Tetraether

membrane

lipids

of

Candidatus

Aciduliprofundum

boonei

a

cultivated

obligate

thermoacidophilic

euryarchaeote

from

deep-sea

hydrothermal

vents

The

lipid

composition

of

Candidatus

Aciduliprofundum

boonei

the

only

cultivated

representative

of

archaea

falling

in

the

phylogenetic

cluster

a

group

of

microorganisms

ubiquitously

occurring

at

hydrothermal

vents

was

studied

The

predominant

core

membrane

lipids

in

this

thermophilic

euryarchaeote

were

found

to

be

composed

of

glycerol

dibiphytanyl

glycerol

tetraethers

-LRB-

GDGTs

-RRB-

containing

cyclopentyl

moieties

In

addition

GDGTs

with

an

additional

covalent

bond

between

the

isoprenoid

hydrocarbon

chains

so-called

H-shaped

GDGTs

were

present

The

latter

core

lipids

have

been

rarely

reported

previously

Intact

polar

lipid

analysis

revealed

that

they

predominantly

consist

of

GDGTs

with

a

phospho-glycerol

headgroup

Introduction

Deep-sea

hydrothermal

vents

are

unique

environments

which

are

thought

to

represent

models

for

both

the

origin

of

life

on

Earth

and

exploration

of

life

on

other

planets

These

vents

contain

ecosystems

which

are

predominantly

fueled

by

geochemical

energy

and

are

host

to

many

newly

described

free-living

microbes

which

are

often

associated

with

actively

venting

porous

deep-sea

vent

deposits

or

chimneys

The

steep

chemical

and

thermal

gradients

within

the

walls

of

these

deposits

provide

a

wide

range

of

microhabitats

for

microorganisms

with

suitable

conditions

for

aerobic

and

anaerobic

thermophiles

and

mesophiles

-LRB-

e.g.

McCollom

and

Schock

-RRB-

Indeed

both

culture-dependent

and

independent

approaches

have

exposed

a

vast

diversity

of

Bacteria

and

Archaea

associated

with

deep-sea

vent

deposits

-LRB-

e.g.

Reysenbach

and

Shock

Schrenk

et

al.

-RRB-

Numerous

Archaea

have

been

isolated

from

these

deposits

but

few

of

these

are

found

in

environmental

rRNA

gene

clone

libraries

and

most

detected

environmental

clones

in

these

environments

have

no

representatives

available

in

pure

culture

In

particular

one

archaeal

lineage

is

widespread

at

deep-sea

vents

namely

the

deep-sea

hydrothermal

vent

euryarchaeotic

lineage

and

is

frequently

associated

with

actively

venting

sulphide

deposits

-LRB-

e.g.

Nercessian

et

al.

Hoek

et

al.

Reysenbach

and

Shock

Takai

et

al.

-RRB-

In

some

cases

it

has

been

reported

as

the

most

dominant

clone

type

in

archaeal

clone

libraries

-LRB-

Hoek

et

al.

-RRB-

yet

the

physiology

of

these

organisms

was

unclear

Recently

Reysenbach

et

al.

-LRB-

-RRB-

isolated

and

cultivated

a

member

of

the

phylogenetic

cluster

Candidatus

Aciduliprofundum

boonei

and

showed

it

to

be

an

obligate

thermoacidophilic

sulphur

and

iron

reducing

heterotroph

capable

of

growing

from

pH

to

and

between

and

°C

This

provided

the

first

evidence

that

thermoacidophiles

may

be

key

players

in

sulphur

and

iron

cycling

at

deep-sea

vents

Archaea

are

not

only

unique

in

their

rRNA

phylogenetic

position

in

the

tree

of

life

but

also

synthesize

specific

membrane

lipids

Analysis

of

cultivated

hyperthermophilic

Archaea

showed

that

their

membrane

is

predominantly

composed

of

isoprenoid

glycerol

dibiphytanyl

glycerol

tetraethers

-LRB-

GDGTs

-RRB-

with

additional

cyclopentyl

moieties

-LRB-

e.g.

Structures

I

VI

in

Fig.

-RRB-

The

structural

differences

from

diacyl

membrane

lipids

of

non-thermophilic

Eukarya

and

Bacteria

i.e.

ether

bonds

and

the

formation

of

a

monolayer

rather

than

a

bilayer

have

been

suggested

to

contribute

to

the

stability

of

membranes

of

hyperthermophiles

at

high

temperatures

and

low

pH

-LRB-

e.g.

De

Rosa

and

Gambacorta

van

den

Vossenberg

et

al.

Macalady

et

al.

-RRB-

This

suggests

that

members

of

the

cluster

may

be

also

synthesizing

GDGT

membrane

lipids

Therefore

in

this

study

we

analyzed

the

lipid

composition

of

the

only

cultivated

representative

of

Aciduliprofundum

boonei

and

examined

both

the

core

GDGT

lipid

composition

as

well

as

its

intact

polar

lipid

composition

Fig.

base

peak

chromatogram

of

GDGT

core

lipids

in

extract

released

after

base

hydrolysis

-LRB-

using

KOH/methanol

mixture

-RRB-

of

the

cell

material

of

Candidatus

Aciduliprofundum

boonei

Inset

shows

the

atmospheric

pressure

chemical

ionization

mass

spectrum

of

GDGT

VII

Note

that

the

position

of

the

covalent

bond

between

the

isoprenoid

hydrocarbon

chains

in

GDGTs

VII

XI

is

tentative

-LRB-

Morii

et

al.

-RRB-

Materials

and

methods

Culture

conditions

Aciduliprofundum

boonei

was

grown

as

previously

described

-LRB-

Reysenbach

et

al.

-RRB-

Cells

were

harvested

at

late

log

phase

of

growth

Cell

pellets

were

freeze-dried

and

stored

frozen

until

analysis

Core

lipid

analysis

Cell

material

-LRB-

mg

dry

weight

-RRB-

of

A.

boonei

was

hydrolyzed

by

refluxing

in

ca.

ml

M

KOH

in

methanol

for

h

The

pH

of

the

hydrolyzed

extract

was

adjusted

to

pH

using

M

HCl/methanol

-LRB-

MeOH

-RRB-

-LRB-

v/v

-RRB-

and

transferred

to

a

separatory

funnel

The

residual

cell

material

was

washed

subsequently

with

ml

MeOH/water

-LRB-

v/v

-RRB-

MeOH

and

dichloromethane

-LRB-

DCM

three

times

-RRB-

The

washings

and

ml

bidistilled

water

were

added

to

the

separatory

funnel

which

was

shaken

vigorously

The

DCM

layer

was

removed

and

the

residual

water/MeOH

layer

was

re-extracted

two

times

using

DCM

The

combined

DCM

layers

were

dried

using

a

sodium

sulfate

column

to

yield

the

hydrolyzed

extract

The

extract

released

upon

hydrolysis

was

condensed

by

rotary

evaporation

Elemental

sulphur

was

removed

with

activated

Cu

The

extract

was

methylated

by

diazomethane

and

filtered

over

a

silicagel

column

using

ethyl

acetate

One

aliquot

of

the

eluate

was

silylated

using

BSTFA

-LRB-

with

TMCS

-RRB-

in

pyridine

-LRB-

v/v

-RRB-

for

min

at

°C

and

subsequently

analyzed

by

gas

chromatography

-LRB-

GC

-RRB-

and

GC/mass

spectrometry

-LRB-

GC/MS

-RRB-

Another

aliquot

of

the

eluate

was

dissolved

in

hexane/isopropanol

-LRB-

v/v

-RRB-

ultrasonicated

and

filtered

using

a

PTFE

filter

prior

to

analysis

for

GDGTs

on

the

high

performance

liquid

chromatography

-LRB-

HPLC

-RRB-

MS.

Part

of

the

GDGT

fraction

was

hydrogenated

by

dissolving

it

in

ethyl

acetate

adding

a

small

amount

of

and

a

drop

of

acetic

acid

and

bubbling

hydrogen

for

h.

Subsequently

the

solution

was

stirred

overnight

and

filtered

over

a

small

pipette

column

containing

-LRB-

top

-RRB-

and

-LRB-

bottom

-RRB-

Ethyl

acetate

was

evaporated

and

the

hydrogenated

GDGT

fraction

was

dissolved

in

hexane/isopropanol

-LRB-

v/v

-RRB-

ultrasonicated

and

analyzed

using

HPLC/MS

A

second

aliquot

of

the

GDGT

fraction

was

separated

using

HPLC

with

a

preparative

device

-LRB-

Foxy

jr

-RRB-

and

the

fractions

collected

were

analyzed

by

flow

injection

analysis

mass

spectrometry

-LRB-

Smittenberg

et

al.

-RRB-

The

fractions

containing

the

unusual

GDGT

-LRB-

see

text

below

-RRB-

were

combined

and

treated

with

HI

and

to

release

the

ether

bound

hydrocarbon

skeletons

-LRB-

Schouten

et

al.

-RRB-

This

fraction

was

analyzed

by

GC

and

GC/MS

The

total

extract

and

the

GDGT

fractions

treated

with

were

analyzed

by

GC/MS

using

a

Finnigan

Trace

GC

ultra

coupled

to

a

Finnigan

Trace

DSQ

mass

spectrometer

A

fused

silica

capillary

column

-LRB-

m

mm

-RRB-

coated

with

CP-Sil

-LRB-

film

thickness

μm

-RRB-

was

used

with

helium

as

carrier

gas

The

oven

was

programmed

at

a

starting

-LRB-

injection

-RRB-

temperature

of

°C

which

rose

to

°C

at

°C

min

and

then

to

°C

at

°C

min

at

which

it

was

maintained

for

min

The

column

was

directly

inserted

into

the

electron

impact

ion

source

of

the

DSQ

quadrupole

mass

spectrometer

scanning

a

mass

range

of

m/z

at

three

scans

per

second

and

with

an

ionization

energy

of

eV

HPLC/MS

analyses

were

performed

according

to

Hopmans

et

al.

-LRB-

-RRB-

Analyses

were

performed

using

an

Agilent

-LRB-

Palo-Alto

CA

USA

-RRB-

series

LC-MSD

SL

equipped

with

an

auto-injector

and

Chemstation

chromatography

manager

software

Separation

was

achieved

on

a

Prevail

Cyano

column

-LRB-

mm

μm

Alltech

Deerfield

IL

USA

-RRB-

maintained

at

°C

Injection

volumes

were

μl

GDGTs

were

eluted

isocratically

with

A

and

B

for

min

followed

by

a

linear

gradient

to

B

in

min

where

A

hexane

and

B

isopropanol

Flow

rate

was

ml/min

After

each

analysis

the

column

was

cleaned

by

back-flushing

hexane/isopropanol

-LRB-

v/v

-RRB-

at

ml/min

for

min

Detection

was

achieved

using

atmospheric

pressure

positive

ion

chemical

ionization

mass

spectrometry

-LRB-

APCI-MS

-RRB-

of

the

eluent

Conditions

for

the

HP

APCI-MSD

SL

were

as

follows

nebulizer

pressure

psi

vaporizer

temperature

°C

drying

gas

-LRB-

-RRB-

flow

l/min

and

temperature

°C

capillary

voltage

kV

corona

μA

-LRB-

kV

-RRB-

GDGTs

were

detected

by

mass

scanning

from

m/z

Intact

polar

lipid

analysis

For

analysis

of

the

intact

polar

lipids

cell

material

of

A.

boonei

were

extracted

using

a

modified

Bligh

Dyer

procedure

-LRB-

Bligh

and

Dyer

-RRB-

To

an

aliquot

of

the

cell

material

a

solvent

mixture

of

phosphate-buffer

-LRB-

M

pH

-RRB-

MeOH/DCM

-LRB-

v/v

-RRB-

was

added

The

mixture

was

sonicated

for

min

after

which

DCM

and

phosphate

buffer

were

added

to

a

volume

ratio

of

After

centrifuging

-LRB-

rpm

min

-RRB-

the

DCM

layer

was

collected

The

residue

was

re-extracted

twice

following

the

same

procedure

The

combined

DCM

layers

were

concentrated

by

evaporating

solvent

under

a

stream

dried

with

and

the

solvent

was

removed

under

a

stream

The

residue

was

dissolved

in

a

mixture

of

hexane/isopropanol

-LRB-

v/v

-RRB-

ultrasonicated

and

filtered

using

a

RC

filter

prior

to

analysis

on

the

HPLC/electrospray

ionization

-LRB-

ESI

-RRB-

MS.

Intact

core

lipids

were

analyzed

according

to

Sturt

et

al.

-LRB-

-RRB-

with

some

modifications

-LRB-

Boumann

et

al.

-RRB-

An

Agilent

series

LC

-LRB-

Agilent

San

Jose

CA

USA

-RRB-

was

used

equipped

with

thermostatted

autoinjector

and

column

oven

coupled

to

a

Thermo

TSQ

Quantum

Ultra

EM

triple

quadrupole

mass

spectrometer

equipped

with

an

Ion

Max

source

with

ESI

probe

-LRB-

Thermo

Electron

Corporation

Waltham

MA

USA

-RRB-

Separation

was

achieved

on

an

Inertsil

diol

column

-LRB-

mm

mm

μm

particles

Alltech

Associates

Inc.

Deerfield

IL

USA

-RRB-

maintained

at

°C

The

following

linear

gradient

was

used

with

a

flow

rate

of

mL

min

A

B

over

min

maintained

for

min

then

back

to

A

for

min

to

re-equilibrate

the

column

where

A

M

ratios

-LRB-

v/v/v

v

-RRB-

and

B

M

ratios

-LRB-

v/v/v

v

-RRB-

For

MS

detection

source

parameters

were

optimized

using

loop

injections

of

standard

phospholipids

-LRB-

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine

1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine

1,2-dipalmitoyl-sn-glycero-3-phospho-rac-glycerol

and

soya

l-α-phosphatidylinositol

Avanti

Polar

Lipids

Alabaster

AL

USA

-RRB-

into

a

stream

of

ml/min

of

eluent

A.

ESI

settings

were

as

follows

capillary

temperature

°C

sheath

gas

-LRB-

-RRB-

pressure

-LRB-

arbitrary

units

auxiliary

gas

-RRB-

-LRB-

-RRB-

pressure

-LRB-

arbitrary

units

-RRB-

spray

voltage

kV

and

source

V.

Mass

detection

was

achieved

on

the

first

MS

scanning

from

m/z

to

Results

and

discussion

Core

lipids

GC/MS

analysis

of

the

lipid

extract

of

hydrolyzed

cell

material

of

A.

boonei

did

not

reveal

substantial

amounts

of

dialkyl

glycerol

ethers

such

as

archaeol

suggesting

that

it

does

not

accumulate

these

compounds

in

substantial

amounts

By

contrast

HPLC/APCI-MS

analysis

of

the

fraction

revealed

the

abundant

presence

of

a

range

of

GDGTs

-LRB-

Fig.

-RRB-

suggesting

that

archaeol

is

effectively

transformed

into

GDGTs

The

main

compound

was

GDGT

II

containing

no

cyclopentyl

moieties

with

lower

amounts

of

GDGTs

III

VI

containing

cyclopentyl

moieties

and

small

amounts

of

GDGT

I

These

GDGTs

are

well

known

compounds

of

thermoacidophilic

Archaea

in

both

the

euryarchaeotal

and

crenarchaeotal

groups

-LRB-

cf.

De

Rosa

and

Gambacorta

Koga

and

Morii

-RRB-

Cultivated

species

of

the

Order

Thermoplasmatales

the

closest

phylogenetic

relatives

to

A.

boonei

-LRB-

Reysenbach

et

al.

-RRB-

also

contain

GDGTs

II

VI

as

their

dominant

membrane

lipids

-LRB-

DeRosa

and

Gambacorta

Uda

et

al.

Macalady

et

al.

-RRB-

Besides

GDGTs

I

VI

another

group

of

later-eluting

compounds

were

present

in

the

HPLC/MS

chromatogram

with

mass

spectra

-LRB-

insert

Fig.

-RRB-

characteristic

of

GDGTs

i.e.

a

base

peak

likely

representing

the

-LSB-

M

H

-RSB-

ions

and

fragment

ions

with

losses

of

Da

-LRB-

hydroxyl

group

as

water

-RRB-

and

Da

-LRB-

part

of

the

glycerol

moiety

-RRB-

-LRB-

Hopmans

et

al.

-RRB-

The

main

compound

had

a

base

peak

ion

of

with

more

minor

compounds

having

base

peak

ions

of

and

respectively

Remarkably

the

distribution

of

these

compounds

is

similar

to

that

of

GDGTs

II

VI

and

their

base

peak

ions

are

all

Da

lower

than

their

GDGT

counterparts

This

suggests

that

these

compounds

are

GDGTs

with

an

additional

double

bond

or

an

additional

cyclic

moiety

To

investigate

the

first

hypothesis

we

hydrogenated

the

polar

fraction

to

remove

any

double

bonds

However

subsequent

analysis

by

HPLC/MS

revealed

no

changes

in

GDGT

composition

suggesting

that

these

compounds

did

not

contain

any

double

bonds

The

unknown

GDGT

was

subsequently

isolated

by

preparative

HPLC

and

treated

with

to

release

the

hydrocarbon

skeletons

-LRB-

cf.

Schouten

et

al.

-RRB-

GC/MS

analysis

revealed

however

no

distinct

peaks

suggesting

that

the

hydrocarbon

skeletons

of

the

unknown

GDGT

do

not

consist

of

regular

isoprenoids

An

explanation

for

these

results

can

be

that

the

hydrocarbon

skeletons

are

covalently

linked

to

each

other

thereby

forming

a

hydrocarbon

after

ether-cleavage

The

molecular

weight

of

such

a

component

is

too

high

to

elute

over

a

regular

GC

column

Examples

of

such

GDGTs

have

been

previously

reported

i.e.

the

so-called

H-shaped

GDGT

-LRB-

Morii

et

al.

-RRB-

that

contains

a

covalent

bond

between

the

two

isoprenoid

chains

We

therefore

analyzed

a

pure

GDGT

VII

standard

obtained

from

Methanothermus

fervidus

-LRB-

a

kind

gift

of

Dr.

T.

Eguchi

-RRB-

by

HPLC/MS

The

mass

spectrum

of

this

standard

was

identical

to

that

of

the

major

unknown

GDGT

in

A.

boonei

and

co-injection

revealed

that

they

have

identical

retention

times

thus

unambiguously

identifying

this

compound

as

GDGT

VII

This

suggests

that

the

other

later

eluting

GDGTs

are

also

H-shaped

isoprenoid

GDGTs

but

with

additional

cyclopentyl

moieties

i.e.

GDGTs

VII

XI

It

must

be

noted

that

the

exact

position

of

the

covalent

bond

in

GDGT

VII

is

tentative

-LRB-

Morii

et

al.

-RRB-

and

thus

also

the

position

of

the

covalent

bonds

in

GDGTs

VII-XI

Lutneas

et

al.

-LRB-

-RRB-

recently

determined

the

structure

of

structurally

similar

isoprenoid

tetracids

in

which

the

covalent

bond

was

between

two

methyl

groups

rather

than

between

a

methyl

group

and

the

aliphatic

chain

as

suggested

by

Morii

et

al.

-LRB-

-RRB-

Hence

future

structural

analyses

are

needed

to

unambiguously

establish

the

position

of

the

covalent

bond

H-shaped

isoprenoid

GDGTs

have

been

only

rarely

reported

in

cultivated

archaea

GDGT

VII

was

first

identified

in

the

hyperthermophilic

methanogen

Methanothermus

fervidus

-LRB-

Morii

et

al.

-RRB-

They

were

further

identified

by

fast

atom

bombardment-MS

in

out

of

species

of

the

Order

Thermococcales

-LRB-

Sugai

et

al.

-RRB-

Until

now

they

have

only

been

identified

in

hyperthermophilic

euryarchaeota

growing

at

neutral

pH

with

optimal

growth

temperatures

°C

The

identification

of

H-shaped

GDGTs

in

A.

boonei

now

shows

that

these

compounds

are

also

synthesized

at

lower

growth

temperatures

-LRB-

°C

-RRB-

and

more

acidic

pH

-LRB-

-RRB-

The

functional

role

of

these

GDGTs

is

not

clear

Possibly

the

introduction

of

a

covalent

cross-link

between

the

alkyl

chains

will

reinforce

the

strength

of

the

monolayer

membrane

to

protect

the

cell

against

membrane

lysis

at

these

high

temperatures

The

similar

relative

distribution

of

GDGTs

II

VI

compared

to

GDGTs

VII

XI

in

A.

boonei

tentatively

suggests

a

possible

biosynthetic

link

i.e.

the

latter

GDGTs

may

be

synthesized

from

GDGTs

II

VI

Intact

polar

lipids

HPLC/ESI-MS

analysis

of

the

Bligh

Dyer

extract

of

cell

material

of

A.

boonei

revealed

one

major

peak

and

one

minor

peak

-LRB-

Fig.

-RRB-

The

mass

spectrum

of

the

major

peak

is

dominated

by

an

ion

at

m/z

with

minor

ions

and

Da

higher

-LRB-

insert

Fig.

-RRB-

These

latter

ions

are

typical

product

of

ammonia

and

sodium

adductions

respectively

to

protonated

molecules

in

the

ESI

interface

and

suggests

that

the

ion

at

m/z

represents

the

-LSB-

M

H

-RSB-

ion

-LRB-

cf.

Sturt

et

al.

-RRB-

As

the

major

core

lipid

is

GDGT

II

with

a

-LSB-

M

H

-RSB-

ion

of

it

suggests

that

the

head

group

has

a

mass

of

Da

in

agreement

with

a

phospho-glycerol

-LRB-

PG

-RRB-

moiety

-LRB-

Sturt

et

al.

-RRB-

The

mass

spectrum

of

the

minor

peak

eluting

slightly

later

is

similar

to

that

of

the

major

peak

except

that

all

ions

are

shifted

Da

lower

In

view

of

the

results

obtained

with

the

core

lipids

it

suggests

that

this

peak

represents

H-shaped

GDGTs

with

a

PG

head

group

PG

moieties

are

not

uncommon

as

head

groups

of

GDGTs

in

hyperthermophilic

Archaea

though

they

are

usually

accompanied

by

a

hexose

head

group

attached

to

the

other

glycerol

moiety

-LRB-

Koga

and

Morii

and

references

cited

therein

-RRB-

However

these

compounds

were

not

detected

in

A.

boonei

although

we

can

not

exclude

that

they

might

be

present

in

minor

amounts

Fig.

base

peak

chromatogram

of

intact

polar

lipids

in

Bligh

Dyer

extract

of

cell

material

of

Candidatus

Aciduliprofundum

boonei

Inset

shows

the

electrospray

ionization

mass

spectrum

of

the

major

peak

in

the

chromatogram

with

tentative

structural

identification

Conclusions

Our

analysis

of

lipid

composition

of

Candidatus

Aciduliprofundum

boonei

the

only

cultivated

representative

of

archaea

falling

in

the

phylogenetic

cluster

showed

it

to

be

composed

of

GDGTs

containing

cyclopentyl

moieties

Surprisingly

GDGTs

with

an

additional

covalent

bond

between

the

isoprenoid

hydrocarbon

chains

the

so-called

H-shaped

GDGTs

were

also

present

in

substantial

amounts

These

GDGTs

mainly

occurred

with

a

phospho-glycerol

headgroup

in

the

membrane

of

A.

boonei

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

glycerol dialkyl glycerol tetraethers
thermoacidophile


Variables affecting the probability of complete fusion of the medial clavicular epiphysis In this study , we have combined data on clavicle fusion from different studies and applied a binomial logistic regression analysis . As such , we aimed to assess whether or not variables such as sex , socioeconomic status , and ethnicity influence the probability of having mature , i.e. , completely fused clavicles at a given age . We further explored whether the method of clavicle examination , i.e. , diagnosis from either a dry bone specimen , an examination of X-rays , or an examination of computed tomography scans , affects the probability of being diagnosed with mature clavicles . It appeared that only ethnicity did not significantly affect this probability . Finally , we illustrated how the logit model may be used to predict the probability of being diagnosed with mature clavicles . Introduction The medial -LRB- or sternal -RRB- clavicular epiphysis matures relatively slowly . In the human skeleton , it is generally the last long bone epiphysis to fuse . Its developmental stage is therefore useful when estimating age at death in the post-pubertal period -LSB- 1 -RSB- or the age of living persons involved in criminal proceedings -LSB- 13 , 15 -RSB- . In the Netherlands , a radiological clavicle examination may be applied to verify the age of young asylum seekers without valid identification documents . Various authors have studied clavicle development to determine the age interval in which fusion of the clavicular epiphyses occurs . Most of the published studies were based on the examination of dry bone specimens -LSB- 3 , 6 , 9 , 10 , 17 , 18 -RSB- . When dealing with living individuals , examiners analyzed either X-rays -LSB- 4 , 13 -RSB- or computed tomography -LRB- CT -RRB- scans -LSB- 5 , 15 , 16 -RSB- . In a number of studies , the results for different ethnic groups were evaluated or the results for males were compared with those for females . In most studies , such comparisons were , however , hampered by a relatively small sample size . In this study , we aim to assess from a large sample whether or not variables such as sex , socioeconomic status , and ethnicity affect the probability of having completely fused clavicles at a certain age . We further aim to compare results from X-ray and CT scan examinations with those from studies based on dry bone specimens . To do so , we have combined data from different studies and performed a binomial logistic regression analysis . We then illustrate how the resulting model can be applied to estimate the probability of completely fused clavicles at a given age . Data Published and unpublished data on the individual subjects , taken from various studies on clavicle development -LSB- 3 -- 6 , 8 , 10 , 13 , 15 , 18 -RSB- were combined . For each individual , information on the calendar age in years was combined with data on the fusion status of the clavicle , i.e. , exhibiting a complete bony fusion of epiphysis and diaphysis -LRB- i.e. , mature -RRB- or not . When data for both left and right clavicles were published , we used the side for which most records were provided . Data on the sex and ethnicity of the individuals were copied when available . In the context of this study , we defined ethnicity as being either of European -LRB- Caucasian -RRB- , Asian , or Afro-American descent . It was further recorded which method was used for determining the fusion status , i.e. , using dry bone specimens , X-rays , or CT scans . The socioeconomic status of individuals was estimated from the human development index -LRB- HDI -RRB- associated with the country in which the study took place . The HDI is a summary composite index that measures a country 's average achievements in three basic aspects of human development : longevity , knowledge , and the standard of living -LSB- 19 -RSB- . The HDI is used by the United Nations Development Programme , and data are available from 1975 onward . To accommodate data from two American studies that took place before this year -LSB- 6 , 18 -RSB- , we extrapolated the HDI by first calculating the linear regression of the HDI on time . In total , data of 3,552 individuals aged 15 -- 33 years were combined . Figure 1 shows the age distribution within the sample . Table 1 provides further information on the number of individuals per category . Fig. 1Age distribution of individuals in the sampleTable 1Number of individuals per categoryCategoryValuesTotal number of individuals3 ,552 SexMales2 ,133 Females1 ,123 Unspecified296EthnicityaEuropean descentb1,316Afro-American descent287Asian descent695Unspecified1 ,254 MethodDry bone specimen1,374X-ray1 ,326 CT scan852aA small number of individuals in McKern and Stewart -LSB- 6 -RSB- were of Afro-American descent . As no individual data were provided , all 374 individuals are classified `` of European descent '' . b `` Of European descent '' includes Caucasian and Latino individuals from North America . Statistical analysis Analysis of the data was performed using the SPSS -LRB- version 10.0 -RRB- software package for statistical analysis . We carried out a binomial logistic regression analysis to assess the effect of various variables on the probability of mature clavicles . In logistic regression , the dependent variable is transformed into a logit variable , i.e. , the natural log of the odds of the dependent occurring or not . This transformation ensures that the estimated probabilities are between 0 and 1 . A logit model is a form of the generalized linear model . Age , sex , ethnicity , and socioeconomic status were considered as potential predictor variables for the probability of mature clavicles . The method of clavicle examination was further considered as a factor influencing the probability of being diagnosed with mature clavicles . Age and socioeconomic status -LRB- i.e. , HDI -RRB- were analyzed as covariates . Sex , ethnicity , and the method of clavicle examination were analyzed as fixed factors . Possible interaction between these fixed factors and the covariate age was also investigated . The initial -LRB- exploratory -RRB- logit model may be described by the following equation : where p denotes the probability of the dependent , α the intercept , β the estimated effect of a covariate or factor-by-covariate interaction term on the logit of the probability , and γ the estimated effect of a fixed factor . Variables to be kept in the model were selected by means of backward elimination using the Wald test . Insignificant interaction terms were successively dropped . After each alteration to the exploratory model , possible significance of the remaining variables was reassessed . A p value lower than 0.05 was considered to be statistically significant . Not all studies that were used as a source for our data provided individual data on sex or ethnicity . Consequently , the described model -LRB- M1 -RRB- could be tested on the data of a subgroup of 2,002 individuals only . No data from CT scans were available for this subgroup . To be able to include data from all available studies , we therefore also tested simplified models . For the first of these simplified models -LRB- M2 -RRB- , we omitted the variable ethnicity and the associated interaction term from the initial model . This model could be fitted to the data of a subgroup of 3,256 individuals . For this subgroup , data resulting from the evaluation of CT scans were drawn from a single study -LSB- 15 -RSB- , as the second available study -LSB- 5 -RSB- did not provide details on the sex of individuals . As a next step , we therefore further omitted the variable sex and the associated interaction term from the initial model . The resulting model -LRB- M3 -RRB- could be fitted to the data of all 3,552 individuals . As a final step , we assessed the significance of the change in − 2 log likelihood between the resulting model and a nested model including the main terms only . We further compare the Nagelkerke pseudo R2 . As such , we compare the difference in model fit . Results Table 2 provides the results for the three different models . Model M1 resulted in a p value higher than 0.05 for ethnicity after all non-significant interaction terms had been removed . Tested for a subgroup of 2,002 individuals , ethnicity therefore did not appear to affect the probability of having mature clavicles . Including this variable in the model does not improve the model fit . Exploring model M1 therefore effectively resulted in model M2 , which could be fitted to the data of 3,256 individuals . Table 2Estimated parameters of logistic regression models for the logit of the probability of mature claviclesModelIndependent variablesBStandard errorp valueM1 N = 2,002 Ethnicitya0 .355 M2 N = 3,256 Age0 .6290.0340.000 Sex -LRB- ref : male -RRB- 0.026 Female0 .3680.166 Human development index2 .7460.5900.000 Method -LRB- ref : dry bone specimen -RRB- 0.000 X-ray − 21.0463.055 CT scan − 7.6242.346 Method × age0 .000 X-ray1.0290 .135 CT scan0 .4230.104 Constant − 18.6381.0020.000 M3 N = 3552Age0 .6300.0340.000 Human development index3 .0120.5840.000 Method -LRB- ref : dry bone specimen -RRB- 0.000 X-ray − 20.7293.060 CT scan − 7.5261.928 Method × age0 .000 X-ray1.0230 .135 CT scan0 .4010.083 Constant − 18.8030.9980.000 aThe remaining parameters of model 1 -LRB- M1 -RRB- are omitted from this section in the table , as final results for a model without the variable ethnicity are provided for model 2 -LRB- M2 -RRB- . From model M2 , it appeared that age , the HDI -LRB- i.e. , socioeconomic status -RRB- , sex , and the method of clavicle examination all significantly affected the probability of having completely fused clavicles . Exploring possible interaction between sex and age did not indicate a significant effect . The interaction term method × age , however , yielded a p value of 0.000 . The subsequent assessment of the significance of the difference in − 2 log likelihood between the resulting model and a nested model omitting the interaction term yielded a p value of 0.000 . The Nagelkerke pseudo R2 reduced from 0.815 to 0.795 after omission of the interaction term . All indicates a better model fit when the interaction term is included . Model M3 , which could be fitted to all available data on clavicle fusion , resulted in comparable model parameters for the dummy variable for CT scans . Confounding by an observer effect is therefore reduced . Application of the model The most appropriate model to predict the probability of being diagnosed with mature clavicles , i.e. , providing the best model fit to the currently available data , appeared to be model M2 . The resulting model -- i.e. , after the removal of non-significant main and interaction terms and using the parameters for covariates and the intercept -- may be described by : Parameters for the appropriate dummies for the categorical variables may be found in Table 2 . From this equation , it follows that the predicted probability may be calculated using the following equation : Based on the currently available data on clavicle fusion , we can now predict the probability of having mature clavicles for different individuals . Here , we provide a few examples . The predicted probability of mature clavicles is 0.016 -LRB- 1.6 % -RRB- for a contemporary 19-year-old German male -LRB- HDI is 0.932 -RRB- . For a 20 - or 21-year-old German male , this probability increases to 0.029 -LRB- 2.9 % -RRB- and 0.054 -LRB- 5.4 % -RRB- , respectively . For females , the predicted probability values are increased to 0.023 -LRB- 2.3 % -RRB- , 0.042 -LRB- 4.2 % -RRB- , and 0.076 -LRB- 7.6 % -RRB- , respectively . However , if the individuals have a lower socioeconomic status , these values are decreased . For contemporary 19 - , 20 - , and 21-year-old males from India -LRB- HDI is 0.611 -RRB- , for instance , they are 0.007 -LRB- 0.7 % -RRB- , 0.012 -LRB- 1.2 % -RRB- , and 0.023 -LRB- 2.3 % -RRB- , respectively . When diagnosed from X-ray or CT scan , the values increase . For the 21-year-old Indian male , it would be 0.040 -LRB- 4 % -RRB- if diagnosed from an X-ray and 0.076 -LRB- 7.6 % -RRB- if diagnosed from a CT scan -LRB- composed using a slice thickness that is far from optimal -RRB- . In Figs. 2 and 3 , we show the differences in predicted probabilities at different ages between the various categories . In Fig. 2 , we compare the predicted probability values for a German male with that for a German female and an Indian male . Figure 3 illustrates the difference in predicted probability for a German male depending on whether dry bone specimens , X-rays , or CT scans are examined . Fig. 2Predicted probability of having completely fused clavicles : a comparison between German males , German females and Indian malesFig . 3Predicted probability of being diagnosed with mature clavicles : a comparison between examination by means of dry bone specimens , X-rays and CT scans Discussion Ethnicity did not appear to significantly influence the probability of having completely fused clavicles , which is in line with the results of Schmeling et al. -LSB- 12 -RSB- . We should , however , keep in mind that the current database is not well balanced . In addition , a small number of individuals taken from McKern and Stewart -LSB- 6 -RSB- were misclassified in our analysis -LRB- see footnote Table 1 -RRB- . Additional data from future studies may contribute to the knowledge on the possible effect of ethnicity , if not strengthen our conclusion that there is no significant effect . The effect of sex on the probability of having completely fused clavicles appeared significant . The estimated model parameter for the dummy variable for females being greater than zero indicates a positive effect on the logit of the probability . The predicted probability of mature clavicles is therefore greater for females than for males of similar ages . This would point to a generally earlier onset of fusion in females . A similar disparity has been reported for epiphysial closure of all other long bones -LSB- 11 -RSB- . There was no indication of significant interaction between sex and age . We may therefore assume similarly shaped probability curves for males and females , indicating an equal number of years during which complete fusion may generally be achieved . A higher HDI , and therefore a higher socioeconomic status , appears to increase the probability of having mature clavicles . This would mean that under less preferable socioeconomic conditions , the process of fusion is generally delayed . This is in concordance with conclusions drawn by Schmeling et al. -LSB- 12 , 14 -RSB- , and was summarized before by Eveleth and Tanner -LSB- 2 -RSB- . The method of clavicle examination appeared to also be significant . The significant interaction term method × age indicates that we should assume a difference in the estimated effect of age depending on the method applied . For the age interval in which mature clavicles have been observed , the predicted probability of being diagnosed with mature clavicles is greater when X-rays or CT scans are used instead of dry bone specimens . It is noteworthy that the difference in the predicted probability is initially very small , but becomes increasingly greater with increasing age during a limited age interval . When keeping in mind that the curve for the predicted probability will be s-shaped , one may image that the difference is again only minimal for the older individuals . A number of theories can be postulated to explain a difference in the predicted probabilities between methods of examination . Inappropriate slice thickness Regarding the results from CT scans , one could postulate that the slice thickness affected the diagnosis . Mühler et al. -LSB- 7 -RSB- concluded that reconstructions composed with a slice thickness of 1 mm should be used . The vast majority of the analyzed data were , however , obtained from reconstructions with a greater slice thickness -LRB- up to 8 mm -RRB- -LSB- 5 , 15 -RSB- . One can foresee that an almost completely fused clavicle may be diagnosed as being mature when details are lost due to the greater slice thickness . Schulze et al. -LSB- 16 -RSB- -- from whom no data were included in our analysis , as no individual data were provided -- observed two 19-year-olds with completely fused clavicles . Both observations were done from reconstructions with a slice thickness of 7 mm -LRB- Schulze , personal communication -RRB- . We therefore think there is a realistic possibility of these cases having been misdiagnosed . Persistent small grooves or notches between diaphysis and epiphysis When comparing the results from their study based on CT scans with two studies based on dry bone specimens -LSB- 6 , 9 -RSB- , Kreitner et al. -LSB- 5 -RSB- hypothesized that persistent small grooves or notches seen in dry bone specimens may be classified as partial fusions of the epiphysis with the clavicular shaft . This would reduce the probability of dry bone specimens being classified as completely fused . Reduced visibility of the epiphyseal disc on X-rays X-rays used in the published studies have not been made with the purpose of examining the clavicles . Possibly , this has hindered a correct classification of the clavicle at times . Interobserver variability As the sample included results from two studies based on X-rays and two studies based on CT scans , we hold it unlikely that what we perceive to be an effect of the applied method is in fact an observer effect . Expectation bias In some X-rays , the -LRB- nearly fused -RRB- epiphyseal disc will have been more difficult to judge than in others . In such cases , knowing the actual age of the individuals could have affected the interpretation of the observation . Clavicles of relatively young individuals would then more often be classified as `` not completely fused '' , while those of relatively old individuals would be classified as `` completely fused '' more often . We know , however , that in at least two out of four studies , one based on X-rays -LSB- 13 -RSB- and one on CT scans -LSB- 15 -RSB- , the examiner was not aware of the age of the subjects when classifying the images . We therefore do not believe that expectation bias could have played a role of any great importance . Statistical artifact In theory , it may be possible that due to an unfortunate coincidence , there were no relatively late maturing individuals examined by X-ray or CT scan.In theory , all of these aspects could have contributed to the significant effect of the variable method . However , if significance of the interaction between method and age is not explained as a statistical artifact , only expectation bias would have resulted in a significant change in the shape of the probability curve . In case of misclassifications of nearly fused clavicles or differences in the classification system among studies , we would have expected a higher number of individuals with completely fused clavicles in the relatively young subjects , even below the age of 20 . If we , however , were to conclude that significance of the interaction term is in fact a statistical artifact , the term should be removed from the model , and model parameters should be recalculated . It is our ambition to investigate the matter further by comparing high quality CT scans , expected to allow a correct diagnosis , with X-rays taken from the same person . Conclusions The probability of having mature clavicles at a certain age is affected by the sex and socioeconomic status of the individual . The probability of being diagnosed with mature clavicles further appears affected by the method of clavicle examination . Predicted probabilities from the model may be used to illustrate acceptance or rejection of individual age claims . Please note that the odds on having mature clavicles given a certain age should not be confused with the -LRB- posterior -RRB- odds of having reached a certain age given that the clavicles have matured . These probabilities are only equal if we assume a prior odds of 1 .

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 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 B 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 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 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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 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 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 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 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

age estimation
medial epiphysis
epiphyseal fusion