UCD Conway Focus Winter 2013

/ 0 " 3

Issue 20,23 3 Winter 2013

4

research

"

Celebrating 10 years of excellence

On September 12th 1 !

2013, UCD Conway %

the 10th

Institute celebrated anniversary

5 6

of its official opening with the launch

of Igniting Discovery, a publication (67 -!

and !

highlighting the research innovation achievements

!

8 ! of Conway researchers in

5

the past decade.

$ '

!

7 Over 700 PhD graduates and 350 1

9

postdoctoral researchers have been 4

: (9 4 :-

educated and trained to exacting % ! 5

international standards in the past ten 6

$

years. These highly-skilled individuals have

taken up employment in the

medical device and

pharmaceutical, ICT

! "

industries as well as in public agencies and

#

academia, in Ireland and abroad.

$ "

% &

The Institute’s research " portfolio includes

research

publications

3,275 $

peer reviewed

" % with the top 1% published in the most

prestigious scientific such as

' (" ) " journals

Nature, Science and Cell. In addition

*+

,UCD Conway researchers currently have

- successful collaborations, leading to .

"

$

publications, with scientists from

" " "

% .

over 1,500

in

" organisations 70 countries.

% Earlier this year, Conway Fellow, Professor Des Higgins broke the barrier of 100,000

/" #

total citations for his work on DNA " -+

+ %

" +% "

"

" sequence comparisons; making him the

"

most highly cited Irish scientist in the past

decade and one of the

most highly cited

" scientists worldwide.

%

The Clustal programme to "

align protein . " " sequences from research group

the

of

Professor Higgins is just one of a number of "

exciting has emerged

% . innovations

that

from the Institute.

Since 2003, Conway *01.,

researchers have disclosed 108 inventions, % '

" filed 139 patents, concluded 21 licence

agreements

and incorporated spin-out compa " 7 UCD

nies from the outputs

of Institute research

"

"

programmes. " 01.% Professor Walter Kolch, Director said,

- 23

“When the Institute was established, its

" " model of multidisciplinary and collabora

tive research was considered a radical

"

" vision of how scientific should carried $% 4 research

be

" out. Today, it is the standard.�

gold

"

" Professor Des Fitzgerald, UCD Vice-Presi

"

"

dent for Research Conway In

added,

“UCD

%

$

"

changing economic and research horizon maintaining

excellence.�

% while research ' ("

"

" "

"

/ 5 " ' '

" " "

% & 6.

Director (" with

" Prof Walter Kolch, pictured PhD students " " ! "

, Paul

(l-r), Anna Sanders, Natalia Volinsky Lavin and Mattia Bramini

"

5 " %7

" "

8 + % + & 9 : 0 : : . . 1 1 + + : . : : ' (" ;% + ' % 23 3 / 22< ==*>,?@A >@

stitute has been a highly effective driver of

0 productivity " and excellence

since research

" " "

"

its establishment. Through its intellectual $

" and infrastructural capabilities, it has risen

to the challenge of providing solutions

to societal needs in the face of a rapidly " "

"

Prof Des Fitzgerald, UCD Vice-President for Research presents the first copy of â&#x20AC;&#x2DC;Igniting Discoveryâ&#x20AC;&#x2122; to Ms Beatrice Conway, daughter of the Institute namesake, Prof Edward J. Conway FRS

) /

Directorâ&#x20AC;&#x2122;s Message

Welcome!

1

8

8

;

In the last 3 months, we marked a milestone in the life of

the Institute with "

our 10th anniversary celebrations. This

" coincided with the 2013 UCD Conway $

Festival of Research & Innovation and by " ("

a visit our distinguished scientific "

$ advisory board.

% . "

B the

in ' As we enter next phase the life

of the we intend continuing to =3 Institute, '

build research excellence and sustain ; 23 ;

" the A " %

4 " C

vibrant environment that ignites discovery,

"

drives innovation and

impacts

positively on

< society for generations.

/ " I extend congratulations to Professor " " " " William Gallagher, Director the first

of

Irish Cancer Society funded Collaborative

"

"

Cancer %Research Centre, BREAST-PREDICT.

This funding award is a milestone for

cancer research Ireland 4

in that will give new hope to patients and their families.

-+ It also

highlights our commitment and mission

$

to carry out research into the " fundamental

" "

" /

molecular of disease.

0

basis

23 3%

Following 0 their " recent successful

applications, I am delighted to welcome

"

" a number of new Conway Fellows and

" their teams whose research programmes

strengthen " + will undoubtedly the research

strategy of the Institute into the future. "

Walter " Kolch

Professor "

% Director B 4 :

-+ + 0 "

<2:4 <7' ' ' 1 '0 / D;+-D E '0 /; 0+ D ; ; +&

+

0 . 0

2013 Conway Festival medal for cilia research

5

5

5 ! 7

9

5 Doctoral candidate, Nils Lambacher

4 Conway

was

awarded the 2013 UCD

' Festival of Research & : Innovation gold medal, sponsored

by Cruinn Diagnostics

: $ '

7A ! for his research on the regulation of

5

! cilium structure in the worm model,

!

Caenorhabditis elegans. '

1 ! 9

7 1

Nils, who is carrying out5 5 his studies under

theB00"$ supervision of Conway Fellow, Dr

Oliver Blacque, impressed the judging

.

0 . his concise

panel with overview of this

and

its innovative

B research project

% potential. . "

D; N allB N

Cilia are present on nearly of our cells

" They

and act like cellular antennae. play fundamental roles in many motility and

" "

sensory functions, including signalling

"

pathways critical to development.

%

Ciliopathies is the collective name for a .

of B diseases . and

range human syndromes

mutations disrupt that arise when genetic "

function.

One

the cilium

structure and such example is nephronophthisis, the

*0' ,

most common cause

of kidney failure

in children affecting 50,000 live births .

" 1 in

in Canada. (" ! " "

D; O B %

The research abstract by Nils Lambacher

+

shortlisted B . was one of 30 for moderated 64 + "

" % "

"

23 3 " Recent publications Conway

from "

"

" "

"

presentation from 130 submissions.

The

winners

"

Crispin %7 other category were Alexander (Ferguson group); Elena +

. Karolina

Woods (Gautier group); Weiner+ " (McCann 6. group);

.

Gorzel Karen Hanrahan group)

and

(Watson Fiona McGillicuddy

"

" (substituted for Orla Finucane).

% .

heard

Delegates at the conference plenary

" lectures from Dr Frank Walsh, CEO,

Wellcome

Ossianix; Dr

Julian Parkhill, Trust Sanger and Professors

% . Institute

"

Cormac Taylor " and Des 10

Higgins,

UCD. Professor Sir Stephen Oâ&#x20AC;&#x2122;Rahilly,

"

University of

Cambridge awarded

was %7 the Ulysses medal, UCDâ&#x20AC;&#x2122;s highest . )

of

his B accolade in recognition outstanding

to "

"

contribution research concerning the mechanisms to molecular

" " leading

diabetes, related metabolic

obesity

and

# "

and disorders. endocrine 5

B

G

In the innovation session, Dr David

"

Oâ&#x20AC;&#x2122;Connell and Professors Stephen

# % Pennington, William Watson, Fionnuala / A 333 0 the

McAuliffe and Gil Lee described journeys undertaken been

to translate

innovative research into 0' % findings +" "

applications to

positively impact society.

They faced discussed challenges "

in commercialising research with the " % .

"

" chaired

Brendan % 0 expert panel,

by Mr 0'

Director of

& Cremen, UCD Enterprise

" including " " "

Commercialisation, Mr Damian % . CEO

Nutritics; -+ + " Oâ&#x20AC;&#x2122;Kelly, & Founder Dr Elizabeth 0' Roper, Partner,

Rock

"

$

Spring Ventures; Dr Ruth McMahon, Commercialisation Specialist,

Enterprise

Ireland Suzi Jarvis, Co-Director,

and Prof %

TCD-UCD Innovation Academy.

64 " " 0'

" sponsors

The primary of the

2013 UCD

Festival of Research $ % .

Conway & Innovation

"

were Cruinn Diagnostics Ltd, Bio-Sciences,

Ltd and

Roche Diagnostics Servier.

7 $ B . % 6 "

$ B "

7% 0 "

" 00

" B

D; N " $ $

* / 5, "

"

% 2013 Conway Festival medal winner, Nils Lambacher (left) and Prof Des Fitzgerald, UCD Vice-President for Research

Revealing chinks in the armour of hepatitis B virus Fellow, Dr Neil Ferguson provide new insights into the structure and thermodynamics of human hepatitis B virus (HBV) replication and show chinks in the armour of HBV that provide new avenues for therapeutic intervention.

00

" " (" " " "

"

%

Their published in

the - findings,

Proceedings of the National Academy of Sciences, show how subtle thermodynamic tuning of HBc structure, using point mutations or small molecules, can prevent capsid assembly and halt viral replication.

References JĂźrgens MC et al (2013). The hepatitis B virus preS1 domain hijacks host trafficking proteins by motif mimicry.

Nature Chemical Biology 9,540-547 Alexander CG et al (2013). Thermodynamic origins of protein folding, allostery and capsid formation in the human hepatitis B virus core protein. PNAS USA 110,E2782-2791 (doi:10.1073/pnas.1308846110)

' &0F "

Each year, 600,000 people die following infection with this blood-borne virus while 350 million people chronic HBV A have

infections.

1 ! 9

7

Despite HBV having only four genes, 5

%

identifying targets and

@ > , â&#x20AC;&#x2DC;druggableâ&#x20AC;&#x2122;

developing therapies have 5 5 improved

been hampered by how challenging HBV 5

$ proteins are to study in vitro. &0F

Ferguson

the

Dr is " deconstructing HBV lifecycle at molecular level

0 % to figure out how component fit together

+ Cparts

" and

facilitate HBV infected

replication 0 within

liver cells.

3

%

" "

Crucial to HBV replication are a series highly coordinated,

*D.GB,

of molecular self 0 % . One

assembly processes. key

stage is the assembly of a

protective protein shell

(virus that envelopes the viral

capsid)

"

$ genome.

Fergusonâ&#x20AC;&#x2122;s team have been able

% to disrupt the sequence of molecular " that

events make the viral core protein

capsids.

" (HBc) form " "

â&#x20AC;&#x153;Capsid formation may be an Achillesâ&#x20AC;&#x2122; heel for HBV. If you prevent the protein " " the virus

to from assembling, is unable mature and become infectiousâ&#x20AC;?, says Dr

Ferguson.

% & " forms,

After the capsid it is wrapped

of

in another shell made viral proteins "

" cells.

that help it escape from infected

. preS1,

The team " % showed that a part of

"

the virion envelope,B" is another potential therapeutic By mimicking target. "

its human counterpart, preS1 tricks the

$

host protein export machinery

" into

participating viral replication

in

but

% without unduly stressing this machinery

$

to the point of cell death.

D.GB

("

first

HBV biomolecular

% This work, the interactions mapped at atomic 4

("

resolution, the " structural and

provides

"

thermodynamic roadmap needed to

"

design antivirals. work aims novel " Future

to therapeutically preS1 functions

$ target

% without impacting normal hepatocyte .

" " functions.

" "

&0F

% B

" "

% 6 "

("

#" "

" "

" 7 % . " "

Connection between folding, allostery & 1 " 0 protein - +

capsid formation.[A]During protein folding, denatured " % HBc chains[D] associate to form an Îą-helical, dimeric intermediate[I2]. This intermediate is a structural stepping stone between alternative native dimers: N2ASS (can assemble into capsids) & N2INC (cannot form capsids). Point mutations & small molecules

? can preferentially stabilise N2INC & ablate capsid / &0F formation.[B]Crystal structures of HBc hexamer

% that corresponds to the 3-fold axis found on capsids B" /

(" +% % (blue/green in [A]) shows HBc has significant structural " F 23 3 >CC >A variation (black arrows) when crystallised under different conditions (blue & magenta).[C]Close up of HBc dimer structure from [B]showing structural variations are propagated throughout the HBc structure, in accord with the structural plasticity found by the Ferguson gp.

Hitting inflammation in the guts Researchers in UCD Conway Institute and Systems Biology Ireland have identified a way that a new class of antiinflammatory drugs may be used to treat inflammatory bowel disease (IBD).

"

;- 1BK

IBD describes a group of commonly occurring and severe diseases including

disease

$ ' Crohnâ&#x20AC;&#x2122;s and ulcerative colitis.

A A

9

A

Treatment options for the hallmark symptom of

chronically inflamed intestinal

â&#x201A;Ź"E+!+00

tissues are

limited, often ultimately 5

7 requiring surgical<

removal of inflamed

F tissue.

' (7 F

5 19.

The team led by Conway Fellow, Professor 5 $ Cormac Taylor have previously shown that a class drugs called hydroxylase inhibitors of* % : % ",

may provide a new approach to the " " "

treatment of IBD. This study now outlines how this occurs at the molecular level.

Hypoxia is a key microenvironmental feature of inflamed tissues that strongly impacts on the inflammatory processes. A family of oxygen-sensing enzymes called HIF-hydroxylases are responsible for controlling the cellular adaptive response to hypoxia.

This new*+: , study, led by Prof Cormac Taylor,

"

" and Dr +:

Scholz

Dr Eoin Cummins Carsten and published in the Proceedings of the

% . " National Academy Sciences, show

of " 2Athat hydroxylase inhibitors work by blocking a * > " @ " , pro-inflammatory pathway termed the IL-1 A " #

" " pathway; a key driver of inflammation. %

These results give significant new . -+ +

"

information as +

to a new level of % crosstalk

%

G between hypoxia and inflammation / "

" " and

provide a mechanism to explain how this new approach to the control of

inflammation may work. â&#x20AC;&#x153;This research stems from studies in our laboratory over the last decade into how cells respond to decreased oxygen levels.

We found that cells can adapt to low oxygen levels much like mountain climbers adapt to low oxygen levels at altitude.

If we use drugs to

mimic this adaptive response in inflammation, it suppresses the +:

inflammatory responseâ&#x20AC;? says Taylor.

% . "

This collaborative project is supported by

Science Foundation Ireland. "

"

"

Reference

Regulation of IL-1βâ&#x20AC;&#x201C;induced NF-ÎşB by hydroxylases

links key hypoxic and inflammatory

+: % ;signaling pathways.

Carsten C. Scholz, Miguel A. S. Cavadas, Murtaza M. % I" % Alex von B Tambuwala, Emily Hams, Javier RodrĂ­guez,

" % Kriegsheim, Philip Cotter, Ulrike Bruning, Padraic G. Fallon, Alex Cheong, Eoin P. Cummins, and Cormac T. Taylor. PNAS 2013 ; published ahead of print October 21, 2013, doi:10.1073/pnas.1309718110

5 ;$

" Addressing critical gaps in breast cancer research G

will claim

55

8 Breast cancer more than

= !

*185,000 lives by 2030 if 10 critical =

! gaps that exist in

$ breast cancer

8 !addressed. research are not urgently

5

!

published

This landmark research, 5

5

= recently in the international open

5

$ access journal, Breast Cancer Research

is a unique collaboration of over 100

G scientists,

internationally recognised / professionals G 0 " clinicians+ and healthcare

'

from the UK and

Ireland.E . *G0' ., + 1 B B " "

During 2012, the leading UK research

("

charity, Breast Cancer $

Campaign,

a series &BD+

facilitated of workshops, each

" "

% covering a specialty area of breast

cancer. Nine expert groups focused on

5

thematic

identifying key gaps in their " "

area and detailing how these gaps could % / be

addressed.

"

% 4 identified

Ten priority gaps cover areas

of genetics, prevention, diagnosis,

and support.

treatment Five key strategic

solutions were

also proposed

" " to address these gaps and help treat and "

support breast cancer patients. These include funding, research infrastructure, multidisciplinary collaborations as well as improved methods of studying the disease and designing clinical trials.

Q

" " % Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT, +

" &BD+ Professor William led "

Gallagher

who $

the working group on Novel Targeted

(" % 0

Therapies, Preclinical Research and

" " Biomarkers commented, â&#x20AC;&#x153;Overcoming

# G0' . these gap areas by prioritising this will

research reduce over-treatment of cancer 4 patients

breast by identifying those

chemotherapy " $ who will benefit from radiotherapy, " and

patient " (" or improve % . "

"

outcomes by tailoring treatment to each

" individual.â&#x20AC;?

;- + ' " "

*Projected breast cancer prevalence in the UK in 2030. " " Møller ;1 Maddams J, Utley M, H. > Projections of cancer B in the % B

"

prevalence United Kingdom, 2010-2040.

British Journal of Cancer 2012; 5 ;$

107: 1195-1202.

?!!

% % ?=3=3! 5 ;$

Critical gaps and translational priorities for the

! research B % successful prevention and treatment of breast cancer.

Breast Cancer Research 2013, 15: R92 doi:10.1186/ 5 ;$ ?

bcr3493

" "

&BD+ 5

G F% / B% + B " /% " " B 23 3 @ *C, 23 KP23C

%

The UK charity aim to raise ÂŁ100 million . G0' .

over the next decade to tackle the critical 5 ;$

;- + ' gaps identified.

In Ireland, the Irish

Cancer Society have similarly committed " " &BD+ in

the proceeds of fundraising, part due the

Cancer

$ Month

to Breast Awareness % .

$ to

campaign â&#x20AC;&#x2DC;Get the Girlsâ&#x20AC;&#x2122;, research with

" the launch of a Collaborative Cancer Research Centre, BREAST-PREDICT

"

in August

2013.

This brings together Irelandâ&#x20AC;&#x2122;s leading .

" breast cancer researchers with the ultimate goal of achieving tailored treatments for individual patients based on the characteristics of their particular tumour. This personalised medicine approach will improve outcomes for breast cancer patients both in Ireland and worldwide.

Pictured (l-r) at the launch of the Irish Cancer Society G0' . / + G Collaborative Cancer Research Centre BREAST 5 ;$

PREDICT: Mr John McCormack, CEO Irish Cancer Society, Professor John Fitzpatrick, Head of Research, Irish Cancer Society; Sharon Burrell, breast cancer survivor and Professor William Gallagher, Director of the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT

Conway Fellow and Director of the Irish < 1

H F

8 5

5

C

5 $ Conway researchers are among

"

" %

Irish Laboratory Award Finalists the finalists in the inaugural Irish G 2%A Laboratory Awards. The initiative was "

%and . celebrate

launched to promote

the

leading people and companies in

science.

Irish

" people "

in " Over 50,000 are employed % research and development, production

and processing. This industry is now - "

worth 50 billion euro to the economy.

"

"

"

: " "

The competitive application process is % 0

judged by a panel of experts drawn from

disciplines.

across a range " of scientific

" "

gala

Winners will be announced at the

in the DoubleTree

" by % dinner Hilton Hotel

Dublin on December 3rd 2013. .

" 8

Category finalists: Prof Cormac (medical

Taylor

laboratory & research laboratory of the year), Dr Alfonso

" % 0

$

Blanco (education laboratory & laboratory staff member David Croucher

" leader of

of the year), Dr (young the " $

year).

%

+

: 6' "

" "

"

"

" 7% . " "

1 " 0 &

' %

?

B

"

% 9 8 + G 4 % )+ ' : % / E / *23 3,

Developing treatments for irreversible blindness

+ ;$

that includes researchers from the or reverse these ocular diseases. For the

A new European research consortium of industry and academic partners will focus efforts for drug discovery : development

5 of novel = ! and eye therapeutics halt or reverse 5 to

7 ocular > ? diseases blindness. leading @ 5 to

8 5 A

The & Development B/ â&#x20AC;&#x2DC;Drug B. Discovery B0,0$ 75

of Novel Eye Therapeuticsâ&#x20AC;&#x2122; (3D-NET) project 9 ! 9 5 8 5 will be funded through a â&#x201A;Ź1.8 million

Marie Curie Industry-Academia Pathways

5 :

55

and Partnerships (IAPP) grant award from the EU Framework 7 Programme.

5

( :- 7 > ? $

Conway Fellow, Dr Breandan Kennedy will lead and coordinate the consortium

University of Valladolid (Spain), KalVista Pharmaceuticals (UK), RenaSci Ltd (UK) and Grupo FarmacĂŠutico . Gadea B

" (Spain).

Millions of people worldwide from

"

suffer

ocular that deteriorate their diseases

"

" quality of life. Conditions such as age "

related degeneration (AMD) or %macular 0

" + diabetic (DR)1 85 are leading 1 retinopathy B

causes of irreversible blindness, and are " F

a growing problem as the worldwide "

population ages. " "

% There is a significant unmet clinical need for more effective treatments to halt

first time, the consortium exchanges industry and academic expertise in the development drugs with " potential to

of

/ / treat and enhances " ocular

disease G"

/ % : the capabilities and infrastructure required to +

G"

test drugs relevant pre-clinical ' these E - in $

models.

$ "

F ) "

The Kennedy laboratory will contribute

"

screening

to the consortium by efficiently

on ocular

compounds for effects

and

" % angiogenesis visual function in the

zebrafish model and validate â&#x20AC;&#x2DC;hit/leadâ&#x20AC;&#x2122; drugs in pre-clinical mouse models of ocular disease.

Welcoming Conway Fellows 1 new " "

# "

Professor Guenther Eissner served as Senior Vice President and Head of Biolgical :

5 5

Research of the Italian pharmaceutical

8

company Gentium (2004-2007)

before being appointed to the Chair for :5 B0,0

C

!

Interdisciplinary Stem Cell Research at the

5 : ( :-D University of Munich. His major research 9 9

$ focus is on endothelial pathology in transplant mesenchymal B medicine and on

"

stem cells in transplant and regenerative

" " medicine.

Prof Eissner is establishing

% . " Fellows collaborative projects with Conway

% .

on lung-specific endothelial cell biology;

cells $

mesenchymal stem and hypoxia/

angiogenesis; endothelium in inflammation "

and oncology; " fibrotide and platelet

% function. + 1 + &

Professor Koelle graduated

Sabine " "

in

Veterinary at the University of J. K Medicine

" L5C:MJ. K Munich, Germany, where she also studied medicine and dentistry. She joins UCD from the University of Vienna where she was Head of the Dept of Anatomy, Histology and Embryology. Her research is focused on reproductive medicine, especially on improving the results of assisted reproduction. Prof Koelle hopes

" imaging +

" to combine modern techniques

"

with molecular analyses in proteomics / 23 3% .

and genomics to create new diagnostic

therapeutic

tools " and " and in subfertility

"

infertility.

School and Sheffield Teaching Hospitals NHS Foundation Trust. Prof Wilson will " " be investigating the pathogenesis of " "

inflammatory musculoskeletal diseases.

% His research interests include the genetic and influences rheumatoid . epigenetic

" J. K in

arthritis; ageing

and epigenetics; and "

pharmacogenetics therapies. " of biological

Prof Sabine Koelle, Professor of Anatomy & Developmental Biology, UCD School of Medicine and Medical Science

+

Professor Kenneth Wolfe joins

% &

UCD

from the

Smurfit Institute of Genetics,

Trinity

%College Dublin where he was Head of Genetics (2011-13). His research area

evolution in

" "

is genome eukaryotes with

"

aJ. K focus on yeasts as a primary system

" " to work

on. Current research projects

Wolfe

% .

underway in the laboratory are

" "

investigating gene gain*

and loss; orphan , gene

evolution; " evolution

genes; order of yeast "

"

% the MAT locus system.

Dr Darran Oâ&#x20AC;&#x2122;Connor has successfully built an independent research group within UCD Conway Institute focused on the discovery, validation and mechanistic anchoring of novel cancer biomarkers. Dr Oâ&#x20AC;&#x2122;Connor has a number of funded collaborations with Conway Fellows through the SFI strategic

Molecular " Therapeutics

research cluster,

" $

for Cancer Ireland and EU FP7 health

programme, AngioPredict.

Prof Gerry Wilson, Arthritis Ireland/UCD Chair of Rheumatology, UCD School of Medicine and Medical Science

+ 23 3 '

'

*'' ,< B E B %

D " + E " "

H + "

Professor Gerry Wilson joins UCD from the University of Sheffield. A graduate of Medicine from Queenâ&#x20AC;&#x2122;s University Belfast, he was Professor in Rheumatology and Honorary Consultant Rheumatologist at the University of Sheffield Medical

4

1

B

% 1 "

B " / .

8

Prof Guenther Eissner, Professor of Translational Systems Biology, Systems Biology Ireland

Prof Kenneth Wolfe, Professor of Genomic Evolution, UCD School of Medicine and Medical Science

* % 8 I" % ,

5 8 * % 8I" % , +

*

% I" % , "

" " % Dr Darran Oâ&#x20AC;&#x2122;Connor, UCD School of Biomolecular & Biomedical Science

! "

# $% & $ '# ()*+* ,- .,/ /.0/ 1# ()*+* ,- .,/ /.0, 2# $ $ 3