

tetrad

analysis

were

used

-LSB-

-RSB-

and

also

carrying

the

gene

were

a

gift

from

Steven

Elledge

-LSB-

-RSB-

was

disrupted

in

two

different

ways

Firstly

the

ORF

was

substituted

with

with

a

PCR

based

method

-LSB-

-RSB-

Primers

tcgttattcgcttttgaacttatcaccaaatattttagtgCGGATCCCCGGGTTAATTAA-3

-LRB-

-RRB-

and

ctggagttcaatcaacttcttcggaaaagataaaaaaccaGAATTCGAGCTCGTTTAAAC-3

-LRB-

-RRB-

which

contain

homology

to

upstream

and

downstream

sequences

of

-LRB-

bases

in

lowercase

-RRB-

were

used

to

amplify

a

bp

sequence

-LRB-

-LSB-

-RSB-

-RRB-

The

PCR

product

was

transformed

into

yeast

and

candidate

colonies

were

selected

for

resistance

Integration

of

the

marker

into

the

locus

was

confirmed

by

PCR

using

two

sets

of

primers

-LRB-

i

-RRB-

forward

-LRB-

-RRB-

with

reverse

primer

-LRB-

-RRB-

and

-LRB-

ii

-RRB-

forward

-LRB-

-RRB-

with

reverse

-LRB-

-RRB-

Disruption

of

was

also

confirmed

by

Southern

blot

-LRB-

data

not

shown

-RRB-

Alternatively

was

substituted

with

using

a

restriction

enzyme

digest

approach

First

was

digested

with

XhoI

and

a

bp

fragment

containing

the

full

gene

was

cloned

into

XhoI

digested

vector

-LRB-

kb

-RRB-

to

create

A

correct

clone

was

identified

by

restriction

digests

was

digested

with

SpeI

to

remove

a

kb

DNA

fragment

containing

the

bulk

of

which

was

replaced

with

a

kb

BamHI

gene

fragment

from

-LRB-

pBSB

KS

containing

a

BamHI

fragment

containing

the

gene

-RRB-

by

blunt

cloning

following

treatment

with

DNA

polymerase

I

Large

-LRB-

Klenow

-RRB-

fragment

-LRB-

New

England

Biolabs

-RRB-

Positive

clones

were

selected

by

restriction

enzyme

digests

to

identify

the

disruption

of

the

bulk

of

with

-LRB-

-RRB-

Disruption

of

was

also

confirmed

by

Southern

blot

-LRB-

data

not

shown

-RRB-

To

disrupt

was

digested

with

XhoI

prior

to

transformation

of

yeast

Spot

tests

Single

colonies

were

inoculated

into

ml

YEPDextrose

-LRB-

YEPD

-RRB-

and

incubated

overnight

with

aeration

at

°C

The

following

day

μl

of

each

culture

was

inoculated

into

ml

of

fresh

YEPD

and

returned

to

°C

Cells

were

grown

for

three

more

hours

and

cell

numbers

were

determined

in

a

haemocytometer

The

cells

were

then

centrifuged

-LRB-

rpm

for

s

in

a

microcentrifuge

-RRB-

washed

twice

with

sterile

water

and

resuspended

in

water

to

a

final

concentration

of

cells/ml

A

five-fold

dilution

series

of

each

of

the

cultures

was

prepared

using

sterile

water

in

a

well

plate

and

μl

spotted

onto

plates

using

a

replica

plating

device

Plates

were

incubated

at

various

temperatures

for

days

before

being

photographed

For

spot

tests

with

strains

containing

or

the

steps

were

as

described

above

but

strains

were

grown

on

selective

medium

-LRB-

URA

-RRB-

All

strains

shown

as

if

on

a

single

agar

plate

were

grown

on

the

same

plate

although

in

some

cases

their

positions

were

moved

using

Adobe

Photoshop

and

Adobe

Illustrator

CS

Unless

otherwise

stated

at

least

two

different

strains

of

the

same

genotype

were

spot

tested

and

representative

examples

are

shown

Yeast

transformation

High

efficiency

transformations

needed

for

gene

disruptions

were

performed

according

to

Gietz

et

al.

-LSB-

-RSB-

For

plasmid

transformations

a

more

rapid

method

was

used

-LSB-

-RSB-

Western

blots

Protein

extracts

were

prepared

by

glass

bead

breakage

in

TCA

essentially

as

previously

described

-LSB-

-RSB-

Bio-Rad

gels

-LRB-

Tris

HCl

-RRB-

Schleicher

and

Schuell

Protan

Nitrocellulose

membranes

and

the

Pierce

Supersignal

West

Pico

Chemiluminescent

Substrate

detection

kit

were

used

in

a

standard

Western

blot

procedure

Rabbit

polyclonal

antibody

-LRB-

dilution

a

gift

from

Dan

Durocher

-LSB-

-RSB-

-RRB-

was

a

primary

antibody

used

with

an

anti-rabbit-HRP

-LRB-

dilution

Dako

-RRB-

as

a

secondary

antibody

used

Mouse

anti-tubulin

-LRB-

dilution

a

gift

from

Keith

Gull

-LSB-

-RSB-

and

anti-mouse-HRP

-LRB-

dilution

Dako

-RRB-

were

used

for

tubulin

loading

controls

Telomere

length

measurement

by

Southern

blot

Strains

were

grown

to

saturation

in

liquid

YEPD

at

°C

DNA

from

each

strain

was

subjected

to

XhoI

cut

The

digested

DNA

was

loaded

on

a

agarose

gel

run

at

low

voltage

overnight

transferred

to

a

Magna

nylon

membrane

and

UV

cross-linked

The

membrane

was

then

hybridised

with

a

Y

TG

probe

-LSB-

-RSB-

A

non-radioactive

detection

kit

was

used

for

the

detection

of

the

hybridisation

-LRB-

Amersham

Arlington

Heights

IL

-RRB-

Synchronous

cultures

strains

with

additional

mutations

-LRB-

see

Supplemental

Table

-RRB-

were

grown

in

YEPD

at

°C

overnight

In

the

morning

cells

were

adjusted

to

a

concentration

of

buds/ml

in

ml

Cultures

were

grown

for

three

more

hours

then

arrested

with

nM

α-factor

for

a

further

h

The

cultures

were

then

released

from

arrest

by

centrifugation

and

washed

twice

in

YEPD

and

placed

at

°C

min

after

the

culture

was

first

centrifuged

Cell

cycle

position

was

monitored

as

previously

described

-LSB-

-RSB-

DNA

was

prepared

and

the

fraction

of

single

stranded

DNA

-LRB-

ssDNA

-RRB-

was

measured

as

previously

described

-LSB-

-RSB-

Asynchronous

cultures

strains

with

additional

mutations

indicated

were

grown

in

YEPD

at

°C

overnight

In

the

morning

cells

were

adjusted

to

a

concentration

of

cells/ml

and

temperature

was

raised

to

°C

Every

min

samples

were

taken

for

cell

cycle

position

and

cell

density

was

re-adjusted

to

cells/ml

Cell

numbers

were

determined

with

a

haemocytometer

Samples

for

Western

blots

were

collected

from

exponentially

growing

cultures

h

after

the

temperature

was

raised

from

°C

to

°C

Cell

cycle

position

determination

A

ml

sample

of

culture

was

centrifuged

for

s

at

high

speed

the

supernatant

was

aspirated

and

cells

were

fixed

at

ethanol

overnight

The

fixed

cells

were

washed

twice

in

water

before

being

resuspended

in

-LRB-

DAPI

μg/ml

-RRB-

Cell

cycle

distribution

was

monitored

by

DAPI

staining

of

nuclei

and

fluorescence

microscopy

For

DAPI

staining

cells

for

each

sample

were

counted

and

classified

as

-LRB-

-RRB-

unbudded

single

DAPI-stained

body

-LRB-

-RRB-

small

budded

single

DAPI-stained

body

with

the

bud

of

the

diameter

of

the

mother

cell

-LRB-

-RRB-

medial

nuclear

division

single

DAPI-stained

body

with

bud

diameter

of

mother

cell

the

arrest

point

-LRB-

-RRB-

late

nuclear

division

two

buds

and

two

DAPI-stained

bodies

the

arrest

point

-LSB-

-RSB-

Results

contributes

to

the

vitality

of

and

mutants

Since

plays

a

role

in

the

checkpoint

response

to

stalled

replication

we

wondered

if

it

also

plays

a

role

at

uncapped

telomeres

The

temperature

sensitive

mutation

causes

a

defect

in

a

telomere

binding

protein

and

cells

containing

this

mutation

accumulate

large

amounts

of

ssDNA

at

telomeres

at

non-permissive

temperatures

-LSB-

-RSB-

Interestingly

deletion

of

checkpoint

proteins

like

and

improves

growth

of

strains

at

semi-permissive

temperatures

-LSB-

-RSB-

This

is

presumably

because

checkpoint

pathways

inhibit

cell

division

by

responding

to

low

levels

of

ssDNA

that

accumulates

at

telomeres

at

semi-permissive

temperatures

Deletion

of

other

checkpoint

proteins

like

the

MRX

complex

which

appears

to

play

a

role

in

telomere

capping

worsens

the

growth

of

strains

-LSB-

-RSB-

Therefore

we

wanted

to

investigate

whether

plays

a

role

at

uncapped

telomeres

and

if

so

whether

it

behaved

like

or

MRX

Fig.

shows

that

deletion

of

dramatically

reduces

the

growth

of

mutant

strains

at

°C

The

effect

of

is

not

as

profound

as

that

of

the

MRX

complex

as

and

display

more

severe

growth

defects

than

even

at

°C

-LRB-

Supplementary

Fig.

-RRB-

Thus

like

MRX

but

unlike

the

majority

of

checkpoint

proteins

contributes

to

the

vitality

of

strains

We

next

wanted

to

investigate

whether

analogous

growth

defects

of

strains

occur

in

strains

is

a

telomere

capping

protein

which

is

also

involved

in

dsDNA

damage

repair

and

in

Non-Homologous

End

Joining

-LRB-

reviewed

in

Ref

-LSB-

-RSB-

-RRB-

Deletion

of

results

in

a

temperature

sensitive

phenotype

at

°C

due

to

telomere

uncapping

which

activates

a

cell

cycle

arrest

-LSB-

-RSB-

Fig.

demonstrates

that

deletion

of

results

in

a

severe

growth

defect

of

mutant

strains

at

°C

and

°C

Thus

contributes

to

the

vitality

of

strains

We

conclude

that

contributes

to

the

vitality

of

two

cell

types

defective

in

telomere

capping

and

are

three

proteins

that

play

similar

although

distinct

roles

in

DNA

replication

-LSB-

-RSB-

Therefore

we

wished

to

address

whether

and

like

contributed

to

the

vitality

of

mutants

Spot

test

analysis

showed

that

although

deletion

of

or

also

confers

some

growth

defects

on

mutants

deletion

of

has

a

stronger

phenotype

-LRB-

Fig.

-RRB-

Therefore

we

decided

to

focus

on

understanding

the

role

of

at

uncapped

telomeres

Growth

defects

of

cells

are

not

due

to

checkpoint

defects

In

budding

yeast

two

independent

roles

have

been

previously

reported

for

One

role

implicates

as

a

mediator

of

checkpoint

activation

under

replication

stress

and

the

other

role

is

as

part

of

the

replication

machinery

-LSB-

-RSB-

Therefore

we

investigated

whether

the

heightened

temperature

sensitivity

phenotype

of

mutant

strains

is

a

result

of

a

replication

defect

a

checkpoint

defect

or

both

mutants

were

complemented

with

either

wild

type

or

an

empty

vector

-LRB-

-RRB-

and

strains

were

grown

at

various

temperatures

Fig.

shows

that

at

°C

complementation

of

mutant

strains

with

either

or

allele

improves

growth

compared

to

the

empty

vector

control

-LRB-

compare

rows

-RRB-

Thus

we

conclude

that

the

checkpoint

role

of

is

not

important

for

the

vitality

of

strains

At

higher

temperature

we

noticed

an

increased

growth

of

cells

carrying

the

allele

compared

to

However

this

phenotype

was

observed

even

in

the

presence

of

the

wild

type

allele

suggesting

that

this

effect

is

due

to

some

type

of

dominant

effect

of

-LRB-

Fig.

compare

rows

and

-RRB-

inhibits

growth

of

and

mutants

encodes

a

nuclease

known

to

contribute

to

ssDNA

production

at

uncapped

telomeres

of

and

strains

-LSB-

-RSB-

If

ssDNA

production

at

uncapped

telomeres

is

responsible

for

the

poor

growth

of

and

mutants

then

removing

should

suppress

their

poor

growth

Fig.

demonstrates

that

at

°C

triple

mutants

exhibit

better

growth

than

strains

showing

that

contributes

to

the

growth

defects

observed

in

strains

Importantly

deleting

also

reverses

the

growth

defect

of

mutants

-LRB-

Fig.

-RRB-

These

data

suggest

that

protects

uncapped

telomeres

from

Effects

of

checkpoint

mutations

on

and

growth

To

understand

if

checkpoint

pathways

are

activated

in

strains

after

telomere

uncapping

we

wanted

to

combine

and

strains

with

checkpoint

mutations

A

genetic

screen

has

revealed

that

is

synthetically

lethal

with

or

checkpoint

mutations

-LSB-

-RSB-

Consistent

with

these

results

we

were

unable

to

recover

viable

offspring

carrying

in

combinations

of

any

of

these

checkpoint

genes

-LRB-

data

not

shown

-RRB-

However

we

were

able

to

combine

with

and

encoding

two

downstream

checkpoint

kinases

-LRB-

analogues

of

human

and

respectively

-RRB-

When

mutants

grow

at

non-permissive

temperatures

and

dependent

parallel

pathways

are

activated

-LSB-

-RSB-

We

deleted

or

and

examined

the

effects

in

mutants

Deletion

of

requires

simultaneous

deletion

of

to

obtain

viable

spores

is

a

small

protein

that

inhibits

the

activity

of

ribonucleotide

reductase

-LRB-

RNR

-RRB-

which

catalyzes

the

rate-limiting

step

of

de

novo

dNTP

synthesis

-LSB-

-RSB-

Normally

is

degraded

in

a

manner

during

S

phase

-LSB-

-RSB-

We

found

that

removal

of

and

improved

the

growth

of

strains

-LRB-

Fig.

-RRB-

However

deletion

of

alone

-LRB-

-RRB-

also

rescued

the

growth

defects

associated

with

deletion

-LRB-

Fig.

-RRB-

Thus

we

were

unable

to

observe

any

strong

role

for

in

maintaining

vitality

of

mutants

other

than

in

degrading

We

suggest

that

the

reason

that

deleting

improves

the

growth

of

strains

is

that

increased

ribonucleotide

reductase

activity

may

stabilise

the

replication

forks

Removal

of

like

removal

of

does

not

rescue

growth

of

strains

indicating

that

does

not

inhibit

growth

of

these

mutants

-LRB-

Fig.

-RRB-

Therefore

we

find

no

evidence

that

inactivating

DNA

damage

checkpoint

pathways

improves

growth

of

mutants

A

deletion

strongly

rescues

growth

of

mutants

at

restrictive

temperatures

-LRB-

Fig.

-RRB-

similarly

to

its

effect

in

mutants

-LSB-

-RSB-

Thus

uncapped

telomeres

qualitatively

behave

like

uncapped

mutants

The

effects

of

in

and

strains

are

consistent

with

earlier

findings

showing

that

the

pathway

is

more

important

in

mutants

-LSB-

-RSB-

is

not

required

for

the

cell

cycle

arrest

after

uncapping

To

directly

test

whether

plays

a

checkpoint

role

in

strains

strains

with

additional

mutations

were

first

grown

at

the

semi-permissive

temperature

°C

Strains

defective

in

telomere

capping

-LRB-

-RRB-

arrest

at

medial

nuclear

division

before

entry

to

anaphase

after

h

at

°C

-LRB-

Fig.

-RRB-

As

expected

when

the

checkpoint

is

compromised

in

or

cells

no

accumulation

at

medial

nuclear

division

is

observed

over

h

-LRB-

Fig.

-RRB-

In

contrast

and

strains

rapidly

accumulate

at

medial

nuclear

division

and

within

h

more

than

of

cells

are

arrested

at

this

point

Consistent

with

these

conclusions

cell

numbers

stopped

increasing

in

and

strains

but

continued

to

increase

in

the

other

strains

-LRB-

Fig.

-RRB-

We

conclude

that

is

not

required

for

the

checkpoint

response

to

dependent

telomere

uncapping

and

are

components

of

parallel

checkpoint

pathways

that

respond

to

induced

telomere

uncapping

-LSB-

-RSB-

It

appears

that

the

pathway

is

more

important

for

arrest

of

mutants

because

of

cells

have

arrested

at

medial

nuclear

division

by

h

whereas

there

is

no

arrest

of

cells

To

test

the

role

of

in

checkpoint

control

in

a

single

cell

cycle

we

combined

with

and

mutations

Over

many

years

we

and

others

have

used

these

mutations

to

determine

the

effects

of

checkpoint

proteins

in

responding

to

telomere

uncapping

-LSB-

-RSB-

encodes

a

protease

that

degrades

the

mating

pheromone

α-factor

Cells

bearing

the

mutation

can

efficiently

arrest

in

phase

of

the

cell

cycle

with

low

levels

of

α-factor

is

necessary

for

mitotic

exit

At

°C

control

strains

released

from

alpha

factor

arrest

accumulate

at

medial

nuclear

division

due

to

telomere

uncapping

However

if

cells

have

escaped

the

checkpoint

like

strains

they

arrest

at

late

nuclear

division

due

to

and

they

are

unable

to

proceed

to

the

next

cycle

The

dependent

cell

cycle

arrest

helps

in

two

ways

it

ensures

that

DNA

damage

checkpoint

defects

are

easily

quantified

because

cells

with

checkpoint

defects

accumulate

at

a

later

stage

of

the

cell

cycle

and

that

DNA

damage

caused

by

is

not

amplified

during

new

rounds

of

DNA

replication

strains

with

additional

mutations

were

arrested

with

α-factor

then

released

from

and

transferred

to

a

non-permissive

temperature

to

induce

telomere

uncapping

and

the

cell

cycle

position

was

monitored

Fig.

shows

that

in

contrast

to

strains

and

strains

arrest

at

medial

nuclear

division

with

similar

kinetics

at

°C

supporting

the

idea

that

does

not

play

a

role

in

checkpoint

activation

after

telomere

uncapping

Interestingly

remain

arrested

at

medial

nuclear

division

-LRB-

Fig.

and

D

-RRB-

which

contrasts

to

the

behaviour

of

that

begin

to

escape

arrest

during

the

period

in

analogous

experiments

-LSB-

-RSB-

This

difference

most

likely

reflects

the

fact

that

mutants

have

more

severe

telomere

capping

defect

than

mutants

and

therefore

activate

a

stronger

checkpoint

signal

In

response

to

both

replication

stress

and

DNA

damage

activation

of

the

checkpoint

machinery

induces

phosphorylation

and

activation

of

kinase

Therefore

we

addressed

the

role

of

in

phosphorylation

after

telomere

uncapping

strains

were

exposed

to

°C

for

h

and

phosphorylation

was

measured

by

Western

blot

phosphorylation

is

observed

in

strains

but

not

in

mutants

-LRB-

Fig.

-RRB-

confirming

a

previous

study

-LSB-

-RSB-

We

conclude

that

activation

of

after

dependent

uncapping

at

non-permissive

temperatures

is

but

contributes

to

telomere

length

regulation

If

plays

a

protective

role

at

telomeres

this

predicts

that

strains

lacking

may

have

short

telomeres

Fig.

shows

that

absence

of

results

in

shorter

telomeres

compared

to

the

wild

type

However

the

telomere

length

defects

of

mutants

are

not

as

severe

as

in

or

mutants

and

this

may

help

explain

why

cells

grow

better

than

cells

-LRB-

Supplementary

Fig.

-RRB-

Our

experiment

is

consistent

with

replication

proteins

having

an

important

role

in

telomere

length

regulation

-LSB-

-RSB-

protects

telomeres

from

extended

ssDNA

accumulation

All

our

data

suggest

an

important

role

of

in

telomere

capping

but

no

role

in

cell

cycle

arrest

To

directly

test

the

role

of

in

telomere

capping

we

measured

ssDNA

accumulation

on

the

TG

strand

at

a

single

copy

locus

approximately

kb

away

from

the

right

end

of

chromosome

V

in

strains

-LRB-

Fig.

-RRB-

strains

were

synchronised

as

in

Fig.

and

D

in

order

to

follow

the

effects

of

on

ssDNA

accumulation

at

non-permissive

temperatures

within

a

single

cell

cycle

We

find

that

mutants

like

strains

accumulate

more

TG

ssDNA

at

kb

from

the

uncapped

telomere

compared

to

strains

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

This

shows

that

like

protects

mutants

from

ssDNA

production

Consistent

with

our

conclusion

increased

ssDNA

accumulation

closer

to

the

telomere

in

telomere

repeats

was

recently

reported

in

and

mutants

using

both

in

gel

and

dot

blot

analyses

-LSB-

-RSB-

Importantly

ssDNA

production

is

reduced

in

strains

in

comparison

to

strains

showing

that

protects

uncapped

telomeres

from

nuclease

action

This

ssDNA

data

is

consistent

with

our

finding

that

contributes

to

the

poor

growth

of

and

mutants

-LRB-

Fig.

-RRB-

Taken

together

we

conclude

that

inhibits

accumulation

of

ssDNA

accumulation

after

telomere

uncapping

and

by

this

criterion

contributes

to

telomere

capping

Discussion

Our

experiments

demonstrate

that

contributes

to

the

vitality

of

budding

yeast

cells

with

uncapped

telomeres

Therefore

behaves

differently

from

many

other

known

checkpoint

proteins

such

as

or

deletion

of

which

improves

the

growth

of

mutants

at

semi-permissive

temperatures

-LSB-

-RSB-

The

effect

of

is

more

similar

to

that

of

the

MRX

complex

another

checkpoint

complex

with

roles

in

telomere

capping

-LSB-

-RSB-

It

seems

that

it

is

the

role

of

at

the

replication

fork

that

contributes

to

the

vitality

of

telomere

capping

mutants

rather

than

its

role

in

checkpoint

activation

Our

findings

are

in

accordance

with

recent

work

that

demonstrated

a

protective

role

of

in

cells

with

or

uncapped

telomeres

or

in

telomerase

deficient

cells

-LSB-

-RSB-

Additionally

our

work

demonstrates

that

the

growth

defects

of

and

mutants

and

enhanced

ssDNA

levels

of

strains

are

suppressed

when

the

nuclease

encoded

by

is

deleted

Therefore

protects

uncapped

telomeres

from

is

recruited

to

the

replication

machinery

as

DNA

replication

initiates

and

is

required

for

normal

rates

of

replication

fork

progression

-LSB-

-RSB-

The

biochemical

role

of

in

replication

fork

progression

is

unclear

which

makes

it

difficult

to

know

its

precise

role

in

telomere

capping

is

also

part

of

a

replication-pausing

complex

formed

when

replication

is

arrested

by

the

S

phase

poison

hydroxyurea

-LRB-

HU

-RRB-

and

required

for

replication

fork

restart

after

HU

However

this

restart

role

for

is

not

universal

since

plays

no

role

in

replication

restart

after

cells

are

treated

with

the

alkylating

agent

MMS

-LSB-

-RSB-

It

is

interesting

that

there

is

evidence

from

budding

yeast

fission

yeast

and

human

cells

that

telomeric

sequences

contain

DNA

regions

that

slow

or

stall

replication

forks

-LSB-

-RSB-

From

this

it

seems

plausible

that

telomeric

DNA

may

be

more

dependent

on

proteins

like

which

contribute

to

fork

stability

and

restart

than

other

chromosomal

regions

That

is

because

the

replication

fork

struggles

to

reach

the

end

of

the

chromosome

in

mutants

where

a

telomere

capping

defect

is

observed

Numerous

studies

on

budding

yeast

mutants

with

DNA

replication

defects

have

demonstrated

interactions

between

DNA

replication

and

telomere

structure

For

example

both

and

-LRB-

large

subunit

of

replication

factor

C

-RRB-

mutants

affect

telomere

length

-LSB-

-RSB-

Here

we

show

that

budding

yeast

mutants

have

short

telomeres

In

S.

cerevisiae

mutants

encoding

temperature

sensitive

DNA

polymerase

α

exhibit

very

long

telomeres

high

levels

of

telomeric

ssDNA

and

elevated

recombination

at

telomeres

-LSB-

-RSB-

Interestingly

the

B

subunit

of

DNA

polymerase

α

physically

interacts

with

which

in

turn

interacts

with

-LSB-

-RSB-

This

shows

there

is

a

very

direct

interaction

between

budding

yeast

telomere

capping

proteins

and

the

replication

fork

machinery

and

suggests

that

telomere

capping

is

intimately

linked

with

DNA

replication

In

this

regard

it

is

perhaps

relevant

that

the

exonuclease

is

involved

in

generating

single

stranded

DNA

at

uncapped

telomeres

-LSB-

-RSB-

and

in

processing

stalled

replication

forks

-LSB-

-RSB-

and

highlights

the

similarities

between

uncapped

telomeres

and

stalled

replication

forks

cells

maintain

a

functional

telomere

cap

-LRB-

low

levels

of

telomeric

ssDNA

-RRB-

when

released

from

arrest

into

the

S

phase

poison

hydroxyurea

HU

stalls

replication

forks

and

stops

late

origins

of

replication

from

firing

However

if

the

same

cells

are

permitted

to

complete

DNA

replication

by

removing

the

S

phase

poison

HU

telomere

uncapping

occurs

and

high

levels

of

ssDNA

are

observed

-LSB-

-RSB-

Therefore

telomere

capping

may

depend

on

a

coordinated

interaction

between

the

chromosome

end

and

the

DNA

replication

fork

Further

studies

examining

the

interactions

between

telomeric

DNA

the

telomere

cap

and

the

replication

fork

will

be

necessary

to

better

understand

these

interactions

B O O O O O O O B B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O B O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O 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Central sensitization : a biopsychosocial explanation for chronic widespread pain in patients with fibromyalgia and chronic fatigue syndrome In addition to the debilitating fatigue , the majority of patients with chronic fatigue syndrome -LRB- CFS -RRB- experience chronic widespread pain . These pain complaints show the greatest overlap between CFS and fibromyalgia -LRB- FM -RRB- . Although the literature provides evidence for central sensitization as cause for the musculoskeletal pain in FM , in CFS this evidence is currently lacking , despite the observed similarities in both diseases . The knowledge concerning the physiological mechanism of central sensitization , the pathophysiology and the pain processing in FM , and the knowledge on the pathophysiology of CFS lead to the hypothesis that central sensitization is also responsible for the sustaining pain complaints in CFS . This hypothesis is based on the hyperalgesia and allodynia reported in CFS , on the elevated concentrations of nitric oxide presented in the blood of CFS patients , on the typical personality styles seen in CFS and on the brain abnormalities shown on brain images . To examine the present hypothesis more research is required . Further investigations could use similar protocols to those already used in studies on pain in FM like , for example , studies on temporal summation , spatial summation , the role of psychosocial aspects in chronic pain , etc. . Introduction Chronic fatigue syndrome -LRB- CFS -RRB- , as defined by the Centers for Disease Control and Prevention -LRB- CDCP -RRB- , is a complex illness characterized by prolonged debilitating fatigue and multiple non-specific symptoms including headaches , recurrent sore throats , fever , muscle and joint pain , and neurocognitive complaints -LSB- 1 , 2 -RSB- . In addition to the chronic fatigue , widespread and persistent pain is common in individuals with CFS -LSB- 3 -- 5 -RSB- . A population-based study revealed that 94 % of the persons diagnosed with CFS report muscle aches and pain and 84 % report joint pain -LSB- 6 -RSB- . Nishikai et al. -LSB- 7 -RSB- reported muscle pain in 85 CFS patients of 114 patients -LRB- 74.6 % -RRB- . Seventy-four patients -LRB- 64.9 % -RRB- complained of arthralgia . In another study , 24 of 44 patients suffered from chronic widespread pain -LSB- 8 -RSB- . Chronic fatigue accompanied by chronic musculoskeletal impairments such as myalgias and arthralgias could be considered an important subclass of CFS -LSB- 9 -RSB- . Evidence supportive of the clinical importance of widespread pain in CFS has been provided -LSB- 10 -RSB- : chronic pain accounts for up to 34 % of the CFS patients ' self-reported activity limitations and participation restrictions . Chronic pain is more disabling than chronic fatigue -LSB- 10 -RSB- . Given these facts , it may be surprising that the etiology of these pain complaints has not been studied extensively in patients with CFS . The systematic literature review by Meeus et al. -LSB- 11 -RSB- shows that only little progress has been made in understanding chronic widespread pain in patients with CFS . A few hypotheses have been proposed , but they have not been studied in depth or relatively little work has been performed to test these hypotheses . In contrast , a large body of scientific literature regarding the etiology of chronic pain complaints in fibromyalgia -LRB- FM -RRB- is currently available . The diagnosis of FM is based on the 1990 American College of Rheumatology criteria . Following these criteria , FM patients present with 11 of 18 positive tender points and with widespread pain -LSB- 12 -RSB- . Validity of both the definition for CFS and FM has been shown -LSB- 12 , 13 -RSB- . Especially , investigations focusing on the phenomenon `` central sensitization '' are presented in force in FM . In CFS , the theory of central sensitization has only been suggested , to our knowledge -LSB- 14 -RSB- . Given the great overlap between CFS and FM -LSB- 15 -RSB- and given the dearth of studies focusing on the explanation for the chronic widespread pain in patients with CFS , it would be interesting to propose a theoretical model for the chronic pain in CFS based on the current knowledge of CFS and on the evidence for central sensitization in FM , giving rise to further research on that matter . Besides the knowledge on chronic pain in FM , it is necessary to gather knowledge on musculoskeletal pain in CFS . The syndromes may overlap , but despite the similarities between the two syndromes , there are also differences . For example , immunological dysregulations such as the abnormal 2 -- 5A synthetase/RNase L pathway -LSB- 16 -RSB- have been revealed in CFS but have never been detected in FM patients . Furthermore , there is not yet any good evidence for similar pain mechanisms in CFS and FM . Some authors already found evidence suggesting differences in pain processing . For example , patterns of functional brain activity in patients with FM are quite different from those in patients with CFS . Patients with CFS , relative to controls , showed significantly lower blood perfusion in the brain stem -LSB- 17 , 18 -RSB- . Patients with FM exhibited significantly lower rCBF levels , during rest , in the thalamus and the caudate nucleus -LSB- 19 -RSB- . Furthermore , Substance P has been found to be elevated in CSF of FM patients -LSB- 20 -RSB- and not in patients with CFS -LSB- 21 -RSB- . Therefore , the knowledge on pain in FM can not be applied on CFS patients without further study . Based on the similarities and differences between the two syndromes , further research on pain in CFS is advised to get an image of pain processing in the two diseases . The goal of this article is to provide a rational basis for future investigations . First , the concept of central sensitisation as a cause of chronic pain will be explained . This theoretical background will then be applied to FM and an overview of the evidence for central sensitization in FM will follow . Finally , based on the theoretical background and the findings in FM , the hypothesis concerning central sensitization in CFS will be unfolded , supported with the present knowledge on CFS . Central sensitization Introduction Pain is a complex perception that is influenced by prior experience and by the context within which the noxious stimulus occurs ; `` nociception '' is the physiologic response to tissue damage or prior tissue damage -LSB- 22 -RSB- . The definition of pain is endorsed by the International Association for the Study of Pain : `` Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage , or described in terms of such damage '' -LSB- 23 -RSB- . There are a host of physiologic mechanisms by which injuries lead to nociceptive responses and ultimately to pain -LSB- 22 -RSB- . However , not all nociceptive signals are perceived as pain and vice versa , not every pain sensation originates from nociception . Nevertheless , acute pain almost always originates from nociceptors in somatic or visceral tissue . Mainly two types of pain receptors are activated by nociceptive input . These include low-threshold nociceptors that are connected to fast conducting A-delta pain fibers , and high-threshold nociceptors that conduct impulses in slow -LRB- unmyelinated -RRB- C fibers . Within the dorsal horn of the spinal cord , these pain fibers synapse with spinal neurons via synaptic transmission . Many neurotransmitters -LRB- i.e. , glutamate , substance P , etc. -RRB- are able to modulate the postsynaptic responses with further transmission to supraspinal sites -LRB- thalamus , anterior cingulated cortex , insular cortex , and somatosensory cortex -RRB- via the ascending pathways -LSB- 22 , 24 , 25 -RSB- . The simplest form of plasticity in nervous systems is that repeated noxious stimulation may lead to habituation -LRB- decreased response -RRB- or sensitization -LRB- increased response -RRB- -LSB- 26 -RSB- . Prolonged or strong activity of dorsal horn neurons caused by repeated or sustained noxious stimulation may subsequently lead to increased neuronal responsiveness or central sensitization -LSB- 25 , 27 -RSB- . Neuroplasticity and subsequent CNS sensitization include altered function of chemical , electrophysiological , and pharmacological systems -LSB- 22 , 28 , 29 -RSB- . These changes cause exaggerated perception of painful stimuli -LRB- hyperalgesia -RRB- , a perception of innocuous stimuli as painful -LRB- allodynia -RRB- and may be involved in the generation of referred pain and hyperalgesia across multiple spinal segments -LSB- 25 , 30 -- 33 -RSB- . While the exact mechanism by which the spinal cord becomes sensitized or in `` hyperexcitable '' state currently remains somewhat unknown , some contributing factors have been proposed . Temporal summation or wind-up `` Wind up '' refers to a central spinal mechanism in which repetitive noxious stimulation results in a slow temporal summation that is experienced in humans as increased pain -LSB- 34 -RSB- . In 1965 , animal experiments showed for the first time that repetitive C-fiber stimulation could result in a progressive increase of electrical discharges from the second-order neuron in the spinal cord -LSB- 35 -RSB- . This mechanism of pain amplification in the spinal cord is related to temporal summation of second pain or wind-up . Second pain , which is more dull and strongly related to chronic pain states , is transmitted through unmyelinated C fibers to dorsal horn nociceptive neurons . During the C-fibres transmitted stimuli , N-methyl-d-aspartate -LRB- NMDA -RRB- receptors of second-order neurons become activated.It is well-known that NMDA activation induces calcium entry into the dorsal horn neurons -LSB- 36 -RSB- . Calcium entry into sensory neurons in the dorsal horn induces activation of nitric oxide -LRB- NO -RRB- synthase , leading to the synthesis of NO -LSB- 37 -RSB- . NO can affect the nociceptor terminals and enhance the release of sensory neuropeptides -LRB- in particular , substance P -RRB- from presynaptic neurons , therefore contributing to the development of hyperalgesia and maintenance of central sensitization -LSB- 38 -RSB- . Substance P -LRB- SP -RRB- is an important nociceptive neurotransmitter . It lowers the threshold of synaptic excitability , resulting in the unmasking of normally silent interspinal synapses and the sensitization of second-order spinal neurons -LSB- 39 -RSB- . Furthermore , SP can extend for long distances in the spinal cord and sensitize dorsal horn neurons at a distance from the initial input locus . This results in an expansion of receptive fields and the activation of wide dynamic neurons by non-nociceptive afferent impulses -LSB- 24 -RSB- . Wind-up can be elicited in human patients if identical nociceptive stimuli are applied to the skin or muscles more often than once every 3 s . The resulting progressive increase of pain sensations represents wind-up and has been demonstrated to result from a central rather than a peripheral nervous system mechanism , because the input from C nociceptors has been shown to decline or stay the same with stimulus repetition -LSB- 40 -RSB- . Endogenous pain modulatory systems The presence of several pain inhibitory and facilitatory centers in the brainstem is well recognized . The dorsolateral funiculus appears to be a preferred pathway for descending pain inhibitory systems -LSB- 41 -RSB- . Experimental evidence for the existence of descending inhibitory pathways , and their connection with central sensitization , include the observations that bilateral lesions of the dorsolateral funiculus in the rat led to a significant decrease in latency for paw withdrawal to noxious stimulus -LSB- 42 -RSB- . Similarly , temporary spinal cord block -LRB- lidocaine -RRB- caused dorsal horn nociceptive neurons to expand their receptive fields and their responsiveness to afferent input -LSB- 43 -RSB- . In addition , selective chemical lesion of serotonergic inhibitory neurons in experimentally `` inflamed '' animals resulted in demonstrable behavioral `` pain '' hypersensitivity -LSB- 42 -RSB- . The foregoing investigations suggest that disruption of one or more of the elements of the inhibitory system can result in , among other things , the equivalent of central sensitization -LSB- 44 -RSB- . One function of the descending inhibitory pathway is to `` focus '' the excitation of the dorsal horn neurons . The effect is to generate a more urgent , localized , and rapid pain signal by suppressing surrounding neuronal activity -LSB- 45 -RSB- . This role is attributed to the `` diffuse noxious inhibitory controls '' -LRB- DNIC -RRB- phenomenon -LSB- 46 -RSB- . According to this model , descending pathways effectively enhance the biologically valuable pain signal by reducing the level of irrelevant `` noise '' in the system.Facilitatory pathways leading from the brainstem have also been identified . There is now behavioral evidence that forebrain centers are capable of exerting powerful clinically significant influences on various nuclei of the brainstem , including the nuclei identified as the origin of the descending facilitatory pathway -LSB- 44 -RSB- . The activity in descending pathways is not constant but can be modulated , for example , by the level of vigilance or attention and by stress -LSB- 47 -RSB- . Brosschot -LSB- 48 -RSB- refers to it as cognitive emotional sensitization . Forebrain products such as cognitions , emotions , attention , and motivation have influence on the clinical pain experience -LSB- 44 -RSB- . Dubner and Ren -LSB- 41 -RSB- rewarded subjects for responding to a randomly delivered transient tissue threatening peripheral stimulus . It was found that sensitization of second-order pain pathway neurons was directly related to the strength of attention . The evidence suggests that selective attention to relevant stimuli activated descending pain modulatory systems , turning the balance in favor of facilitation . The dominance of descending facilitation then led to sensitization of second-order neurons -LSB- 44 -RSB- . Behavioral variables such as attention to a potentially threatening stimulus result in sensitization of dorsal horns spinal cord neurons . Moreover , behavioral modulation associated with selective attention to a perceived threat utilizes the same forebrain and brainstem structures and mechanisms as are involved in the development , amplification , and maintenance of persistent pain after actual tissue damage and inflammation -LSB- 41 -RSB- . Certain cognitive styles and personality traits have been associated with amplification of pain and its extension in the absence of tissue damage . These include somatization , catastrophizing , and hypervigilance -LSB- 49 -- 52 -RSB- . Thus , via descending pathways behavioral and cognitive therapies might also effect synaptic transmission in the spinal cord and thereby have the capacity to prevent or reverse long-term changes of synaptic strength in pain pathways -LSB- 47 -RSB- . Evidence in FM Exaggerated pain is common in whiplash and FM patients . Theoretically , peripheral mechanisms can account for the pain hypersensitivity . In FM , however , there is no evidence for peripheral sensitization as the cause of hyperalgesia , given the absence of real tissue damage . Peripheral sensitization is defined as a reduction in the threshold of nociceptive afferent receptors caused by a local change in the sensitivity of sensory fibers initiated by tissue damage -LSB- 53 -RSB- . Peripheral sensitization almost always depends on local inflammation , which may lead to decreased nociceptor thresholds . Despite extensive investigations , no tissue pathology , structural abnormalities , or evidence for a source of chronic stimulation of pain afferents have been detected in fibromyalgic patients -LSB- 54 -RSB- . Furthermore , FM pain lacks a distinct spatial localization -LSB- 55 -RSB- . This suggests that pathophysiological central mechanisms contribute to or are responsible for FM pain -LSB- 56 -RSB- . This has led to the hypothesis that the central nervous system is hyperexcitable in these patients . Central hypersensitivity could explain exaggerated pain in the presence of minimal and undetectable tissue damage , in that the nociceptive signal is amplified by the hyperexcitable neurons -LSB- 57 -RSB- . Pain measurements Studies in patients with chronic pain after whiplash injury and with FM have demonstrated exaggerated pain responses after sensory stimulation of healthy tissues -LSB- 55 , 58 -- 63 -RSB- . For example , at same level of thermal stimulation , FM patients perceived pain as 49 and 52 % more intense than healthy controls and patients with low-back pain -LSB- 61 -RSB- . After-sensations at 15 s after heat taps were regarded as painful on 83 % of FM patients , compared to 37 % pain reports of healthy controls . The late after-sensations -LRB- 2 min after heat stimulation -RRB- were rated as painful in 55 and 5 % of FM and control subjects , respectively -LSB- 55 -RSB- . It was not mentioned in the results if all FM patients were subject to increased sensitivity , but there were always significant differences between the FM patients and the healthy controls.Although most comparisons were made to pain-free subjects , some researchers , like Julien et al. -LSB- 61 -RSB- , even compared the pain responses of FM patients to other pain patients , such as patients with chronic low-back pain . They could also report significant differences . Despite the lack of research into the contribution of psychological factors in wind-up , these results suggest that input to central nociceptive pathways is abnormally processed in patients with FM . Intramuscular electrical stimulation has been used to assess the efficacy of temporal summation of painful muscle stimuli . Temporal stimulation was found to be more pronounced and to cause stronger pain and larger referred areas in FM patients compared to controls -LSB- 60 -RSB- . The increased efficacy of temporal summation in FM has even so been reproduced with cutaneous heat stimulation -LSB- 55 -RSB- and with cold and heat taps -LSB- 59 -RSB- . Facilitated temporal summation in patients with pain suggests that the efficacy of central processing is increased -LRB- central sensitization -RRB- in these patients -LSB- 64 -RSB- . In addition , after-sensation was greater in magnitude , lasted longer , and was more frequently painful in patients with FM -LSB- 55 -RSB- . The prolonged decay together with the augmentation provides evidence for the presence of central sensitization -LSB- 24 , 25 -RSB- . Immersion of the arm in circulating noxious cold water resulted in a 49 % more intense pain in FM patients compared to healthy controls -LSB- 61 -RSB- . Further evidence of central sensitization in FM is seen by enlarged referred pain areas . Sörensen et al. -LSB- 60 -RSB- found that fibromyalgic patients experienced stronger pain and larger referred areas after intramuscular injection of hypertonic saline.Moreover , spatial summation effect during increase of the stimulation area was found only in patients with FM and not in healthy controls or in patients with chronic low-back pain -LSB- 61 -RSB- . This indicates that pain inhibitory systems are not optimally recruited in patients with FM . FM patients perceived pain at the same intensities and unpleasantness during the ascending and the descending sessions -LRB- fingertip to shoulder and shoulder to fingertip immersion -RRB- . In healthy controls and patients with low-back pain , the noxious stimulation of a large surface area results in an activation of a large population of nociceptive afferents that induce endogenous inhibitory responses , resulting in a decreased response in the dorsal horn neurons , and subsequently leads to lower pain intensities afterward.Given that FM patients experience similar pain intensities in the descending session after noxious stimulation of the whole arm , this study clearly demonstrated that FM patients present with a lack of activation of endogenous inhibitory systems -LSB- 61 -RSB- . Secondly , Lautenbacher and Rollman -LSB- 65 -RSB- showed that tonic painful and non-painful thermal stimulation of the foot increased the pain thresholds to electrical stimulation applied to a non-tender point -LRB- inner forearm -RRB- in healthy controls but had no effect on patients with FM . Kosek and Hansson -LSB- 66 -RSB- found that tourniquet ischemic pain in an arm increased the pressure pain threshold in healthy controls but not in FM patients , suggesting once more a deficiency in the latter of a pain-inhibitory phenomenon termed `` diffuse noxious inhibitory control '' -LRB- DNIC -RRB- -LSB- 66 , 67 -RSB- . Similarly , aerobic exercise has been shown to decrease wind-up pain in normal subjects but increased it in FM patients , suggesting the possibility of reduced endogenous analgesic mechanisms -LSB- 68 -RSB- . Also , isometric exercise -LRB- i.e. , hand-grip exercise -RRB- resulted in increased thermal pain ratings and decreased pain thresholds , both ipsilateral and contralateral to the exercised extremity -LSB- 69 -RSB- . These investigations support a general hypothesis that FM reflects a disorder affecting modulation of pain sensitivity -LSB- 66 , 67 -RSB- . In one such scenario , tonic DNIC is present in the normal situation , and its pathological absence results in the spontaneous pain and evoked pain sensitivity associated with FM -LSB- 34 -RSB- . Measurements of excitability The results of the above-mentioned studies , however , are based on pain reports of the patients and thus subjective in nature , and it was not clear whether this hypersensitivity was the result of central mechanisms or whether the hypersensitivity was the cause of hypervigilance . Banic et al. -LSB- 57 -RSB- could provide objective evidence by quantifying the minimal intensity of transcutaneous electrical stimulation of the sural nerve required to evoke flexion reflex in the biceps femoris . This study clearly demonstrates that spinal cord neurons are sensitized in chronic pain after whiplash and in patients with FM ; because the stimuli were delivered at random time intervals and the latency of EMG response was measured , voluntary symptom amplification could be ruled out . Moreover , the electrical stimulation bypasses peripheral receptors . Cognitive emotional sensitization Patients with FM or other pain disorders often receive the message that it is `` all in their head '' . One construct that has been hypothesized to explain the pain amplification in FM is that of hypervigilance -LSB- 34 -RSB- . This hypothesis of hypervigilance has been argued by McDermid et al. -LSB- 70 -RSB- . FM showed an increased aversiveness to non-painful stimuli such as loud noise . Also , Crombez et al. -LSB- 71 -RSB- reported significant correlations between hypervigilance and pain intensity in FM patients . Furthermore , exposure to stressful situations , including noise , lights , and weather , is known to exacerbate symptoms of FM , including pain -LSB- 72 -RSB- . In addition , FM patients with catastrophic thoughts report increased pain intensities -LSB- 73 -- 75 -RSB- . Hassett et al. -LSB- 74 -RSB- found catastrophizing -LRB- 27 % of the variance -RRB- and depression -LRB- 30 % of the variance -RRB- to be significant predictors of pain . Finally , kinesiophobia and fear of pain are related to pain severity in patients with FM -LSB- 76 -RSB- . The foregoing relations between emotions or cognitions and reported pain severity support the hypothesis of cognitive emotional sensitization in FM . Central abnormalities in FM FM patients differ from healthy persons in regional cerebral blood flow -LRB- rCBF -RRB- distribution in several brain structures involved in pain processing and pain modulation both at rest and during experimental pain induction . Patients with FM exhibited significantly lower rCBF levels , during rest , in the thalamus and the caudate nucleus -LSB- 17 -RSB- . Dysregulation of thalamic activity and hypoperfusion of the caudate nucleus may contribute to the abnormal pain modulation , given the results of previous investigations -LSB- 77 -- 79 -RSB- . In addition to nociceptive transmission , the thalamus also plays an important role in pain modulation . Animal studies proved that thalamic stimulation induces analgesia and lesions of the thalamus cause hyperalgesia -LSB- 77 , 78 -RSB- . Similarly , stimulating the caudate nucleus decreases pain behavior -LSB- 79 -RSB- . During pain induction in patients with FM , the absence of significant thalamic activation and a bilateral activation of the somatosensory cortices and the right anterior cingulated cortex was seen -LSB- 80 -RSB- . The patterns of cortical activation may be characteristic of patients with allodynia , and pain-induced activation of the right anterior cingulated cortex is associated with the use of maladaptive coping strategies -LSB- 81 -RSB- . Patients recruited by Bradley et al. -LSB- 80 -RSB- indeed reported significantly greater use of emotion-focused coping strategies -LRB- e.g. , praying and hoping -RRB- during pressure stimulation . In addition , increased right frontal brain activity seemed related to increased pain sensitivity -LSB- 82 -RSB- . Furthermore , FM patients are characterized by relatively high cerebrospinal fluid levels of substance P -LSB- 20 -RSB- . This finding indicates that abnormal brain activity in persons with FM is associated with abnormal CSF levels of a neuropeptide involved in pain transmission -LSB- 80 -RSB- . In addition , it is shown that a subgroup of FM patients present with mycoplasma infections -LSB- 83 -RSB- . Infection triggers the release of the pro-inflammatory cytokine interleukin-1β , which is known to play a major role in inducing cyclooxygenase-2 -LRB- COX-2 -RRB- and prostaglandin E2 expression in the central nervous system -LSB- 84 -RSB- . Peripheral nerve terminals can be sensitized by elevated COX-2 amounts and prostaglandin E2 . Peripheral infections are even able to activate spinal cord glia , leading to the release of NO and proinflammatory cytokines , enhancing the pain response -LSB- 85 -RSB- . Physiological symptoms , such as pain , can be explained by these mechanisms -LRB- `` sickness response '' -RRB- . Central sensitization in CFS ? Direct evidence supporting the central sensitization hypothesis in CFS patients is currently lacking . But the present knowledge concerning CFS is suggestive of a central process similar to that seen in FM , given the great overlap between the two diseases and the observed similarities . First , lower pain thresholds at different sites -LRB- hyperalgesia -RRB- are reported in patients with CFS , compared to controls -LSB- 86 , 87 -RSB- . Similar to FM , the lack of peripheral tissue damage and the lack of a distinct localization of the pain complaints are suggestive of a central abnormality responsible for the chronic widespread pain . To our knowledge , there are no investigations that focused on abnormal wind-up , temporal summation , or spatial summation in CFS , to collect evidence for central sensitization in CFS . Yet , evidence of a dysfunctional central anti-nociceptive mechanism in CFS has been proved by Whiteside et al. -LSB- 88 -RSB- . They reported a decrease of pain threshold in patients with CFS after graded exercise , while healthy controls present an increased pain threshold . These findings are similar to those of Vierck et al. -LSB- 68 -RSB- in FM patients . Secondly , the frequently reported opportunistic infections -LSB- 89 , 90 -RSB- may lead to the `` sickness response '' and complies with the central sensitization hypothesis , as explained above . Indeed , elevated NO levels have been documented in CFS patients -LSB- 91 -RSB- . As earlier mentioned , NO plays an important role in the history of central sensitization and , therefore , central sensitization caused by NO would be likely in patients with CFS . The release of excessive amounts of Substance P in the CSF , however , could not be documented in patients with CFS , in contrast to FM patients -LSB- 21 -RSB- . The CFS patients in this study , however , did not experience chronic widespread pain , and it is not clear if they fulfilled the 1994 CDC criteria -LSB- 2 -RSB- . Given that 70 % of the CFS patients do fulfill the ACR criteria for widespread pain -LSB- 12 -RSB- , it is striking that the patients included in the investigation of Evengard et al. -LSB- 21 -RSB- -LRB- focused on the source of pain -RRB- did not report these pain complaints . Further research on that matter should analyze the CSF of CFS patients -LRB- fulfilling the Fukuda criteria -RRB- suffering from chronic widespread pain . A third important argument in the central sensitization theory for CFS concerns the cognitive , psychological , and behavioral changes in patients diagnosed with CFS . CFS patients often present with depression -LSB- 6 , 92 -RSB- , catastrophizing -LSB- 93 , 94 -RSB- , somatization -LSB- 95 , 96 -RSB- , and kinesiophobia or fear avoidance -LSB- 97 -- 99 -RSB- . In CFS , it is known that these psychosocial aspects are important factors in maintaining the complaints of CFS . These cognitions and emotions are able to influence pain perception via modulation of the descending pathways -LSB- 44 -RSB- . Furthermore , these `` cognitive styles '' and `` personality traits '' have earlier been mentioned to be associated with the amplification and the extension of pain -LSB- 49 -- 52 -RSB- . Finally , brain imaging already provided evidence for altered brain activity in CFS . Patterns of functional brain activity in patients with FM are quite different from those in patients with CFS . Patients with CFS , relative to controls , showed significantly lower blood perfusion in the brain stem -LSB- 17 , 18 -RSB- . Patients with FM exhibited significantly lower rCBF levels , during rest , in the thalamus and the caudate nucleus -LSB- 19 -RSB- . However , the areas are different in FM and in CFS ; both the affected areas could be related to central pain processing . Low brain stem rCBF levels may contribute to abnormal function of the locus ceruleus in patients with CFS . The locus ceruleus is involved in controlling descending anti-nociceptive pathways from the brain to the spinal dorsal horn -LSB- 56 -RSB- . In consequence , pain experiences of patients with CFS may be related to low resting state levels of functional activity in the brain stem -LSB- 80 -RSB- . Conclusion Chronic widespread pain can be the consequence of central sensitization . Central sensitization is known as an increased central neuronal responsiveness and causes hyperalgesia , allodynia , and referred pain and hyperalgesia across multiple spinal segments , leading to chronic widespread pain . Possible triggers for sensitization of the spinal cord have extensively been discussed , such as wind-up or temporal summation , dysregulated descending inhibitory pathways , and upregulated facilitatory modulation . Wind-up or temporal summation is the result of repetitive noxious stimuli , leading to an increase in electrical discharges in the dorsal horn . Inhibitory modulation can be impaired by abnormalities in the central nervous system and the facilitatory pain pathways can be stimulated by certain behavioral and cognitive factors . This theoretical background can be applied to FM . In FM , studies already provided evidence for central sensitization as the cause of chronic pain . Temporal summation was found to be more facilitated , and the inhibitory pain modulation seemed impaired in FM patients . These findings can explain the chronic spontaneous pain in FM . Furthermore , some central abnormalities could be examined/objectified in FM : 1 -RRB- hyperexcitability of the spinal cord , 2 -RRB- decreased perfusion of pain-related brain structures , and 3 -RRB- high levels of substance P in CSF . In addition , FM patients often present with pain hypervigilance , maladaptive coping strategies , and catastrophic thoughts , leading to cognitive central sensitization . Based on the knowledge on central sensitization , on FM and on CFS , it is suggested that chronic widespread pain in CFS is the consequence of central sensitization . There are arguments and probable mechanisms that could explain this phenomenon in CFS . Also , in other chronic pain populations , central sensitization may play a key role . In fact , there are many similarities between CFS patients and other chronic pain populations such as patients with chronic low-back pain , whiplash , FM , etc. . The psychosocial factors , for example , have been proved to contribute to pain perception in these different pain populations . But the specific nature of CFS such as the immunological abnormalities , elevated NO amounts , preceding infections etc. , invites further research , in particular , on the possible contributory role of these abnormalities to pain processing in CFS . In FM , many researches have been conducted to prove the theory of central sensitization . In CFS , however , it sticks to `` supposing . '' To give a scientific basis to the theory , the protocols applied in FM investigations could be used for patients with CFS . It would , for example , be interesting to test the efficacy of temporal summation in CFS . The inhibitory control of pain could be another point of interest . The influence of exercise on pain tolerance has already been studied in CFS -LSB- 88 -RSB- , however , on a relatively small sample . On the contrary , spatial summation has , to our knowledge , never been investigated in CFS . Furthermore , the role of depression , hypervigilance , kinesiophobia , catastrophising , etc. on chronic pain in CFS requires further research . To obtain more direct and objective information on central sensitization , the protocol described by Banic et al. -LSB- 57 -RSB- could be used to test the sensitivity of the central nervous system . Clearly , there are many possible research areas to test the hypothesis , but there is still a long way to go to elucidate the nature of the chronic pain in CFS .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O

central sensitization
chronic fatigue syndrome
chronic pain
fibromyalgia