A novel platelet function test (The Platelet Monitoring Biochip)

MCT Research Talks –13th March 2017 

Jonathan Cowman reports

Cardiovascular disease (CVD) is the leading cause of death and disability in the world (approx. 1.9 million deaths per year within the EU). Platelet’s play a key role in this process and hence is why antiplatelet therapy such as aspirin is effective in reducing its incidences. Platelet function testing has a role in identifying those that are at high risk of a CVD related event (example a heart attack) and also identifying those patients that do not respond to their medication. There are a number of platelet function tests on the market however these tests suffer from a number of disadvantages such as expense, high sample volume, requirement for trained lab personal and single drug test capability.

Jonathan Cowman

My current research under the supervision of Prof. Dermot Kenny (RCSI) and Dr. Niamh Gilmartin (DCU and DIT) is to work alongside our multi-disciplinary team to produce a cost effective, rapid, small sample volume platelet function assay which can detect the effect of multiple antiplatelet drugs in a single patient blood sample. The project is known as the Platelet Monitoring Biochip (PMB). The PMB device consists of 6 micron sized fibrinogen dots, which are micro contact printed to a Zeonor (plastic) surface, a bright-field imaging system and a custom designed platelet analysis software. Blood is added to the device and rocked for 30 minutes to allow platelets to adhere to the fibrinogen dots. The device has 3 channels, a control (no agonist well), an adenosine diphosphate (ADP) well and an Arachidonic acid (AA) well which can be used to detect P2Y12 platelet inhibition and aspirin effect simultaneously. The PMB device provides a fast, easy and low cost way to determine the effectively of antiplatelet therapy against multiple agonists in whole blood. The device is currently in operation in RCSI Beaumont.

Diagnostic gene sequencing in adults with epilepsy and intellectual disability

MCT Research Talks – 20th February 2017

Sinead Heavin reports

Sinead Heavin, PhD Post-Doctoral Researcher

Epilepsy is a common neurological disorder that affects ~40,000 people in Ireland. There are many different types of seizures which are caused by uncontrolled electrical impulses in the brain. Anti-epileptic drugs control seizures for ~50% of people with epilepsy but up to ~30% of patients remain uncontrolled despite treatment with multiple drugs. Epilepsy is caused by a number of factors include stroke, trauma and infections. However, more recently we have learned that epilepsy can be caused by genetic mutations. Some epilepsies are heritable while others arise de-novo. Many patients with an intellectual disability (ID) also have epilepsy. Many of these patients lack a specific diagnosis due to limited testing and available investigations. We sequenced a cohort of 99 adult patients with epilepsy and ID on a custom gene panel of ~150 genes. A likely pathogenic variant was identified in 20 patients in 19 different genes, including SCN1A, DCX and DEPDC5, well-known epilepsy genes. Furthermore, we identified copy number variants in two patients which are likely causative. Further work is needed to investigate the phenotype-genotype correlations identified in this study and any potential treatment options that may arise.

miR-155, a master regulator of the immune response

MCT Research Talks – 13th February 2017

Dr. Claire McCoy

Background

I have recently arrived as a Lecturer in Biochemistry/Immunology within the Molecular and Cellular Therapeutics Department at RSCI. I am a dedicated and passionate Biochemist/Immunologist who obtained a BA (Mod) in Biochemistry from Trinity College Dublin in 2001.

Dr Claire McCoy

In 2006, I completed my PhD at the University of Dundee, Scotland after which I conducted my postdoctoral training in innate immunology with Prof Luke O’Neill. In 2010, I received a Marie Curie Mobility Fellowship where I gained scientific independence and re-located to the Hudson Institute of Medical Research in Melbourne, Australia. In 2014, I was awarded an Australian NHMRC project grant enabling me to lead an independent research team, conducting my research specifically on the regulation of microRNAs in innate immune cells, with a particular focus on inflammatory diseases such as Multiple Sclerosis.

Specific Research
My lab aims to understand how microRNAs regulate inflammation in disease. Our particular focus is how the pro-inflammatory microRNA, miR-155, plays a fundamental role in one immune cell subset called the macrophage. Macrophages are the sentinel cells of our immune system and quickly respond to infection to clear invading microbes. However, in chronic inflammatory diseases and autoimmunity, the presence of macrophages largely contributes to the damage, tissue destruction and symptoms associated with these diseases. Our research has shown that miR-155 is a key driver of this response. My lab aims to identify the molecular and functional mechanisms that underpin inflammatory macrophages, with the aim that miR-155 inhibition will lead to real therapeutic potential.
Multiple Sclerosis (MS) is a progressive degenerative disease where the prevalence in Ireland far exceeds the global average. Disease onset occurs between 20-40 years, an age critically affecting working and family life. To this day, there is no known cause and no cure for MS. Although, the early disease can be managed by current drug therapies, there is no treatment at the later progressive stages of disease, and no known treatments to repair the damage caused to the central nervous system. My research aims to uncover the role of macrophages in MS, and the contribution of miR-155 in this effect.

Awards
Claire McCoy is the recipient of a prestigious Marie Curie International fellowship and an Investigator Project Grant from the National Health and Medical Research Council (NHMRC), Australia. Altogether my research has attracted €800K in both national and international funding. I have published >21 highly cited and seminal publications in Nature Reviews Immunology, Nucleic Acids Research, Journal of Leukocyte Biology and Journal of Biological Chemistry. I am book editor for Springer Science, USA, as well as peer reviewer for international journals and funding agencies.

I will be talking about my research at 12pm TR4, Monday 13th Feb. The title of my talk will be ‘miR-155, a master regulator of the immune response’.

ALL WELCOME!

Dr. Claire McCoy

Lecturer in Biochemistry/Immunology,
Royal College of Surgeons in Ireland,
123 St Stephens Green,
Dublin 2,
Ireland.
Tel: 01-4025017
Email: clairemccoy@rcsi.ie

The Immune-Clock laboratory of Dr. Annie Curtis, a recent recruit to RCSI

MCT Research Talks – 30th January 2017

Last week’s departmental talks encompassed a Deep Dive into Clock biology in Macrophages affecting the Inflammatory Response. This area is the focus of the Immune-Clock laboratory of Dr. Annie Curtis, a recent recruit to RCSI.

Jamie Early, my PhD student

Jamie Early (PhD student of the Curtis Lab) currently residing in the Luke O’Neill Laboratory presented his findings on the role of the circadian clock in suppressing inflammation in macrophages and if the anti-oxidant transcription factor and redox sensor NRF2 plays a role. His talk was titled ‘The macrophage clock is a key controller of the anti-oxidant and inflammatory response via the transcription factor Nrf2’.

Second up, we had Mariana Cervantes (PhD student and visiting scientist from the Instituto Politecnico Nacional (IPN) in Mexico) present her talk titled ‘The macrophage clock is having a profound impact on mitochondrial dynamics- what are the implications for inflammation?’

Mariana Cervantes

Mariana is interested in how mitochondria alter their morphology, either fusing together to form networks or fragmenting into smaller units termed fission. She is trying to uncover if the clock is regulating this process and if so what are the implications for the inflammatory response.

This work is part of a collaboration between RCSI and  Luke O’Neill laboratory at TCD and is funded through Science Foundation Ireland

Microcalcifications in breast cancer: Exploring their molecular formation and biological significance

MCT Research Talks – 23th January 2017

Survival rates for breast cancer have risen significantly over the past few decades, in large part due to a considerable increase in the number of tumours detected via mammography at an early, more easily-treated stage. The presence of microcalcifications on a mammogram constitutes an important diagnostic clue to radiographers, with approximately 30% of invasive breast tumours and up to 90% of cases of ductal carcinoma in situ (DCIS) being detected by the presence of calcifications. Some studies have also suggested that the presence of calcifications may act as a prognostic factor, as patients presenting with breast tumours with associated calcifications have a worse prognosis than those without.
Despite their importance in breast cancer diagnosis, the exact mechanism by which microcalcifications are formed remains largely unexplored. Our group previously established the first in vitro model of mammary cell microcalcification (1) which we have recently extended to the human the breast cancer cell line MDA-MB-231. When cultured with a cocktail of osteogenic-reagents for a prolonged period, these cells produce deposits of calcium phosphate.

Figure 1. Alizarin Red S stained MDA-MB-231 cell monolayer, grown in DMEM (Control) or DMEM supplemented with osteogenic cocktail and dexamethasone (OC+Dex). Red staining indicates presence of calcified deposits.

Using a combination of histological staining, quantitative measurement of calcium content, alkaline phosphatase activity and analysis of gene expression, we can monitor the changes in cell phenotype leading to onset of mineralisation. The nature of our model allows for easy manipulation of cell culturing conditions and by adding various inhibitory compounds or cytokines to our culture media, we can identify the key pathways and targets necessary for calcification production. In doing so, we hope to build up a comprehensive understanding of the cellular and molecular basis underlying the formation of these important diagnostic clues.

Recommended reading:

Cox RF, Hernandez-Santana A, Ramdass S, McMahon G, Harmey JH, Morgan MP.  Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation. Br J Cancer. 106(3):525-37 PMID: 22233923 (Jan 2012)

Shane O’Grady, Maria Morgan

The role of the anorectic neuropeptide CART in breast cancer

MCT Research Talks – 16th January 2017

Breast cancer currently affects 1 in 8 women in Ireland, with over 3000 reported cases each year. The most common subtype of breast cancer, known as Estrogen Receptor positive (ER+) breast cancer, accounts for roughly 70% of all breast cancers diagnosed. The most common drug used to treat this disease (Tamoxifen) works by preventing estrogen from driving the growth of the cancer cells, however, roughly 1 in 3 women will be resistant to tamoxifen treatment, highlighting the need for further research into this field. A number of years ago, though mining of publically available datasets, we identified a gene known as CART to be a marker of poor prognosis in ER+ breast cancer. CART (The Cocaine- and Amphetamine-Regulated Transcript) is a neuropeptide involved in processes such as feeding and drug reward. We have identified that high expression of CART in breast cancer patients correlates with poor overall survival, and also a poor response to tamoxifen. We also demonstrated that CART could influence the activity of ERα in a ligand-independent manner [1]. Our current research focuses on combining proteomic (mass-spectrometry) and transcriptomic (RNA-seq) approaches in order to fully understand the role CART plays in ER+ breast cancer. We aim to modulate the expression of these identified targets in order to investigate whether any of these targets could slow the growth of breast cancer cells in vitro. Combining these approaches, we hope to identify novel therapeutic opportunities for patients with ER+ breast cancer.

Recommended reading:

[1] DJ Brennan, DP O’Connor et al., The Cocaine- and Amphetamine-Regulated Transcript mediates ligand-independent activation of ERα, and is an independent prognostic factor in node-negative breast cancer. Oncogene 2012, 31, 3483–3494; doi:10.1038/onc.2011.519

Brian Mooney, Darran O’Connor

The cerebrovascular nature of neurological disorders

MCT Research Talks – 9th January 2017

Guest speaker: Dr. Matthew Cambell

At MCT, we are very excited to announce our visiting guest speaker Dr Matthew Campbell from the Smurfit Institute of Genetics in Trinity College Dublin. Dr Campbell is a prestigious and young emerging principal investigator in Ireland who has made significant contributions in the fields of Neuroscience, Inflammation and the Vasculature System, core research areas that are also conducted within the MCT department.

Dr. Matthew Cambell

Dr Campbell aims to elucidate and address questions associated with dysfunctional vasculature within neural tissues. Recently published in Nature Medicine, Dr Campbell made a significant discovery uncovering the role of the NLRP3 inflammasome in the development of one of the most common forms of central retinal blindness, AMD. His lab is now pursuing a range of novel therapeutic solutions for the treatment of AMD and recently reported on the translational potential of human IL-18 as an immunotherapy.

Dr Campbell interests also focus on the blood-brain barrier, where he recently reported for the first time on the auto-regulated diffusion of amyloid-β in Alzheimer’s disease. More recently, he has identified molecular mechanisms underlying the development of chronic traumatic encephalopathy (CTE) to concussive injuries in athletes and military personnel. He spearheads a project involving the use of RNA interference (RNAi) to modulate levels of distinct tight junction proteins at the blood-brain barrier. This led to a novel form of patented technology that was termed “Neural Barrier Modulation” which could have broad applications for a range of neurological conditions.

Dr Campbell is the recipient of Ireland’s most prestigious prize for young researchers, the “President of Ireland Young Researcher Award (PIYRA)“, in addition to the international Genentech/ARVO fellowship. He will be speaking today on ‘the cerebrovascular nature of neurological disorders’ at 12 pm in Tutorial Room 2/3. Lunch will be provided for all after the talk.

All welcome!!

Written by Dr Claire McCoy, Lecturer in Biochemistry, MCT, RCSI.

Using genotype data to infer population structure and history

MCT Research Talk – 12th December

The Human Genetic Variation Research Group 

The Monday 12th December MCT Seminar Series will feature presentations from Amy Cole and Edmund Gilbert, of the Human Genetic Variation Research Group at RCSI. Led by Prof. Gianpiero Cavalleri, this research group studies large genetic datasets to investigate population structure, natural selection and the genetic basis of human disease.

Andean native in a small village on the outskirts of Cuzco

Amy Cole’s research focuses on identifying adaptive genetic variants in high altitude populations.  There are more than 140 million people living at high altitude who are exposed to two primary environmental extremes; hypobaric hypoxia and cold. At altitudes >2500 m individuals have between 11-14% effective oxygen availability, instead of the 21% available at sea level. Previous studies have identified genetic signals of selection across the genome, which have facilitated an adaptive phenotype for survival in this hypoxic environment.  Studying these indigenous high altitude populations will enable us to shed light on genes and molecular mechanisms involved in the response to hypoxia. This insight can help shed light on a number of illnesses associated with hypoxic states in low altitude populations, such as pneumonia, chronic obstructive pulmonary disease, asthma and cancer.

Research group en route to Cerro de Pasco

Today Amy presented research on a whole genome sequencing project on native high altitude Quechua individuals, recruited from the city of Cerro de Pasco, Peru, during a field trip in 2015.  Amy recently completed a three-month lab placement at MD Anderson Cancer Center with Professor Chad Huff’s research group. Here Amy performed a number of computational analyses to identify regions of the genome that are under selection in this cohort.

Edmund Gilbert’s research involves investigating the genetic structure and diversity found within the Irish. As an island population on the west of Europe, the Irish population is, from the genetic perspective, relatively homogenous compared to populations of the European mainland. As a results of this elevated homogeneity, the Irish population is well suited to studies of genetic disease. Such studies have recently shifted focus towards rare variants, which are more geographically stratified than more common variants. Therefore understanding the population structure within Ireland is key for the optimal design of genetic disease causing rare variant identification within the Irish.

Today Edmund will be presenting research investigating the extent of fine-scale population structure found within Ireland. He has been using SNP-array genotype data from the genetic ancestry DNA cohort called the “Irish DNA Atlas”. The Atlas is a cohort of individuals with Irish ancestry from three generations ago who have all eight of their great-grandparents born within 50 km. Edmund will be presenting analysis based on the suite of software known as fineStructure; investigating both fine-scale structure as well as the genetic ancestry of this structure.

Amy Cole, Edmund Gilbert, Gianpiero Cavalleri

Targeting drug resistance in neuroblastoma

MCT Research Talks 5th December 2016  rcsi-logo

Cancer Genetics Group

Neuroblastoma is a childhood cancer caused by the abnormal growth and development of neural crest cells (1). The disease commonly affects children age 5 years or younger. Approximately 50% of children have cancer cells that have migrated to distant sites in the body and formed tumour masses at the time of diagnosis. The main challenge in treating neuroblastoma is to combat tumour metastasis and development of resistance to multiple chemotherapeutic drugs. Despite major advances in available therapies, children with drug resistant and/or recurrent neuroblastoma have a dismal outlook with 5 year survival rates of less than 20%.

Research of Prof. Stallings lab is focused on elucidating the molecular events that contribute to the development and progression of neuroblastoma (2).  A major area of research involves the identification and functional analysis of microRNAs that contribute to chemotherapy resistance in neuroblastoma, along with the development of microRNA-mediated therapeutics.

The main research projects were presented at the Departmental meeting on December, 5th.

Microscopic examination of drug resistant neuroblastoma cells KellyCis83. Cells look healthy and can be kept for another 2-3 days to form a more dense population.
Microscopic examination of drug resistant neuroblastoma cells KellyCis83. Cells look healthy and can be kept for another 2-3 days to form a more dense population.

The first talk by Olga Piskareva has explored how current concepts of development of drug resistant, tumour microenvironment and cell-to-cell communication can be applied to reconstruct relapsed or drug resistant neuroblastoma microenvironment using 3D tumour models.

TEM analysis of exosome fractions. Vesicle sizes range from 30 to 200nm in Kellycis83 (A) and Kelly (B) neuroblastoma cells. (C) EVs which appear larger than the 100 nm upper limit for exosomes (D) Close up of the dried exosome preps identify the typical bowl shaped morphology associated with TEM images of exosomes. (3)
TEM analysis of exosome (EV) fractions. Vesicle sizes range from 30 to 200nm in Kellycis83 (A) and Kelly (B) neuroblastoma cells. (C) EVs which appear larger than the 100 nm upper limit for exosomes (D) Close up of the dried exosome preps identify the typical bowl shaped morphology associated with TEM images of exosomes (3).

The second talk was presented by Ciara Fallon. Ciara is our StAR PhD student. She has selected the project ‘Exosome mediated drug resistance in high-risk neuroblastoma’ as her first choice.  At the moment she is doing her lab placement in Cancer Genetics group as a part of the RCSI StAR PhD Programme. Built upon results of the former BioAt PhD Student Ross Conlon (3), Ciara’s project is focused on the validation of exosomal miR-548d-5p as a regulator of cell viability and proliferation in cisplatin sensitive and resistant neuroblastoma cell lines.

Finally, the last, but not least was a talk by John Nolan. His talk entitled “MiRNA-124-3p Reduces Cell Viability in Cisplatin Resistant Neuroblastoma Cell Models” was focused on the results submitted to the Royal College of Surgeons for the Degree of Doctor of Philosophy. His studies cover the development of cross resistance to other drugs, investigation of common altered proteins and signaling pathways in cisplatin resistant neuroblastoma cell lines and validation of miRNA that can target these proteins and stop cell proliferation. Part of the results was published last year in Cancer Letters (4).

The work carried out in Prof. Stallings lab is supported through the research grant to Prof. Ray Stallings and PhD fellowship to John Nolan by National Children’s Research Centre, Crumlin Hospital. ncrc

References:

  1. Davidoff, A. M. Neuroblastoma. 2012 Semin. Pediatr. Surg. 21, 2–14.
  2. Piskareva, O., Stallings, R. Neuroblastoma. In: Epigenetic Cancer Therapy edited by Grey S., Elsevier 2015.
  3. Conlon, R., Analysis of microRNA bearing exosomes in models of drug resistant neuroblastoma. PhD Thesis. Dublin: Royal College of Surgeons in Ireland; 2015.
  4. Piskareva, O., Harvey, H., Nolan, J., Conlon, R., Alcock. L., Buckley, P., Dowling, P., O’Sullivan, F., Bray, I., Stallings, T.L. The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro. 2015 Cancer Letters, 364:142-55.

Reported by Olga Piskareva