Cystic Fibrosis (CF) is a progressive, genetic disease that causes persistent lung infections and limits the ability to breathe over time. CF is caused by mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene which encodes a chloride channel responsible for helping conduct chloride and other ions across epithelial membranes. Loss of functional CFTR channel disrupts ionic homeostasis resulting in mucus production that clogs the lungs and results in a vicious cycle of chronic infection/inflammation. 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 and function correctly as a chloride channel. The Coppinger research lab is focused on understanding the basic mechanisms of CF disease with a focus on the ΔF508 mutation and translating these findings into diagnostics/therapies. We are particularly interested in two areas of research 1. Using basic science technologies to identify novel signalling pathways in CF to discover new CFTR corrector therapies in ΔF508 CF models. We have recently discovered the PI3K/Akt/mTOR signalling pathway to be dysregulated in CF models and a possible therapeutic avenue worth further exploration in CF. Additionally, we are interested in 2. Investigating how diminished ΔF508 CFTR activity leads to heightened inflammatory cell recruitment and CF airway pathogenesis. Exosomes are nanovesicles (40–100 nm) actively secreted by cells and are crucial mediators of intercellular communications. We hypothesised that exosomes may be released from ΔF508 CF patient bronchial cells/fluids and play a role in regulating immune cell function. Preliminary data has confirmed this hypothesis and also indicated exosomal signatures may possibly serve as markers of disease progression in CF. These studies are in collaboration between several groups at the National Children’s Research Centre, Royal College of Surgeons in Ireland, Beaumont Hospital University College Dublin, Cystic Fibrosis Unit, St Vincent’s Hospital.
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:
Dr Justyna Surowka, Medical University of Lublin, Lublin, Poland
(Current Erasmus Post-doc with the O’Connor group) presented “Assessment of chosen immune cell populations in patients with ovarian cancer”
Despite the decades of studies on developing new therapeutic strategies, ovarian cancer remains one of the malignancies with the highest mortality rate. Therefore, new therapies, among them immunotherapy, are in demand. Recently, Kurman and Shih proposed a new classification of ovarian cancer. It is based on molecular and histopathological differences between tumors and divides them into two subtypes: type I and type II ovarian cancer. However, there are no studies exploring functions of an immune system in those types of ovarian cancer. We demonstrated that each type of ovarian cancer can induce a unique phenotype of dendritic cells and differentiation of Tregs, both associated with immunosuppressive function, which may be an obstacle while developing effective anticancer dendritic cell vaccination.
Dr Sudipto Das presented “Dissecting the epigenome of metastatic colorectal cancer”
The talk highlighted the experimental and analytical pipelines that have been established in the lab in order to develop single-base pair resolution DNA methylation maps derived from difficult-to-handle FFPE (Formalin Fixed Paraffin Embedded) tissue. We next applied these optimized approaches to primary tumour samples derived from 58 metastatic colorectal cancer (mCRC) patients and 10 matched normal samples, with an aim to unravel the methylation alterations across both conventional gene regulatory regions such as promoters as well as alternative regulatory elements such as enhancers of protein-coding and non-coding genes. Intriguingly, we have now identified a DNA methylation specific signature consisting of 377 differentially methylated loci that differentiates tumour and normal and in parallel provides us with three distinctive clinical clusters, which show a significant overlap with prognostically relevant consensus molecular sub-types of CRC. However, further work is warranted to ascertain the precise function of the signature as well as their role in predicting patient response to treatment.
The second part of the talk detailed about the ongoing genomics focused on “n-of-1” genomic studies which essentially involves atypical cancer presentation in patients, with the idea of understanding the biology of such unusual clinical phenotypes and moreover to identify any potential therapeutic targets.
On November 14th, we welcomed almost 50 secondary school students at our Department for Lab Safari. The event was designed to encourage young people to consider a career in Science, Technology, Engineering, Maths and Medicine through hands-on experience and demonstrations prepared by our researchers. We developed 6 different workstations focused on Cancer biology and biomarkers, Drug Discovery, Multiple Sclerosis, Human Genetics and Immunology/Body clock
The event was opened by Prof. Tracy Robson, Head of MCT, sharing her career path in research and lessons that she learnt. Dr Avril Hutch, Head of RSCI Equality and Diversity Unit, also spoke about stereotypes in STEMM careers and having an awareness of unconscious bias.
Our workstation was led by Caragh Stapleton, Katherine Benson and Edmund Gilbert, centered around human genetics. Our activity set out to teach participants about inherited traits and demonstrate how variation in our DNA influences our physical attributes. We investigated a number of traits including PTC taster (using PTC taste strips), colour blindness, widows peak, tongue rolling, attached earlobes, bent little finger, eye colour and red hair. Each participant noted whether or not they had the given trait and we then discussed the hypotheses of the genetic variants influencing the different traits.
Our workstation was led by Olga Piskareva and John Nolan. We explained the concept of biomarkers and the importance of discovering novel biomarkers for neuroblastoma, a childhood malignancy. Various chromosomal aberrations can be biomarkers of neuroblastoma aggressiveness. One of the strongest predictors of rapid neuroblastoma progression is MYCN status. We selected several neuroblastoma cell lines with known MYCN status providing a good illustration of biomarker’s quantity. Using immunodetection, we visualised the differences in the MYCN presence.
Our workstation was led by Annie Curtis, Mariana Patricia Cervantes Silva, George Timmons and Cathy Wyse. The theme of our activity was on the body clock and immune function. We discussed with the students why they get jet lag and what that has to do with their body clock. Students then moved to the first station where they got a chance to add colouring to macrophages, so we had red, yellow, blue and green macrophages and were able to look at their coloured macrophages under a microscope. Then they moved to the next station where they got to see the master clock which resides in the hypothalamus of the brain under a microscope. Finally, we displayed some images of activated macrophages and explained their function.
Cancer Cell Biology
Our workstation lead by Sudipto Das, Gillian Moore and Stephanie Annett, focused on showcasing the various laboratory-based approaches applied regularly to identify and investigate novel gene or protein-based biomarkers of cancer progression. Within our workstation, we highlighted three key areas including how samples following biopsy from a cancer patient are used to construct tissue microarrays which are used for assessing the importance of a certain protein in cancer. This was followed by demonstrating a particular tissue culture-based method used to study anti-cancer properties of drugs and finally displaying an array of microscopic images of blood vessels developing in a given tumour.
Our workstation was led by Claire McCoy, Remsha Afzal and Conor Duffy. The research focus at our lab safari station was Multiple Sclerosis (MS). We explained how the causes of MS are unknown, but that it is characterised by an influx of immune cells into the brain and spinal cord. Our research aims to investigate one type of immune cell called the macrophage. We aim to understand the damage macrophages cause in MS and if we can reverse this to provide an alternative tool for MS therapeutics. We really enjoyed explaining our research at the Lab Safari, where we showed students how MS impacts on brain function and showed them examples of activated macrophages under the microscope.
Our workstation was led by Dermot Cox and Padraig Norton. Students were given a brief history of drug discovery. Then they were introduced to the basic concepts of how a drug binds to its target and the different ways in which a drug can bind. Students were then shown a demonstration of molecular docking on a computer whereby a small molecule, or drug candidate, was virtually docked into a target binding site using the software.
The event was led by Dr Maria Morgan, Anne Grady, Prof. Tracy Robson, Dr Olga Piskareva and John O’Brien. Guides on the evening included Olwen Foley, Camille Hurley, Mary Ledwith, Seamus McDonald and Shane O’Grady.
Registration is closed and all places on the MCT Lab Safari have now been filled.
We can’t wait to meet our potential future scientists tomorrow at 5pm! The lab coats are ready and the MCT labs have never looked so clean. We have prepared some great experiments and demonstrations to showcase our ongoing research. Everyone will have an opportunity to explore our labs and perform some hands-on science activities.
MCT Lab Safari Programme:
4.50pm: Arrival and registration – Front Hall SSG
5pm: Welcome and brief introductions
5.05pm: Meet a leading scientist and hear her career journey
5.15pm: Visit our research laboratories in small groups safari style
6.30pm: Q&A with refreshments
We are looking forward to seeing you all tomorrow!
EVER WONDERED WHAT THOSE PEOPLE IN WHITE LAB COATS ACTUALLY DO?
COULD YOU BE ONE?
Join us for an opportunity to see exactly what
happens in a research laboratory. The Department
of Molecular and Cellular Therapeutics (MCT)
at RCSI is opening its doors for a science week
interactive tour. Visitors will get to interact with
high-profile scientists, explore our labs and
perform some hands-on science activities. Anyone considering a career in science is
welcome but we particularly encourage young
women/girls to attend, to promote the full
participation of girls and women in science.
As some of you know, I have joined RCSI as a StAR research lecturer in June. My plan is to establish a lab on ‘MechanoVascular Biology and Microscopy’. What do I mean by this?
The first part ‘MechanoVascular Biology’ sets the scope. I am interested in how cells in the cardiovascular system use mechanical forces to achieve their tasks. As mechanical and chemical cell functions are tightly related, both play important roles in health and disease. Most research has focused on one or the other aspect, but not both. The novel research field of ‘mechanobiology’ takes an integrative approach to better understand how physical forces co-regulate chemical processes on the molecular level. In my previous work at ETH Zurich, I have studied how fibroblasts sense matrix stiffness and respond to it. Here at RCSI, I want to study platelets in the context of thrombosis and, over the years, investigate their interplay with endothelial cells.
The second part ‘Microscopy’ highlights one of the major working horses in my lab. Following the credo ‘seeing is believing’, watching cells can tell you a lot about how they do things. I use microscopy to test hypothesis but also to discover unexpected behaviour. Over the years, I have developed several new microscopy techniques to look at sub-second dynamic processes, directly measure cellular tractions, or determine the nanoscale architecture of multi-protein structures. These are great tools to better understand how the processes starting from platelet activation and ending with the consolidation of the thrombus are regulated in space and time. For this we will use in vitro models, but I am keen to move in the future towards in vivo imaging.
By now, you may have noticed from my scientific viewpoint and my enthusiasm for technology that my background is in physics. I studied physics with a specialization on biophysics at the Technical University Munich. My PhD work at the Max Planck Institute of Biochemistry focused on electrical stimulation of neurons with extracellular electrodes. After a short postdoc at the Ludwig Maximilians University Munich where I studied bi-molecular binding kinetics in living cells, I moved to ETH Zurich in Switzerland. That’s where I have started with mechanobiology and super-resolution fluorescence microscopy, which I know bring over to RCSI.
A long way is lying ahead of me to cross the bridge towards clinical research. I look forward to having many inspiring discussions with you, already thank you for the ones we had so far, and hope that I can make a valuable contribution to the research here at RCSI!
Looking forward to seeing you at MCT Research Talks on 16th October 2017 at 12.00 TR4!
Last Monday while in Amsterdam with my Mam and two sisters, a friend of mine sent a text to let me know that the 2017 Nobel Laureates in Physiology and Medicine were Hall, Rosbash and Young. They were awarded the Nobel for their work in identifying the key genes that create circadian or body clock rhythms in the fruit fly. My feet literally were stuck to the ground, it was thrilling to know that these gentlemen would get the recognition that they so deserve, but also what this will mean for the field of science that I am so passionate about. The body clock is the molecular timekeeping system that exists in practically every organism on the earth and in every cell in our body. Simply put, it allows the cell to tell what time of day it is. Why is that important? We live on a spinning planet and because of the earth’s rotation to the sun, all life on earth has been subjected to daily periods of light and heat, dark and cold. The body clock allows us to anticipate and respond to these 24-hour predictable environmental changes and synchronises our physiology to it. For example, the body clock increases cortisol levels in the body ahead of awakening, this helps us to become active once we wake. The body clock also increases expression of digestive enzymes in the intestinal tract during daylight hours (this is why curry chips at 3am is never a great idea!).
Back in the 80’s Hall, Rosbash and Young independently isolated a gene called Period, they showed how the gene encodes a protein PER that builds up in cells at night and degrades during the day. This daily rise and fall of PER essentially allow the cell to track time of day. How thrilling it must have been for them to observe this daily change in the mRNA levels of Period gene (Figure 1- black line), all that is changing along the x-axis is the time of day.
So what does this mean three decades later? We have made great strides in understanding how the molecular clock works. We now know that the clock keeps time by a series of transcriptional-translational feedback loops. We also know that the clock controls 40% of all coding genes within the body. The body clock controls all aspects of our physiology from metabolism to immunity.
Many diseases, such as osteoarthritis and cardiovascular disease, are highly time of day dependent. Moreover, it appears that disruption of our body clocks, caused by our non-stop 24/7 lifestyle and exposure to artificial light at all times of day, is partly responsible for the increase in chronic inflammatory diseases. Unfortunately, most cell culture systems are not synchronized with the time of day, and this, in my opinion, is one of the main reasons that many researchers unknowingly neglect this field. Finally, we are making great strides in attempting to time specific treatments to the right time of day, an area called chronotherapy. Therefore, it is my hope that this increased awareness of the body clock will bring more researchers into this fascinating field. If we don’t fully understand how our body clock controls physiology and disease we will certainly be left in the dark.
Annie Curtis is a Research Lecturer and runs the Immune Clock laboratory at MCT and is fascinated by all things body clock related.
I have recently arrived as a Postdoctoral Researcher in Dr Claire McCoys Lab in the Molecular and Cellular Therapeutics Department (MCT) at RSCI. I specialise in immune signalling pathways and inflammatory complexes underlying infectious and inflammatory diseases, including multiple sclerosis (MS), sepsis and highly virulent strains of influenza.
After obtaining my BSc in Biotechnology in 2005 from Dublin City University I was awarded a postgraduate scholarship from the Irish Research Council and went on to complete my PhD in Immunology in 2009 (DCU). I conducted my postdoctoral training in innate immune signalling with Prof Luke O’Neill in Trinity College Dublin with a strong focus on understanding the mechanisms regulating a key inflammatory complex in immune cells known as the inflammasome.
I am excited to be joining MCT and the McCoy Lab. Like Claire, I have a passionate interest in medical research and chose to work in inflammation because it has a central role in the progression of a broad range of diseases. I am also passionate about community engagement, science communication and educating the next generation about the importance of medical research and the role of inflammation in disease.
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.
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.