Conway Focus Issue 17 Summer 2012

Elaine QuinnCommunications & Education Officer

UCD Conway Institute of Biomolecular & Biomedical ResearchUniversity College Dublin

Belfield, Dublin 4Ireland

E: [email protected]: (+353-1) 716 6706F: (+353-1) 716 6701

W: www.ucd.ie/conway

Investing in Your Future

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Research by Conway Fellow, Dr OliverBlacque has revealed new informationabout a gene implicated in Joubertsyndrome and related cerebellar disorders(JSRDs), characterised by blindness, boneabnormalities, cystic kidneys,developmental delay and loss of muscletone and control. The findings of thisresearch, funded through a ScienceFoundation Ireland President of IrelandYoung Researcher Award (PIYRA) to OliverBlacque, were recently published in theJournal of Cell Biology.

One of seven genes associated with JSRDs,Arl13b codes for a protein already known toplay roles in the formation and/or functionof cilia, which are hair-like projectionsextending from the surface of the cell.However, the precise molecular details ofwhat exactly Arl13b is doing in cilia haveremained unclear.

Blacque’s team studied the gene in the ciliaof tiny worms (Caenorhabditis elegans)while collaborators in the University ofTokyo conducted parallel experiments incultured human cells. Together, theyconfirmed that Arl13b proteins uses lipidanchors to associate with the ciliarymembrane.

Much of the work on the project was carriedout by UCD doctoral candidate, SebihaCevik. She and her colleagues went on todemonstrate in C. elegans how disrupting

the function of worm Arl13b causes theciliary membrane to bulge and becomemisshapen as well as affecting the ability ofother proteins to properly distribute withinthe ciliary membrane.

They also found that a fully functionalArl13b protein is needed for the normalfunctioning of a protein transport system incilia. This intraflagellar transport system(IFT) makes contact with the ciliarymembrane. Based on these results, theyproposed a new working model for Arl13b,where it functions at the ciliary membraneto regulate important ciliary membraneproperties such as shape, transmembraneprotein distributions and IFT.

Up to only 20 years ago, many believedmost cilia to be redundant cellularorganelles that have fallen victim tomammalian evolution - a type of cellularappendix. We now know that these cellularantennae present on nearly all of our cellsand serve fundamental roles in manymotility and sensory functions, includingsignalling pathways critical to development.

It is now not surprising that defects incilium structure and function are associatedwith an ever expanding range of humandiseases and syndromes, collectively calledciliopathies, which have overlapping clinicalfeatures such as polycystic kidneys andlivers, retinal degeneration, boneabnormalities, hydrocephalus, as well as

complex traits including obesity, diabetes,mental retardation and even cancer.

Dr Oliver Blacque hopes that these findingswill ultimately lead to a greaterunderstanding not only of JSRDs, but alsoof closely related ciliopathies such asMeckel Gruber and Bardet-Biedl syndrome,as well as perhaps more commonphenotypes associated with ciliumdysfunction such as mental retardation andobesity.

He notes “That Arl13b associates with ciliarymembranes and is required for ciliumstructure/function in both worms andmammals demonstrates the remarkableevolutionary conservation of how this smallG-protein functions.”

ReferenceJoubert syndrome Arl13b functions at ciliarymembranes and stabilizes protein transport inCaenorhabditis elegans. Cevik S, Hori Y, Kaplan OI,Kida K, Toivenon T, Foley-Fisher C, Cottell D, Katada T,Kontani K, Blacque OE. J Cell Biol. 2010 Mar 22;188(6):953-69

May2010Issue10

I am delighted to welcome you to theConway Focus and look forward to workingwith you in the coming months!

As an institute with an increasinglyinterdisciplinary research focus andexpertise in specialist technology platforms,we are uniquely placed to participate in theeducation and training of the nextgeneration of research scientists. The newlylaunched PhD programme in Bioinformaticsand Systems Biology recently attracted 80EU and 120 non-EU applicants for up to 15studentships.

We introduced 4 new advanced coretechnology modules on a pilot basis thisacademic year; those covering principlesand practical aspects of imaging and appliedproteomics began in late March and will rununtil mid-June with a further two modules ingenomics and flow cytometry taking placeover week-long periods during the summermonths.

We hope to determine from this pilot ifdemand for training from within UCD as wellas from external academic centres andindustry will dictate stand-alone delivery ofthese advanced modules or inclusion within

larger programmes such as the new MSc inImaging commencing in September 2010.

I hope that a number of new initiatives,including the early-stage researcher forumdescribed in this issue, will not onlystrengthen the community of Conwayresearchers and forge new interdisciplinarylinks but will celebrate and heightenawareness of our research and innovationsuccesses.

Professor Walter KolchDirector, UCD Conway Institute

Director’s Message

Worm research reveals cilia disease gene function

UCD CONWAY INSTITUTE OF BIOMOLECULAR & BIOMEDICAL RESEARCH

Arl13b at ciliary membrane

Centre of Excellence hosts international workshop

As a European Centre of Excellence, theDepartment of Rheumatology in St Vincent’sUniversity Hospital was chosen to host aninternational two-day workshop on March26th & 27th 2010. Sponsored by RochePharma, the Participate workshop gavetwenty rheumatologists from centres inEurope and Australia the opportunity towitness the care of patients with rheumatoidarthritis (RA) at St Vincent’s.

The Participate programme focused onadvanced treatment regimens for RA in theclinic, including practical aspects of biologictherapy carried out in the accredited infusionsuite that provides optimal care for RApatients. In addition to lectures from ConwayFellows, Professors Oliver FitzGerald andDouglas Veale, delegates heard from clinicalnurse specialists about patient education,screening and monitoring of biologicaltherapies.

Addressing the international guests, Ms MaryDuff, Director of Nursing and Ms. KayConnolly, Assistant Director of Nursing for theBone & Joint Unit expressed their hope thatthe experience of the multidisciplinary teamfrom St Vincent’s would provide a template fordelegates to reproduce similar care andfacilities for the management of RA patients intheir respective countries.

Launch of Conway postdoctoral & graduate forum

A new fortnightly forum for Conway graduateand postdoctoral researchers will be launchedin May 2010. This initiative will give early-stage researchers the opportunity to talk aboutscience and issues that concern them in aninformal environment with refreshmentsprovided! Content for the forum will be

decided by the members but it is envisagedthat it will include a mix of technologyoverviews, informal and formal researchseminars, PhD viva rehearsals and careerdevelopment talks from within and beyondacademia. Forum committee members, PhDstudent Mr Thomas Schwarzl

([email protected] ) and postdoctoralresearchers, Dr David Gomez([email protected]) and Dr Craig Slattery([email protected] ) invite suggestions forthe forum schedule.

Frog peptide structure selected for journal cover

An image of the frog peptide structure centralto the work of a UCD Conway research teamwas selected as the front cover image for theApril 2010 edition of the scientific journal,Biochimica et Biophysica Acta (BBA);Proteins & Proteomics.

Peptides in the skin of many species of froghave anti-bacterial and anti-fungal propertiesas well as the ability to lyse or rupturemammalian cells. This innate immunityprotects it from attack. These properties offrog skin peptides are exciting from theprospect of being developed intotherapeutically valuable anti-infective and anti-cancer agents.

Conway Fellow, Dr Chandralal Hewage and histeam carried out molecular modelling of theXT-7 peptide and its analogue [G4K]XT-7 in a

variety of solutions in order to understand thestructural basis for the difference in biologicalactivity.

This particular XT-7 peptide, isolated from theskin of Silurana tropicalis, shows potential fordrug development as it has impressive activityagainst the growth of bacteria and Candidaalbicans. However, this potential as an anti-infective agent is restricted by its haemolyticactivity.

A change to the amino acid structure of theXT-7 peptide can create an analogue thatretains anti-infective properties but without thecomplicating haemolytic activity. The singleamino acid substitution (glycine replaced bylysine) improves the therapeutic potential ofthis naturally occurring peptide.

Cover of April 2010 edition of Biochimica et BiophysicaActa (BBA); Proteins & Proteomics.

May 2010.qxd:Layout 1 copy 07/05/2010 13:12 Page 1

Irish biopharmaceutical developmentcompany, Sigmoid Pharma recently scoopedthe overall Innovation of the Year award atthe inaugural Irish Times All-IslandInnovation Awards. The life-science SME,founded by UCD pharmacology graduate, DrIvan Coulter, has actively collaborated withConway Fellow, Professor Cormac Taylorsince being granted a Science FoundationIreland industry research partnership awardin 2004.

The Irish Times award recognises the stellarinnovative performance of Sigmoid Pharma intheir application of research and development.The company has developed two new drugtechnologies, LEDDS™ and SmPill™, which notonly enhance the solubility and permeability ofthe drug but can deliver it to targeted locationsin the gastrointestinal tract, including thecolon.

The Sigmoid Pharma-Taylor collaboration isfacilitating the development of a newtherapeutic approach for the treatment ofinflammatory bowel disease (IBD) by bringingtogether the research methodology of theTaylor group with the proprietaryliquid/emulsion drug delivery system,LEDDS®, developed by Sigmoid Pharma.

Commenting on the award, Professor Taylorsaid, “We are delighted for Dr Coulter and histeam and indeed it reflects well on ourpartnership. A key driver of successfulscientific research in 2010 is meaningfulinteraction of academia with industry that

results in both academic outputs and theapplication of research toward clinical productdevelopment with indigenous companies.”

Commenting on the Taylor collaboration, DrCoulter said, “The collaboration with the Taylorlaboratory clearly demonstrates the potentialthat can be realised through focusedapplication of basic research. The developmentof a smart economy will depend on like-mindedcollaborative partners identifying opportunitiesand realising basic and applied researchsynergies. This collaboration, initiated througha SFI research supplement, highlights thepotential that innovative indigenous companiesworking with academia can realise.”

Taylor’s collaboration with Sigmoid Pharma hascertainly been successful by these metrics withthree co-authored publications to date in two ofthe highest ranking scientific journals in thefield, Gastroenterology and Proceeding of theNational Academy of Sciences, as well as thegeneration of proprietary innovative productsand the creation of high-skilled jobs.

More than 15,000 people in Ireland andmillions of people worldwide are living with thesymptoms IBD today. Current therapeuticoptions for this chronic debilitating disease arevery limited with surgery often being the onlyviable option. The Sigmoid product, currentlyin a phase II clinical study, uniquely enablesoral delivery of a powerful drug withoutcausing systemic side-effects.

Under normal conditions, the gastrointestinal

tract is lined with cells that block the contentsof the gut from leaking into the intestine. In anIBD patient, this barrier is broken and thecontents of the gut leak out into surroundingareas. The research team in UCD Conway useda model of IBD and, by applying a new class ofdrugs known as hydroxylase inhibitors, wereable to almost completely reverse thesymptoms of the disease.

“When we applied these new drugs to our IBDmodel, the gut was tricked into thinking that itwas being deprived of oxygen. This activatedprotective pathways, which in turn preventedthe death of the cells that line thegastrointestinal tract,” explained ProfessorTaylor. “Our pre-clinical trial data can nowinform the next phase Sigmoid Pharma productdevelopment pipeline”.

In addition to the product that is currently inphase II clinical studies, Sigmoid Pharma isdeveloping a LEDDS™-enabled formulation ofthe hydroxylase inhibitordimethyloxaloylglycine (DMOG) to bring abouteffective, safe and targeted delivery of thispotential therapeutic agent.

Conway collaborator scoops Irish Times Innovation Award

Conway researchers led by Dr. TaraMcMorrow and Professor Michael Ryan havesuccessfully secured €485,500 in funding aspart of an overall award to the SystemsBiology towards Novel Chronic KidneyDisease Diagnosis and Treatment (SysKid)consortium under the European Union FP7programme.

Syskid (www.SysKid.eu) was established tofurther our understanding of chronic kidney

disease (CKD) and improve patient treatment,with particular focus on CKD arising fromdiabetes and hypertension. The multi-disciplinary research consortium has 25partners (16 universities, 9 industry partners)from 15 countries and the project is due to runfor five years.

The UCD Conway team, including postdoctoralresearchers Dr. Craig Slattery and Dr. NataliaMartin, will utilise cell culture and animal

models to identify processes involved in earlyCKD progression and investigate novelstrategies for preventing and slowing theprogression of the disease. These studies willbe integrated with clinical and epidemiologicalstudies across the consortium using systemsbiology methodologies in order to develop newdiagnostic strategies and treatment options toprevent CKD development. [email protected] for details of a PhDposition currently available.

Success in EU FP7 call

Bioinformatic analysis of HIV progression studies

Meta-analysis by UCD researchers led byConway Fellow, Professor Denis Shields hasdetermined that there is no clear evidencethat particular defective viral sequences inthe HIV-1 nef gene or certain regions of theprotein play a significant role in diseaseprogression.

HIV-infected patients can be categorised onthe basis of the number of years it takes forthem to progress to AIDS. Some patientsmaintain stable CD4 lymphocyte counts anddo not go on to develop AIDS even after morethan 10 years of infection.

Scientists are curious as to why these long-term, non- progressor (LTNP) patients do notdevelop AIDS. This may reflect differences inthe host, viral genetics or environmentalfactors that could potentially be exploited forthe treatment of the disease.

Studies in model organisms have shown thatinfection with nef-deficient virus shows anattenuated course of infection and some

studies in humans have shown that nonprogression was associated with grossdeletions in the nef gene.

However, much of the research in the area todate has been based on observational or casestudies rather than rigorous, systematicscientific evaluation. This work by doctoralcandidate Ravindra Pushker set out todetermine in a substantially larger sample setif any association exists between diseaseprogression and particular amino aciddifferences or deletions within the nef gene.

On a per patient basis, there was no excess ofLTNP patients with one or more defective nefsequences when compared to progressors.While the high frequency of amino acidreplacement at particular residues is indicativeof rapid evolvement, permutation testingshowed that residues with more replacementsthan expected were not statistically significant.

The researchers conclude that current dataprovides no evidence that individual residues

of HIV nef are critical in determining the rateof disease progression. Professor DenisShields believes that searches for modifiers ofprogression rates should now focus on otherhost and viral factors. “Systematic evaluationof large quantities of data by bioinformaticscan be just as important in refutinghypotheses that are current in the literature, asit is in discovering unknown patterns”, he said.

This research was funded through ScienceFoundation Ireland and University CollegeDublin.

Reference:Meta-analysis to test the association of HIV-1 nef aminoacid differences and deletions with disease progression.Ravindra Pushker, Jean-Marc Jacquē, Denis C. Shields.Journal of Virology, Apr 2010 p3644-3653

GlycoExtractor advances glycan data manipulation

Glycomics needs the supporting frameworkof extensive, well-curated databases andflexible analytical tools. Currently, this fieldof study lacks the established,comprehensive and centralised datacollections, standards and informationreporting protocols that exist withingenomics and proteomics.

Researchers, Dr Natalia Artemenko and DrMatthew Campbell from the National Instituteof Bioprocessing Research & Training (NIBRT)led by Conway Fellow, Professor Pauline Ruddhave developed an open access, web-basedinterface that facilitates extraction, queryingand sharing of HPLC-glycan data generated inhigh throughput processes.

Glycosylation is the most common andstructurally diverse post-translationalmodification of proteins. More than half of allgene products have been shown to beglycosylated. With many specific glycans orglycoforms showing potential as cancerbiomarkers, there is an increasing need todevelop high throughput, highly sensitive androbust strategies that detail the glycome of

cells, tissues and fluids.

Commercial packages that support HPLCinstruments cannot facilitate the extraction oflarge quantities of data. In an effort to alleviatethis bottleneck within high-throughputglycomic projects, the NIBRT team havedeveloped a web-based tool that interfaceswith the Waters chromatography softwarepackage to improve and automate the exportof data to various file formats for subsequentanalysis. The modular architecture of the toolalso facilitates the manipulation of data fromother data management systems.

The NIBRT team are now working to integrateGlycoExtractor with the EUROCarbDBframework to provide a comprehensive high-throughput HPLC data analysis platform forthe storage and annotation of experimentaldata. The integration of next generationbioinformatic tools will further thedevelopment of unified approaches forhandling large scale glycomics data initiativeswith applications in biomarker discovery

This research was partially supported by

EUROCarbDB, a research infrastructure designstudy funded by the EU 6th FrameworkProgramme. Prospective users can access theGlycoExtractor tool onhttp://glycobase.nibrt.ie:8080/DemoGlycoExtractor/mainPage.ReferenceGlycoExtractor: A web-based interface for highthroughput processing of HPLC-Glycan dataNatalia V. Artemenko, Matthew P. Campbell, and PaulineM. Rudd Journal of Proteome Research, 2010, 9 (4), pp2037–2041

NIBRT researchers Dr Matthew Campbell and Dr NataliaArtemenko who developed GlycoExtractor

Zebrafish model gives insights into diabetic blindness

New research led by Conway Fellow DrBreandán Kennedy indicates that treatmentof diabetic blindness should look atprotecting the neurons responsible for colourvision in the eye and not just targeting theblood vessels as is currently the practice.

Nearly 2.5 million people worldwide are blinddue to diabetic retinopathy. This secondarycomplication of diabetes activates the growthof new leaky blood vessels in the eye and isresponsible for the death of photoreceptors,the neurons that send visual messages to ourbrain.

Until now, it was unclear if the changes tovessels and neurons occurred independently ofone other and which type of retinal neuron is

most likely to die as a result of the raisedglucose levels seen in diabetes.

Dr Kennedy and his team found that new bloodvessels and the neuronal cell death in diabeticretinopathy can arise independently of eachother. In addition, they identified that conephotoreceptor neurons, those involved incolour vision and used in daylight, are mostaffected by the high glucose levels.

The research team made their observationsusing a novel zebrafish model of diabetes,which resembles the early stages of diabeticretinopathy in humans. It is an excitingdevelopment for the group who now hope tofurther extend their research and establish amodel of late stage diabetic disease.

Commenting on the research, Dr Kennedysaid, “By establishing a robust model for earlyand late stage diabetic retinopathy, we wouldhope to better understand the progression ofthe disease and pave the way for identifyingnew drug targets for its successful treatment”.

The research was funded through ScienceFoundation Ireland and the Health ResearchBoard.

Reference:

Predominant cone photoreceptor dysfunction in ahyperglycaemic model of non-proliferative diabeticretinopathy. Yolanda Alvarez, Jenneth Chen, AlisonReynolds, Nore Waghorne, John J. O’Connor, BreandanKennedy. Disease Models & Mechanisms (2010)




















































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Tracking the secretory pathway in cellsSecretion is a fundamental process essential to almost every cell type in the body; delivering hormones into the blood stream, digestive enzymes into the gut, and signalling molecules between cells.

Similar to any supply chain logistics, proteins and lipids are transported along a complex pathway from the point of manufacture through stages of packaging, labelling and transportation through the cell before delivery to the cell surface.

Technology did not permit scientists to catalogue this journey through cell organelles until recently. For the first time, using a combination of genetic and sophisti-cated microscopy techniques, over 8 million individual cells have been assessed to reveal those genes that influence the transport network or secretory pathway.

“This study is the first genome-wide assess-ment of the secretory process in a human cell system”, explains Conway Fellow, Professor Jeremy Simpson, UCD School of Biology & Environmental Science, co-author of the research paper published in Nature Cell Biology.

“In order for us to understand the impact on the body when this fundamental process of secretion goes wrong, we must first deci-pher the functional network of membrane trafficking pathways within the cell.

Previous studies on the secretory process have been carried out in more simplistic organisms such as the fruit fly (Drosophila) or with a more narrow focus such as on a specific group of genes. Now, using high content screening, we have been able to target 22,000 human genes and track the journey of a specific, fluorescently-tagged protein as it travels through, and out of, over 8 million individual cells.”

The UCD team, in collaboration with sci-entists in the European Molecular Biology Laboratory (EMBL) in Heidelberg, system-atically silenced each of our 22,000 genes to observe to what extent this affected the cell’s ability to transport a protein. They found that 15% of human genes somehow influence the secretory pathway including genes that provide a link to other events in and around the cell.

From analysis of more than 700,000 micro-scopy images, they found 554 proteins that influence secretion with more than 143 influencing the early stage of the secretory pathway or morphology of the Golgi, a cellular structure responsible for modifying proteins after they have been made. Membrane traffic pathways connect membrane bound organelles in a carefully ordered sequence to ensure the correct complement of proteins and lipids exists within the cell to maintain cellular balance or homeostasis.

Newly synthesised proteins and lipids in the

endoplasmic reticulum are modified as they pass along the secretory pathway through the Golgi apparatus to the cell surface.

The secretory pathway has the capacity to cope with a wide variety of cargo molecules, and as such utilises extensive regulatory ma-chinery in the process. This study focuses on particular regulatory elements in the early stage of the pathway called the cytoplasmic coat protein complexes.

The major part of funding for this research has come from Science Foundation Ireland, the EU-funded network of excellence, ‘Systems Microscopy’, and the EU-FP6 Mito-Check consortium.



















































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Director’s MessageWelcome!

This issue coincides with preparations for the Euroscience Open Forum Dublin 2012 when those who most influence science, society and policy will arrive in the capital for the largest open forum of its kind. It is anticipated that the international conference will attract more than 5,000 scientists, business leaders, government officials and media.

UCD Conway Institute will have a presence at the event as part of the University’s research showcase. We look forward to presenting the quality and breadth of our research programme as well as highlighting the core technology expertise and customised service available to academics and industrial partners in conjunction with our dedicated core technology directors and their staff.

As the international spotlight turns to focus on Irish science, I am delighted to see the impact of Conway research as our scientists continue to publish in highly regarded journals. In particular, I would like to congratulate Professor Jeremy Simpson on his Nature Cell Biology research article that is highlighted in this issue.

Professor Walter Kolch Director

The primary cilium is a highly specialised sensory and signal transduction hub that plays a central role in diverse cellular processes such as differentiation, polarity and cell cycle progression. Primary cilia are absent in several cancer cell types such as breast and pancreatic cancers.

Research from the UCD Renal Disease Research Group (www.ucd.ie/renal) led by Conway Fellow, Dr Tara McMorrow and collaborators, Professor Michael Ryan and Dr Oliver Blacque, has demonstrated that known renal carcinogens disrupt cilia structure and function in tubular epithelial cells.

In a study, recently published in the

American Journal of Physiology-Renal Physiology, the team demonstrated that exposure to carcinogens (ochratoxin A and potassium bromate) significantly reduced the percentage of ciliated renal cells. Transcriptomic analysis identified several important signalling pathways involved in mediating these effects.

The work was carried out in conjunction with scientists in Austria, Switzerland and The Netherlands as part of the EU FP6 project, CarcinoGENOMICS.

Dr McMorrow plans to expand this research by characterising mechanisms of primary cilium loss during carcinogenesis and explore the application of this research as a novel screening method for potential chemical carcinogenicity.

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Carcinogen exposure causes cilia loss in renal cells



















































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Conway scientists at every level were recognised for their research through personal and funding awards in the last quarter.

Conway Fellow, Dr John O’Connor was selected by the Royal Academy of Medicine in Ireland to deliver the 2012 Conway Review Lecture and receive the Academy silver medal in recognition of his work on the effect pro-inflammatory agents have on synaptic signalling before and during hypoxic insults to neurons.

Research Fellow, Dr David Croucher received funding under the first Science Foundation Ireland starting investigator research grant (SIRG) programme with co-funding from the International Marie Curie COFUND scheme. David will look at the fundamental behaviour of signalling pathways in breast cancer using systems biology approaches.

Research Fellow, Dr Silke Ryan won the 2012 James B. Skatrud new investigator award at the annual conference of the American Thoracic Society. Silke works on obstructive sleep apnoea syndrome

Recognising excellence in researchand the role of intermittent hypoxia in the development of cardiovascular complications in OSAS by selectively activating inflammatory pathways.

Postdoctoral Fellow, Dr Maria Prencipe won the 2012 DAMC Young Investigators Research Symposium laboratory-based oral presentation award. Maria’s research focuses on finding potential therapeutic targets for treatment-resistant prostate cancer cells and she presented work on a candidate biomarker, serum response factor (SRF).

Reference:

Simpson, J.C et al. Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway. Nature Cell Biology, advance online publication 3 June 2012, doi: 10.1038/ncb2510. Data available at www.mitocheck.org

In cells where different genes are silenced (middle, bottom), the site where the secretory processes begins (green) changes compared to normal cells (top).

Reference Radford, R et al. Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle. Am J Physiol Renal Physiol (2012) 302(8):F905-16.

Hair-like structures known as cilia (red), located on the surface of kidney epithelial cells, may be damaged or lost by carcinogen exposure.

New insight to CO2 sensing and signalling

There is a Jekyll and Hyde quality to impact of hypercapnia or elevated levels of CO2 in the body. In inflammatory conditions such as chronic obstructive lung disorder (COPD) or cystic fibrosis, hypercapnia is associated with a worse prognosis for patients as it results in them being susceptible to infection.

In contrast, hypercapnia and associated acidosis can be associated with improved patient outcome in instances where clinicians reduce mechanical damage to the lungs of patients with acute respiratory distress syndrome using a therapeutic hypoventilation strategy.

With CO2 gaining recognition as an intracellular signalling molecule that can affect both inflammatory and immune

responses, it is becoming increasing important to decipher the exact molecular pathway by which it operates.

New findings from researchers led by Conway Fellow, Professor Cormac Taylor provide the beginnings of a molecular understanding of how elevated CO2 can affect immune/inflammatory signalling.

Senior author, Dr Eoin Cummins explains, “We identified a novel modulation of a key innate immunity/inflammatory pathway (NF-κB). A key transcription factor in this pathway, RelB is processed and localised differentially under conditions of elevated CO2 that may influence NF-κB dependent signalling.”

The group used murine and human models of lung injury to show that RelB is cleaved and sent to the nucleus of cells in response to elevated arterial CO2.

Science Foundation Ireland funded this research including a short term travel fellowship to Dr Cummins to Northwestern University, USA.



















































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Reference Oliver, KM et al. Hypercapnia Induces Cleavage And Nuclear Localisation Of RelLB Giving Insight Into CO2 Sensing And Signalling. Journal of Biological Chemistry (2012) doi:10.1074/jbc.M112.347971



















































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Gremlin key to tackling pulmonary hypertensionIrish biopharmaceutical developmentcompany, Sigmoid Pharma recently scoopedthe overall Innovation of the Year award atthe inaugural Irish Times All-IslandInnovation Awards. The life-science SME,founded by UCD pharmacology graduate, DrIvan Coulter, has actively collaborated withConway Fellow, Professor Cormac Taylorsince being granted a Science FoundationIreland industry research partnership awardin 2004.


















































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UCD researchers have identified, for the first time, a molecular mechanism that accounts for the unique vascular remodelling of the pulmonary circulation in response to hypoxia or low levels of oxygen.

The findings, published in the journal, Circulation may focus efforts in tackling pulmonary hypertension, a feature of chronic hypoxic lung diseases such as chronic obstructive pulmonary disorder (COPD) and emphysema that significantly impacts on patient morbidity & mortality.

First author and PhD candidate, Edwina Cahill explains that “diseases such as emphysema, chronic bronchitis and fibrosis of the lungs cause abnormally low levels of oxygen in the lung. When the lung is starved of oxygen, blood pressure in the arteries of the lung increases causing damage to blood vessels and making it more difficult for the heart to pump blood. In many cases, this can lead to heart failure and premature death”.

While investigating the factors that contribute to high blood pressure in the arteries, the UCD team found that, with reduced oxygen levels in the lung, the body produces elevated quantities of a protein called gremlin, which suggests a link between elevated levels of gremlin and pulmonary hypertension.

The research team focused on gremlin 1 given its ability to efficiently block the actions of the BMP signalling pathway in pulmonary hypertension.

Under hypoxic conditions, gremlin 1 expression is increased as is its secretion from the vascular endothelium. It inhibits BMP signalling and contributes to the development of pulmonary hypertension.

Conway Fellow, Professor Paul McLoughlin who led the project believes that, “Given that hypoxia induced increase in gremlin 1 is selective for the lung, it offers an attractive potential target for therapy as antagonising its action may have minimal effects in other organs.”

This research was funded primarily through the Health Research Board and Science Foundation Ireland.



















































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Identifying proteins critical to bovine embryo survivalUCD researchers led by Conway Fellow, Professor Mark Crowe describe, for the first time, the protein landscape of the uterine environment in any domesticated species before implantation.

With more than 30% of reproductive failure in mammals arising due to the loss of an embryo in the preimplantation period, it is vital to understand how the uterine environment functions to support the embryo during the early establishment of pregnancy.

As the sole supply of vitamins, minerals, enzymes and other nutrients for the developing conceptus before implantation, the histotroph (uterine secretions) is critical for survival of the early embryo and may

be a promising source of biomarkers of uterine function.

Describing the study, Dr Giuliano Elia, UCD Conway Proteomics Core Director said, “Using label-free liquid chromatography-tandem mass spectrometry, we performed shotgun proteomics of the bovine histotroph proteome at two key preimplantation stages of the estrous cycle in high fertility cattle. We examined the proteomic changes occurring between days 7 and 13 to identify specific proteins likely to contribute to embryo survival.”

Functional analysis of 34 differentially expressed proteins revealed distinct biological roles believed to be involved in early pregnancy. These included

remodelling of the uterine environment for implantation; nutrient metabolism; embryo growth, development & protection; maintenance of uterine health and maternal immune modulation.

A Science Foundation Ireland strategic research centre (SRC) award funded this research.

Reference Mullen, M et al. Proteomic characterisation of histotroph during the preimplantation phase of the estrous cycle in cattle. Journal of Proteome Research (2012) 11, 3004-3018 doi/10.1021/pr300144q



















































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New test can better predict successful IVF embryos

Research from Conway Fellows, Dr Lorraine Brennan and Dr Fionnuala McAuliffe outlines a new way to measure the potential success rate of an embryo before it is transferred back into the womb during in vitro fertilisation (IVF).

The fluid within a woman’s ovaries that surrounds the egg or oocyte holds metabolic information that can improve predictions on which embryo is more likely to lead to pregnancy.

“We analysed samples of the follicular fluid surrounding the immature ovum or egg before it was retrieved for IVF,” says Dr Brennan. “There were clear metabolic differences between the follicular fluids from women who successfully achieved pregnancy as a result of IVF to the fluids from the women who did not.”

An accurate and validated method of embryo assessment and selection for transfer might allow implementation of single embryo transfer as the standard

procedure while maintaining a high pregnancy rate and minimising multiple pregnancies.

Reference Cahill, E et al. Gremlin Plays a Key Role in the Pathogenesis of Pulmonary Hypertension. Circulation (2012) Published online before print January 13, 2012, doi: 10.1161/ circulationaha.111.038125

Professor Steve Pennington pictured with Dr Ben Collins, Agilent UCD Newman Fellow (now based at the Institute of Molecular Systems Biology, ETH Zurich).



















































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It is not currently possible to predict accurately and at an early stage whether there are toxicity issues with candidate drugs. This shortcoming of existing toxicology evaluation methods can not only create a bottleneck in the drug development pipeline but can sometimes lead to the withdrawal of drugs from the market.

UCD researchers have reported in Molecular & Cellular Proteomics on a proof-of-principle study that may benefit the pharmaceutical industry in the future by providing a roadmap for large scale pre-clinical toxicology biomarker verification studies.

The study involved the molecular profiling of models that had been exposed to known toxic insults in an effort to derive the associated biomarker signatures.

Forty-eight candidate biomarkers of liver toxicity were assembled from a discovery proteomics screen of liver in a hepatotoxicant treated rat model using label free liquid chromatography mass spectrometry (LC-MS); a previous transcriptomics study of the sample and

from literature sources.

The team developed and optimised a selected reaction monitoring assay (SRM) in order to quantify the proteins in this putative biomarker panel. This revealed a panel highly enriched for proteins that had been changed significantly as a result of toxicant exposure.

Dr Ben Collins, first author and Agilent UCD Newman Fellow, explains, “The idea was to use transcriptomics and proteomics and to combine the data to provide earlier markers of toxicity. Although this study focused on one hepatotoxic compound, there is sufficient flexibility in the approach used to allow medium to high throughput for large scale verification studies involving large numbers of well-defined toxicants and ultimately for more sensitive toxicology evaluation for drugs under early development.”

Team leader and corresponding author, Professor Steve Pennington adds, “We are now working to extend this approach to more readily accessible sample types such as blood and are applying it in other studies to develop diagnostics for chronic conditions such as cancer, cardiovascular

Predicting toxicity in the drug development pipeline

disease and arthritis. With support from Agilent Technologies, we are now establishing a dedicated laboratory to undertake these SRM-based validation studies.”

Representative confocal image of immunofluorescent (FITC) staining of gremlin showing labelling in the alveolar wall is suggestive of localisation predominantly in the capillary endothelium.

Reference Collins, BC et al; Development of a pharmaceutical hepatotoxicity biomarker panel using a discovery to targeted proteomics approach. Molecular & Cellular Proteomics (2012) doi: 10.1074/mcp.M111.016493

Reference Wallace M et al. An investigation into the relationship between the metabolic profile of follicular fluid, oocyte developmental potential and implantation outcome. Fertility and Sterility 97; 5, 1078-1084.e8 May 2012



















































Reference:



Nature Communications; from bovines to brainsProblems with diagnosing bovine TB in cattle infected with the common livestock disease, liver fluke, may be contributing to the spread of bovine TB, according to new research published in Nature Communications involving Conway Fellow, Professor Grace Mulcahy.

A study of 3,000 dairy herds in England and Wales found that liver fluke infection reduces the sensitivity of skin tests used to diagnose bovine TB (BTB).

Consequently, cattle infected with both liver fluke and BTB may not be identified by existing bovine TB surveillance schemes and may continue transmitting BTB if they are moved from their farm of origin.

In the same issue, a collaborative research study that included Conway

Fellow, Professor Sean Callanan outlined a method of safely manipulating blood vessels in the brain to reduce cerebral oedema.

By allowing periodic opening of tight junction channels between cells lining the blood vessels in the brain, the blood vessels in the brain become marginally and reversibly permeable to tiny molecules. This allows the fluid in the brain, largely comprising water, to efficiently drain back into the blood.

The team also observed cognitive improvement in murine models of focal cerebral oedema after administering short interfering RNA directed against claudin-5.

These observations may have consequences for early intervention in cases of traumatic brain injury, or indeed any neurological condition where cerebral

H&E stained section showing a region of cerebrum revealing cerebral damage to the level of the hippocampus. Courtesy of Brian Cloak, UCD

Reference Claridge, J et al. Fasciola hepatica is associated with the failure to detect bovine tuberculosis in dairy cattle. Nature Communications 22 May 2012 doi: 10.1038/ncomms1840

Campbell, M et al. Targeted suppression of claudin-5 decreases cerebral oedema and improves cognitive outcome following traumatic brain injury. Nature Communications 22 May 2012 doi: 10.1038/ncomms1852

oedema is the hallmark pathology.

Documents

Conway Focus Issue 17 Summer 2012