A new strategy to study neuroblastoma

MCT Research Talks

Neuroblastoma is a cancer of the nervous system that primarily affects children aged 5 and younger. Although neuroblastoma accounts for only 5% of childhood cancers, it is responsible for approximately 15% of childhood cancer deaths. For children with high-risk neuroblastoma – children in which cancer has spread significantly – the outlook is extremely poor. Approximately 1 in 5 of these children will not respond to treatment, and of those that do, 50% will develop drug resistance leading, in many cases, to death.

Dr Olga Piskareva, an NCRC supported scientist and Honorary Lecturer at RCSI, has recently published a study describing a new way to grow cancer cells in the lab. Traditionally, researchers grow cancer cells in the flasks on the flat surface. This is not the way cells grow in the human body. Dr Piskareva in collaboration with Dr Curtin and Prof O’Brien has designed a new way to grow cancer cells that recreate their growth in 3 dimensions as in the human or mice body. They used special cotton wool like sponges as a new home for cancer cells and populated them with cancer cells. At the next step, they gave cells the drug at the different amount and checked what happened. In this system, cells responded only to the drug at doses used in the clinic or mice models.

This new strategy to grow cells on sponges should help to understand cancer cell behaviour better and accelerate the discovery and development of new effective drugs for neuroblastoma and other cancers. This, in turn, will make the outlook for little patients better and improve their quality of life.

Dr Olga Piskareva and her research group

More details about our research can be found in Dr Piskareva’s blog!

Reported by Olga Piskareva

The Irish DNA Atlas

The Irish DNA Atlas, a study of Irish genetic history and diversity led by researchers at the Royal College of Surgeons in Ireland (RCSI) and the Genealogical Society of Ireland (GSI), has recently published in findings into the genetics of Ireland in the Nature Publishing journal Scientific Reports (The Irish DNA Atlas: Revealing Fine-Scale Population Structure and History within Ireland). The Irish DNA Atlas is a cohort of individuals with four generations of ancestry from specific regions in Ireland, recruitment is organised and managed by Seamus O’Reilly at the GSI. Mr O’Reilly helps potential recruits finish, or double-check, family history and pedigree charts for the recruitment process, and mails out sample kits and paperwork for their return to RCSI.
The researchers, led by Professor Gianpiero Cavalleri at RCSI, have found; i) different groups of Irish individuals, clustered by genetic similarity alone; ii) the genetic differences between these groups are incredibly small, iii) members of each of these groups share ancestries from similar regions in Ireland (see image below); iv) a migration event(s) is observed in the north of the island of Ireland that dates somewhere in the 17th and 18th centuries and is from Britain; v) a number of genetic barriers within in Ireland, notably; in the north, and between Leinster and Munster; and finally vi) a significant level of Norwegian-like genetic ancestry throughout Ireland is observed for the first time and this is associated with a genetic migration into Ireland around the turn of the first millennium.
Using the Irish DNA Atlas in conjunction with a dataset of British individuals with regional ancestry (the People of the British Isles Study) the project was able to clusters 2,103 individuals from Ireland and Britain based on genetic similarity as 30 distinct genetic groups (see image 1 for clusters within Ireland). People within the same group are more genetically similar to each other than they are to individuals in other groups. When each Irish individual is colour coded by the group and is placed on a map based on where their great-grandparents were born, we generate a map shown below. Shown to the left are the geographic spread of the identified clusters and on the right a map of Irish kingdoms that represent proto-Provinces circa 800AD.

Analysing the Atlas, the broadest groups within Ireland are either; nearly 100% made up of Irish/Northern Irish individuals (i.e. from the island of Ireland), or are a mix between Irish and mainland British individuals. In the case of the latter, this suggests that those (Irish and mainland British) individuals have shared Irish and British genetic ancestry. The Irish individuals within these mixed groups are mainly from the north of Ireland (predominantly those who are blue crosses in the image above), and the British members are predominantly from the north of England and the south-west of Scotland.
These groups/clusters of near 100% Irish membership are interpreted as mainly ‘Gaelic’ Irish, and the genetic differences between these groups are incredibly small. The groups/clusters are grouped geographically and most are remarkably faithful to the boundaries of the Provinces in Ireland (shown on the left map). We compare these clusters and kingdoms from around 800AD in the above image for illustrative purposes. The reflection between the genetic and historical groups suggests that these Provinces and the kingdoms they represent have subtly impacted the genetic landscape of Ireland. Of particular note is within Co. Clare, which has historically been both parts of Munster and Connacht. Individuals with ancestry from Co. Clare reflect this by showing a mix of genetic groups found within both Munster and Connacht.
In addition to identifying different genetic groups within Ireland, the research sought to investigate whether previous migrations into Ireland had a detectable genetic impact on the genetics within Ireland. Having already identified groups of Irish individuals mainly in the north of Ireland who appeared to a mixture of Irish and British genetics, the researchers tested whether this could be due to a specific event creating these mixed groups. They estimated that these mixed groups are from a number of admixture events in the past, dating around the 17th and 18th centuries.
As well as migrations from Britain, the researchers asked whether evidence of migrations from wider afield, i.e. from continental Europe, could be found. A surprisingly larger amount of Scandinavian – specifically Norwegian – looking ancestry in all our Irish clusters was detected (see below image). This image shows along the horizontal axis each of the 30 genetic groups identified in Ireland and Britain. Along the vertical axis is the average proportion of the genome that’s the closest similarity is found in each of the 10 reference European populations. Ireland and Wales share a lot of French-like ancestry, but Ireland shows a lot of Norwegian-like ancestry compared to England or Wales. In fact, in this Norwegian respect, Ireland shows a similarity to Orkney.

This similar pattern of elevated Norwegian-like in Ireland and Orkney is interesting as Orkney is a region with strong evidence of Norwegian Viking genetic migration and mixture. Therefore the researchers investigated whether this Norwegian ancestry in Ireland was due to a mixture event dating from the time of the Viking activities in Ireland. They dated the ancestry to sometime around 1000 AD, which agrees with a ‘Viking Hypothesis’. This result was perhaps the most surprising using the Irish DNA Atlas, as previous work with Y-chromosomes found no evidence of Norse genetics within Ireland. However now, with whole-genome data, the extent of Norwegian mixture within Ireland is able to be shown.
This research has been funded through a Career Development Award from Science Foundation Ireland. RCSI is ranked among the top 250 (top 2%) of universities worldwide in the Times Higher Education World University Rankings (2018) and its research is ranked first in Ireland for citations. It is an international not-for-profit health sciences institution, with its headquarters in Dublin, focused on education and research to drive improvements in human health worldwide. RCSI is a signatory of the Athena SWAN Charter.

Reported by Edmund Gilbert

A great week for Body Clock Research in MCT RCSI

Last week was another superb week for circadian research in the Molecular and Cellular Therapeutics Department. The Curtis Laboratory published our first big paper on the immune body clock in Nature Communications. This study originated back in 2013. I was still a postdoc in Prof. Luke O’Neills laboratory at Trinity College and was intrigued by some of the studies that showed that multiple sclerosis (MS) was affected by the circadian disruption. A key study showed that teenagers who work shift work before the age of 18 are more susceptible to multiple sclerosis in later life. I wondered if we would see any differences in multiple sclerosis if we disturbed the immune body clock. I approached Prof. Kingston Mills also at Trinity College, who is one of the world leaders of multiple sclerosis and has a key mouse model that recapitulates certain features of MS, called experimental autoimmune encephalomyelitis (EAE). The first experiment we conducted was to see if a mouse which does not have the molecular clock in macrophages was more susceptible to disease, and low and behold it was! This project was driven by one of the most talented researchers that I have ever had the pleasure of working with, Dr. Caroline Sutton, who is a senior postdoctoral fellow in Prof. Mills lab. This project is a great example of collaboration between multiple labs, Mills, O’Neill and my own new group here at RCSI.

And if that wasn’t enough! We also hosted the circadian expert Prof. Qing-jun Meng for our second institutional seminar series on Thursday. Prof. Meng is a world expert on clocks in the musculoskeletal system at University of Manchester. I met Qing-jun in 2013, and have followed his research intensely. He has made seminal discoveries on the impact of the clock on cartilage and invertebral disk function and how this leads to diseases of ageing, such as osteoarthritis and lower back pain. He had the audience enthralled for an hour with his rhythmic images of cells glowing with 24-hour rhythms, and his use of Google searches. It was an absolute pleasure to have Qing-jun with us for the day, and I hope that we can have him back again in the near future.


Some news features on the article can be found here:




Written by Annie Curtis

A possible therapeutic avenue in Cystic Fibrosis

Cystic fibrosis (CF) is an inherited chronic disease that primarily affects the lungs and digestive system. CF is caused by mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene, a chloride channel responsible for helping conduct chloride and other ions across epithelial membranes. The loss of a functional CFTR channel disrupts ionic homeostasis resulting in mucus production that clogs the lungs and pancreas and results in a vicious cycle of chronic infection and inflammation as the disease progresses.

There are almost 2,000 different variants in the CFTR gene and 70 % of CF patients contain a mutation at position 508, which results in the loss of Phe508 and disruption of the folding pathway of CFTR. ΔF508 CFTR is a trafficking mutant that is retained in the endoplasmic reticulum (ER) and unable to reach the plasma membrane. Efforts to enhance exit of ΔF508 CFTR from the ER and improve its trafficking are of utmost importance for the development of treatment strategies. Clinically, progress has been made in recent years identifying therapeutics that target CFTR dysfunction in patients with specific mutations. However, small molecules that directly target the most common misfolded CFTR mutant, ΔF508, and improve its intracellular trafficking in vitro, have shown modest effects We performed a study aimed to identify new therapeutic targets that will help address the unmet clinical need for CF patients homozygous for  the ΔF508 mutation.We aimed to understand the protein interactions regulating CFTR transport using mass spectrometry-based proteomics. Using mass spectrometry based protein interaction profiling and global bioinformatics analysis we revealed mammalian target of rapamycin (mTOR) signalling components to be associated with ∆F508 CFTR.  Our results showed upregulated mTOR activity in ΔF508 CF bronchial epithelial cells. In addition to a well described role in several cancer subtypes, excessive activation of the mTOR pathway has been reported to be involved in age-related misfolding diseases. There are a range of inhibitors that target the PI3K/Akt/mTOR pathway and after screening a selection of inhibitors, we identified 6 different inhibitors that demonstrated an increase in CFTR stability and expression. Mechanistically, we discovered the most effective inhibitor, MK-2206 exerted a rescue effect by restoring autophagy in ΔF508 CF cells. These findings highlight this pathway as a possible therapeutic avenue worth further exploration in Cystic Fibrosis.

Judith Coppinger and her team: Mark Ward and Zivile Useckaite

We aimed to understand the protein interactions regulating CFTR transport using mass spectrometry-based proteomics. Using mass spectrometry based protein interaction profiling and global bioinformatics analysis we revealed mammalian target of rapamycin (mTOR) signalling components to be associated with ∆F508 CFTR.  Our results showed upregulated mTOR activity in ΔF508 CF bronchial epithelial cells. In addition to a well-described role in several cancer subtypes, excessive activation of the mTOR pathway has been reported to be involved in age-related misfolding diseases. There are a range of inhibitors that target the PI3K/Akt/mTOR pathway and after screening a selection of inhibitors, we identified 6 different inhibitors that demonstrated an increase in CFTR stability and expression. Mechanistically, we discovered the most effective inhibitor, MK-2206 exerted a rescue effect by restoring autophagy in ΔF508 CF cells. These findings highlight this pathway as a possible therapeutic avenue worth further exploration in Cystic Fibrosis.

This study was a collaboration between several groups at University College Dublin, Cystic Fibrosis Unit, St Vincent’s Hospital, Royal College of Surgeons in Ireland, Beaumont Hospital and the University of Mainz, Germany. Ongoing work in this area is taking place at the National Children’s Research Centre. Further details can be found here in a recent publication on this work.

Teasing out the mechanisms of biofilm formation for the treatment of S. aureus infections on indwelling devices: the role of the surface protein SdrC

Antibiotic resistance has become a great challenge in the healthcare setting. In particular antibiotic resistant strains of Staphylococcus aureus pose further challenges. Methicillin resistant S. aureus (MRSA) is widespread in healthcare facilities and in the wider community and multi-drug resistant strains have been identified. S. aureus is normally present on the surface of the skin where it causes no harm. However, it can easily colonize open wounds causing infection. Systemic infections can result from these wound infections leading to severe problems such as sepsis and infective endocarditis. Infection after surgical implantation of devices, such as joint replacements, can result in the formation of biofilms coating the devices that are difficult to treat. Biofilms are an accumulation of bacteria on a surface which often persist as most antibiotics do not easily penetrate them. In biofilms, bacteria interact directly with the foreign surface, with host proteins coating the surface and can also accumulate through interactions directly with each other.

Thus, there are multiple mechanisms involved in biofilm formation. It is important to fully understand all the mechanisms of biofilm formation in order to be able to disrupt their formation and persistence. In our recent paper, we have characterized the direct binding interaction (cell-cell adhesion) through the S. aureus surface protein, serine-aspartate repeat protein C (SdrC). Our study also reveals the mechanism of interaction between SdrC and inert surfaces. Furthermore, we have demonstrated how a small peptide can be used to block these interactions preventing biofilm formation suggesting a possible approach that could be used to treat SdrC dependent S. aureus biofilms. This study is the result of a multi-disciplinary collaboration across research institutes in Ireland and Belgium with Dr. Brennan (RCSI) contributing to the molecular modelling, Prof. Joan Geoghegan, Prof. Timothy Foster and Leanne Hayes (Trinity College Dublin) leading the molecular biology and microbiological functional studies and researchers from University Catholique de Louvain characterizing the interactions quantitatively using atomic force microscopy.

Marian Brennan

More details can be found in “Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC”.


MCT researchers shed light on the ancestry of the Irish Travellers from the perspective of DNA

Edmund Gilbert reports

A new study, led by Prof. Gianpiero Cavalleri at MCT and Prof. Jim Wilson at the University of Edinburgh, has examined the population history of the Irish Travellers and has confirmed that the Irish Travellers share a common Irish origin with the settled Irish population. The work has also for the first time estimated the date which this divergence occurred.

A roadside camp in County Mayo 1972. Courtesy of George Gmelch

The Irish Travellers are a small nomadic population, making up about 0.6% of the total population on the island of Ireland, or between 29,000 and 40,000 individuals. Within the population cousin marriages (consanguineous marriages) are common, and the population is socially isolated from the surrounding settled Irish population.
The researchers, who also include MCT PhD student Edmund Gilbert, Shai Carmi of the Hebrew University of Jerusalem, and Sean Ennis of University College Dublin, used SNP-array based genotype data to compare the population genetics of the Irish Travellers to neighbouring Irish and British populations, as well as world-wide groups and European Roma Gypsies.
The study found that although the Irish Travellers were genetic closest to the settled Irish population, they showed significant differences. The study also confirmed the lack of recent shared genetic ancestry between the Irish Travellers and Roma Gypsies. The Irish Travellers, therefore, represent a subset of Irish genetic diversity, and the significant differences can be attributed to genetic drift, brought on by hundreds of years of genetic isolation and a decreasing population size. The analysis showed Irish Travellers also exhibit within-population sub-structure with four apparently distinct groups emerging, and interestingly these groups mirror different forms of the Shelta language and sociological groups within the Irish Travellers.

Galway John Ward making tinware and Galway 1971. Courtesy of George Gmelch

The dating of the origin of the Irish Travellers is of considerable interest, but this is a distinct date from the genetic origins of each population. This study has estimated a time of genetic divergence of the Irish Travellers and the settled Irish population using genomic tracts of shared identity. This method estimated the divergence to about 12 generations (360 years) ago, which is far older than common belief that the Irish Traveller population arose from the time of the Great Famine. The size of the dataset limited the authors to exploring the relatively simple model of one divergence event, future work is required to expand the study to explore more complex demographic models. The Irish Traveller population was shown to have high proportions of the genome where both maternal and paternal copies are identical, at similar levels to other consanguineous populations around the world.
The research was also welcomed by author and Traveller activist, Michael McDonagh said, “As a Traveller who has spoken on the history and identity of Irish Travellers to many groups ranging from children to academics, you sometimes rely on anecdotal information in trying to put across a serious message about Irish Traveller history. I am delighted that now we have qualified evidence that substantiates the argument I have made for many years, which is that Travellers did not descend from the Famine in Ireland. This research allows us to bring Irish Traveller history back many and gives us a factual identity.”

Recent MCT Papers In High Impact Journals With Commentaries

CNV and Schizophrenia Working Groups of the Psychiatric Genomics Consortium; Psychosis Endophenotypes International Consortium. [including Waddington J]. Contribution of copy number variants to schizophrenia from a genome-wide study of 41,321 subjects. Nat Genet. 2017 Jan;49(1):27-35. PMID: 27869829

John Waddington writes about his group’s participation in a global research endeavour that has produced a series of articles in Nature, Nature Genetics and Nature Neuroscience on the pathobiology of schizophrenia The Psychiatric Genomics Consortium (PGC) unites investigators around the world to conduct meta- and mega-analyses of genome-wide genomic data on psychiatric disorders, to achieve goals that cannot be reached by individual or indeed most national programmes. Its website (www.med.unc.edu/pgc) provides information about the organization, implementation and results of the PGC. This consortium began in early 2007 and has rapidly become a collaborative confederation of investigators from 38 countries. There are samples from more than 900,000 individuals currently in analysis, and this number is growing rapidly. The PGC is the largest consortium and the largest biological experiment in the history of psychiatry.
From 2007-11, the PGC focused on several disorders, from schizophrenia through to autism, attention-deficit hyperactivity disorder, bipolar disorder and major depressive disorder. These now extend to large studies of eating disorders, substance use disorders, obsessive-compulsive disorder/Tourette’s Syndrome and post-traumatic stress disorder. Initially, the PGC focused on common single nucleotide polymorphisms (SNPs) but has expanded to include copy number variation (CNVs) and uncommon/rare genetic variation. The PGC has received funding from many sources. It has relied heavily on the goodwill of its members and their donated effort, with the establishment andgenotyping of its primary studies funded by a wide range of national, international and commercial entities.

We have been involved in the Schizophrenia Working Group of the PGC for many years, from its progenitor organisation the International Schizophrenia Consortium, primarily through substantial funding from the Wellcome Trust for five centres across the island of Ireland (one led by us) to contribute DNA from well characterised patient populations to a national dataset curated at Trinity College Dublin. This dataset has then been shared with the PGC. The resultant studies have evolved from the first investigation of CNVs in schizophrenia (3,391 cases, 3,181 controls; Nature 2008; 455: 237-41), through to the largest GWAS study on schizophrenia undertaken to date (36,989 cases, 113,075 controls; Nature 2014; 511: 421-7), to this most recent publication (21,094 cases, 20,227 controls; Nature Genetics 2017; 49: 27-35), the largest study of CNVs in schizophrenia yet undertaken. This has been, and continues to be, an exemplary, rewarding journey of global collaboration to illuminate the pathobiology of the intractable human condition that is psychotic illness.


Chion A, O’Sullivan JM, Drakeford C, Bergsson G, Dalton N, Aguila S, Ward S, Fallon PG, Brophy TM, Preston RJ, Brady L, Sheils, O, Laffan M, McKinnon TA, O’Donnell JS. N-linked glycans within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance. Blood. 2016 Oct 13;128(15):1959-1968. PMID: 27554083.

Jamie Marie O’Sullivan writes about this study:

Von Willebrand factor (VWF) is a large multimeric plasma sialoglycoprotein that plays two critical roles in normal haemostasis. First it mediates platelet adhesion to exposed subendothelial collagen at sites of vascular injury. Second, VWF acts as a carrier molecule for procoagulant factor VIII, thereby protecting it from premature proteolytic degradation and clearance. Deficiency of either VWF (von Willebrand disease, VWD) or FVIII (Haemophilia A) is associated with a significant bleeding phenotype. Conversely, elevated plasma levels of the VWF-FVIII complex constitute a dose dependent risk factor for both venous and arterial thrombosis. Consequently, understanding the factors that determine how plasma VWF levels are regulated is not only of basic scientific interest, but also direct clinical importance.

Despite the fact that type 1 von Willebrand disease constitutes the most common inherited bleeding disorder worldwide, the mechanisms underlying the reduced plasma VWF levels in these patients is not well established. However, emerging evidence suggests that reduced circulatory half-life may be important in a significant number of patients. However the biological mechanisms underlying VWF clearance from the plasma remains elusive. Work is on-going within the group of Professor James O’Donnell to investigate the importance of specific VWF domains in modulating half-life in vivo.

In the October issue of Blood the group published novel findings demonstrating a critical role for VWF glycosylation in determining its circulatory half-life. By examining the clearance of various truncated fragments of VWF, we observed that the A domains of VWF contain a receptor-recognition site important in mediating VWF binding to macrophages in vitro, and in regulating VWF clearance by macrophages in vivo. Furthermore site-directed mutagenesis demonstrated a key role for N-linked glycan structures at position N1515 and N1574 within the A domains in protecting VWF against macrophage-mediated clearance. More specifically, the protective effect of these large complex N-linked glycan structures may be due to steric hindrance, with shielding of cryptic binding sites for macrophage clearance receptor low density lipoprotein receptor-related protein 1 (LRP1).

Defining the mechanisms underpinning VWF clearance is not only of direct translational relevance, but may also have important implications for the development of novel therapeutic agents with significant commercial appeal. For example, the estimated cost of clotting factor concentrates to the global market is predicted to exceed €10 billion per annum by 2020. It is therefore perhaps unsurprising that major pharmaceutical companies (including Baxter®, Bayer®, Novo Nordisk® and Octapharma®) are actively pursuing development of longer-acting coagulation factor products (including VWF and FVIII) as a priority. Our novel findings further scientific understanding in relation to the molecular mechanisms involved in regulating coagulation glycoprotein clearance from plasma.

McGettrick AF, Corcoran SE, Barry PJ, McFarland J, Crès C, Curtis AM, Franklin E, Corr SC, Mok KH, Cummins EP, Taylor CT, O’Neill LA, Nolan DP. Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion. Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):E7778-E7787. PMID: 27856732.

Dr. Anne McGettrick and Sarah Corcoran at Trinity College Dublin lead a team of researchers including Dr. Annie Curtis (now at MCT) on a study that uncovered how the African parasites trypanosomes evade the immune system in their hosts, The biochemists have unearthed a metabolic by-product of trypanosome activity known as indolepyruvate modulates the inflammatory response of the host and evades immune detection. This discovery may offer excellent possibilities for developing anti-trypanosome drugs and therapies because inhibiting the production of indolepyruvate may be key in fighting the parasite. Full details can be found here https://www.tcd.ie/news_events/articles/solving-a-putrid-camel-pee-riddle-may-aid-millions-affected-by-sleeping-sickness/7385