How things work in science: targeting cell components.

How do researchers study cells? How do we get the nitty gritty?

We use many methods to tag and chase various cell components. One of my favourites is fluorescent microscopy. It allows the use of nearly all spectrum of colours from blue to purple in one go. However, we prefer to narrow it down to 2-3 colours and avoid their overlap.

How does it work? First, we use DAPI or Hoescht, which are blue fluorescent dyes used to stain DNA. This way, we tag the nucleus of the cell. Then, we tag a protein of interest. In our case, it was MYCN, a protein that acts as a transcription factor. MYCN amplification is associated with poor prognosis in neuroblastoma. As a transcription factor, it binds to genomic DNA and is located in the nucleus. We used a specific antibody that was labelled with a green fluorescent dye. Look at the image below. The green colour pattern overlaps with the blue colour. Then, we tagged the cytoskeleton, a complex of various proteins that hold the cell architecture and dynamics. We used phalloidin with red fluorescence. It is a highly selective bicyclic peptide and a popular choice for staining actin filaments.

Neuroblastoma organoids stained with DAPI, Phalloidin and anti-MYCN antibody. This work was done during the Fulbright journey to Ewald’s Lab at Johns Hopkins

Now, we can enjoy visualising cells and test different research questions. For example, how do cells respond to a drug? Or how do neuroblastoma cells spread?

Written by Olga Piskareva

How things work in science: Scaffolding

At the Cancer Bioengineering Group, we use different types of scaffolds to mimic the 3D structure of tumours outside the body. We use these scaffolds to test new therapeutics and understand the tumour microenvironment. But I bet you didn’t think we had this in common with spiders?

Spiders make their webs by producing silk from specialized glands in their abdomen. They release the silk through spinnerets located at the back of their abdomen, then use their legs to manipulate the silk strands into intricate patterns, depending on the species and purpose of the web.

The process of web building begins with a scaffold. The specialized glands that spiders use are called spinnerets, and they produce liquid silk proteins that solidify into a thread when they come into contact with air. Using their many legs, spiders can manipulate the threads by changing the speed and tension they enforce on the silk, thus controlling thickness, stickiness and strength. They first lay a framework of non-sticky threads, known as scaffolding. And layer by layer, different species of spiders will add their own artistic sticky silk design to the scaffold depending on their aim. Take the deadly redback spider for example, these guys have a utilitarian approach to web building relying on their webs mainly for shelter and capturing prey. As such, they don’t put much effort into producing irregular and messy homes. In comparison, the orb-weaving spider produces “Mona Lisa”-like designs, with complex geometric patterns and intricate designs. The differences in effort seem to come from the environments in which the webs are located, with the redbacks choosing more sheltered environments and thus not needing much strength to their webs. Whereas orb-weaving spiders are more adapted to a range of environments, from forests to grasslands to urban gardens. So, while the redback gets a lot of attention for their neurotoxic venom, they need to step up their artistic skills to match that of their orb-weaving colleagues.

The redback spider and its webs are reminiscent of an aggressive tumour, which is erratic, dangerous, and unpredictable. We want to find the “anti-venom” for such tumours so we can wipe them out for good.

Watch this amazing web-building timelapse by BBC Earth.

Written by Ellen King

How things work in Science: Tìr na nÒg

In humans, NANOG, SOX2, and OCT4 are transcription factors that maintain the undifferentiated state of embryonic stem cells (ESCs). NANOG was first discovered in 2003 by Chambers et al. and Mitsui et al. as a transcription factor in ESCs responsible for cellular self-renewal. More importantly, it enables continuous self-renewal of cancer stem cells, leading to metastasis when the regulatory genes involved do not function normally. These have been identified as cancer stem cells, with NANOG being a marker of “stemness”. In multiple cancer types, NANOG has various effects, including cellular expression of mesenchymal phenotype, cellular invasion/migration, repressed apoptosis, drug resistance, and increased angiogenesis. In pathways, NANOG either promotes or represses the expression of other genes that lead to cancer-favoured cellular behaviour. Overall, a higher expression level of NANOG is usually indicated in cases of poor prognosis.

NANOG is even more interesting due to its eponym, which comes from Tìr na nÒg. A Celtic myth of the Land of Youth, where the Tuath Dé resided in a supernatural land of paradise. This land offered beauty, health, joy, and everlasting youth to the inhabitants. As the myth goes, the Tuath Dé were gods of the land, and the god that ruled, Manannán mac Lir, was the first ancestor of humans. In various Celtic legends, humans are invited by the gods to visit Tìr na nÒg on great adventures.

However, time passes much slower in Tìr na nÒg, making it precarious for humans to return to their own world. As is the fateful tale of Oisín, who fell in love with the Tìr na nÒg goddess, Niamh. He travelled with her to Tìr na nÒg, where they lived happily in paradise. Upon a visit back to Ireland, Oisín realized that all his family had died over the years. When Oisín found a group of men who were struggling to move a giant rock, he stopped to lend them a hand while on his horse. However, the weight of the rock caused his saddle strap to snap. He fell from his horse, and when he touched the ground, he suddenly aged 300 years all at once.

Written by Alysia Scott

Sources:

Gawlik-Rzemieniewska, Natalia, and Ilona Bednarek. “The role of NANOG transcriptional factor in the development of malignant phenotype of cancer cells.” Cancer biology & therapy vol. 17,1 (2016): 1-10.

The Story of Tír Na NÓg.” Celtic Titles, 10 Feb. 2022

Congratulations to Dr Ciara Gallagher!


Huge congrats to a newly minted Dr Ciara Gallagher!  She defended her PhD on March 8 – International Women’s Day. Your enthusiasm and perseverance are truly fascinating! May this be the stepping stone towards a brighter future, Ciara!

We thank examiners Dr Marie McIlroy (RCSI) and Prof Jan Škoda (Masaryk Uni) for the time and expertise they provided.

We also thank the Irish Research Council for their generous support!

Dr Ciara Murphy (Chair), Dr Olga Piskareva (Supervisor), Dr Ciara Gallagher, Prof Jan Skoda (examiner), Dr Marie McIlroy (Examiner)

Ever wonder how scientists figure out a specific protein’s role in cancer?

Researchers use various methods, but I employ gene knockdown in my experiments. Basically, I use small RNA molecules that specifically target and degrade the mRNA of my gene of interest. This leads to a decrease in the corresponding protein levels, enabling me to observe the effects on neuroblastoma cell behaviour.

I feel a bit like Sherlock Holmes, you know? I’m selectively putting my suspect protein – the one I’m eyeing – under the spotlight to see how it’s pulling the strings on the cell’s behaviour. It’s like I’m in a cellular mystery, complete with a gene knockout magnifying glass 🔍🧬🕵

So, what I’ve been up to these past months is knocking down my protein and trying to find answers to the following questions:

Can neuroblastoma cells survive? And if not, how do they meet their demise? Do they go on a growth spree and start proliferating? Are they capable of migration? And here’s the twist – when my protein of interest takes a dip, do other proteins decide to change their expression levels?

The picture below can probably help you get an idea of what I’ve done so far. Do you see those brighter spots in Pictures A and B? Those are dead cells. Their number indicates the proportion of dead cells after a treatment. Picture A has just a few; the majority are healthy and well-spread cells. This is our negative control, a condition when we show neuroblastoma cells that have been transfected, but no gene knockdown happened. Transfection is the term for introducing small RNA molecules. Now, in Picture B, when we knocked down the protein, it caused the death of the cells, and you can clearly see that from all those many little bright spots.

We have found answers to many of the previous questions, but new questions have arisen, and we can’t wait to answer them!

Written by Federica Cottone

International Childhood Cancer Day – 15 February 2024

We are celebrating #ICCD2024 with a Bake Sale and a Quiz. To earn a piece of cake, you have to answer a question correctly! Have a look at some:

  • Which civilisation first described cancer?
  • Where did the word cancer come from?
  • Do children get cancer?
  • What is the most common type of cancer in children?
  • Can the Human Papillomavirus (HPV) vaccine prevent cancer?
  • Can neuroblastoma begin to develop before birth?
  • What is the name of the nerve cell in which neuroblastoma begins to grow?
  • Can a child have a genetic predisposition to neuroblastoma?
  • What % stands for the incidence of neuroblastoma: 8 or 15?
  • What % stands for the neuroblastoma-related deaths: 8 or 15?
  • Does neuroblastoma first appear in the brain?
  • What does the letter N stand for in the gene MYCN?
  • How often does childhood cancer occur compared to adults?
  • How often does hereditary cancer happen in general?
  • Do you think that children are small adults when we talk about anticancer treatment?

How things work in Science: Classifiers

For our next little series introducing a different thing in science and how it works every week, I decided to focus on classifiers. With artificial intelligence becoming more and more prominent in our daily lives as of late, I thought this would be a good lead into the explicitly science-focused topics to come. So, what is a classifier? How does it work? And why does it matter?

At their core, classifiers are algorithms designed to categorize input data into predefined classes or categories. They learn patterns and relationships from labelled training data to make predictions on new, unseen data.

Once features are extracted, identified and quantified from labelled or annotated input data, mathematical models are employed for pattern recognition and predictions.

These models can range from simple decision trees to complex neural networks, each with its own strengths and weaknesses.

Training these models is an iterative process. That means to produce one good classifier, lots of classifiers were created in the process: Every time the pattern recognition is run, the annotated data is categorised by the classifier and compared to the annotation class. Prediction errors are corrected, and performance is optimised. This whole process is one iteration. How many iterations are required for a well-trained classifier varies widely and is largely dependent on the input data and application. For my tissue classifiers, it took up to 20,000 iterations.

Classifiers use these models to categorise unseen data into categories the user-defined at the start. In the figure, you can see my annotated histological slides from which the classifier extracted patterns to then classify the rest of the slide and entirely unseen slides into tumour (red), stroma (green) and background (blue) classes.

From identifying fraudulent transactions, filtering out junk mail, targeted advertising, and facial recognition to unlock your phone or diagnosing diseases, classifiers play a vital role in automating decision-making processes and driving advancements across a wide range of industries. Keep your eyes peeled, and you can find more classifiers in action all around you.

Written by Ronja Struck

Women in Science: Dr. Margaret Jane Pittman

In the world of scientific research, Dr. Pittman’s name stands out. She was a bacteriologist and one of the founders of vaccines against infectious diseases such as typhoid, cholera, whooping cough and meningitis. Dr. Pittman achieved another incredible feat as the first woman to give rise to a National Institutes of Health Laboratory in the US. This accomplishment dispelled the persistent gender stereotypes that had long prevented women from pursuing careers in STEM and showcased her extraordinary credentials and experience.

Dr. Margaret Jane Pittman was born on January 20, 1901, and grew up in Prairie Grove, Arkansas. Dr Margaret Pittman graduated magna cum laude from Hendrix in 1923. In 1925, she took her first course in bacteriology. At the time, it was propitious. Later in the nineteenth century, bacteriology became a distinct biological science. She got her Ph.D. from the University of Chicago in 1929 and started working for the National Institutes of Health in 1936. She developed, assessed, and standardised immunisation programmes against cholera, whooping cough, typhoid, and other diseases during most of her career. She continued to work at NIH as an unpaid guest. She occasionally served as a consultant for the World Health Organisation and as a guest scientist in several countries, including Spain, Scotland, Egypt, and Iran.

Her work and relentless efforts have revolutionised the world of vaccines and immunisation. Dr Pittman’s Haemophilus influenzae study has significantly influenced the medical field. Based on her research, numerous lives have been saved due to the creation of potent vaccinations, which has led to a notable drop in diseases linked to Haemophilus influenza. Pittman’s collaborations with esteemed scientists like Drs. Jonas Salk and Albert Sabin were essential in creating vaccinations against polio. They collaborated to carry out a great deal of research and clinical testing, which led to the development of the first effective polio vaccine. This enormous accomplishment was a game-changer in the global battle against polio, saving countless lives.

CREDIT: NATIONAL MUSEUM OF AMERICAN HISTORY
Margaret Pittman and fellow NIH physician Sadie L. Carlin are “reading” an agglutination reaction, part of the test for potency of commercially prepared anti-meningitis serum during the meningitis epidemic of 1935-1937 (1937).

As a woman in a male-dominated field, Margaret Pittman faced numerous challenges throughout her career. In an era when gender bias was pervasive, women struggled with standardisation and opportunities for advancement in scientific research. Pittman’s journey was the same, as she faced challenges that would have prevented her from moving further. The legacy of Dr. Margart Jane Pittman endures today, as her contributions continue to influence the direction of immunisation research. She stated that revolutionising problems with pertussis immunisation makes this work one of my best accomplishments that “despite the problems that have occurred with the pertussis vaccine… I consider this work one of my best accomplishments.”. Her partnerships and contributions have established a standard for upcoming generations and cleared the path for additional developments in vaccine research. By honouring her outstanding accomplishments, we can ensure that her influence on public health continues to be a driving force in the battle against infectious illnesses.

Written by Rabia Saleem

Congratulations to Dr Cat Murphy!

November 22, 2023 – Catherine was officially coined Dr Catherine Murphy. A Big Day for Catherine, her family and me.

Catherine joined our team in July 2019 to carry out a research project funded by Neuroblastoma UK. In this project, she aimed to use 3D culturing to engineer a novel experimental model and study the biology and immunology of neuroblastoma, an aggressive childhood cancer. There was the full spectrum of challenges and hard work spiced up with the uncertainty of the COVID-19 restrictions!

The PhD journey is never a straight line. It has a range of colours with 50+ shades for each. There are black alleys and hidden cul de sacs. Between July 2019 and June 2023, some days were sunny and bright, and some had scattered showers, gale winds and stormy snow, with sunshine developing elsewhere. The journey was spiced up with publications, conferences, travels, days out and fundraising events with the team.

Of note, she was behind our Twitter activities and blogging #AskCat, making our team visible! All these together have moulded into a new multi-skilled professional – Dr Catherine Murphy!

Well done to Catherine! Wish you the best of luck in your new adventure!

Women in Science: Mary Golda Ross

A woman from a background of adversity cannot be shaken when confronted with resistance. Mary Golda Ross was, “the kind of person who would walk through a door and stick in her foot to make sure that it stayed open for others”. She followed her passion and held her own to become the first Native American female engineer in the 1940s. Ross was a pioneer of rocket science and was an integral part of, still to this day, classified research on interplanetary space travel.

Mary Golda Ross was the great-great-granddaughter of John Ross, the chief of the Cherokee nation. About 70 years before Mary was born, John was tirelessly resisting the seizure and erasure of Cherokee lands and culture by the U.S. Government. In 1838, due to the Indian Removal Act, tens of thousands of Cherokee were forcibly removed from their homes and forced to march over 1,200 miles (about 1,930 kilometers) into present-day Oklahoma. Thousands died during the journey. Mary was born in Park Hill, Oklahoma in 1908. As a student in the Cherokee Nation, Mary was very bright and caught on to mathematics quickly. During her university classes, Mary was ostracized for her interest in STEM as she found herself alone on one side of the classroom with the men on the other. As was the case with many women in science at the time, she both learned and excelled academically with a massive lack of support.

After receiving her bachelor’s degree, Mary held teaching positions and became a statistical clerk at the Bureau of Indian Affairs. While working, she took astronomy classes at the present-day University of Northern Colorado and earned her master’s degree in mathematics in 1938. A full 100 years after the Trail of Tears. Soon after, World War II had started and Mary moved to California to help with the war efforts, as 1 in 4 women worked outside the home during this period. She was hired as a mathematician in 1942 by Lockheed Aircraft Corporation, an aerospace engineering company. Her first project was analyzing the effects of pressure on the design of the P-38 Lightning, the fastest fighter jet in the world at the time, reaching speeds of 400 mph (640 km/h) in a level flight. Mary’s ambition did not stop there as she began to wonder how to become involved in space travel.

When the war ended, Lockheed saw her brilliance and sent her to UCLA where she earned a professional certification in engineering in 1949. Carrying on her engineering work in 1952, Mary helped found Lockheed’s Advanced Development Program, otherwise known as Skunk Works. She was the only woman engineer on this team, and most of the research from this program is still classified. However, it is known that Mary developed technology for space exploration and orbiting satellites. Mary’s work also helped develop the Agena spacecraft used in the Gemini and Apollo space missions. In 1966, Mary was a primary author for NASA’s Planetary Flight Handbook Vol. III, a still relevant source for space travel. Even suggesting the possibility of travelling to Mars and Venus.

Mary retired in 1973 and was influential in both the American Indian Science and Engineering Society and the Society of Women Engineers. She worked hard to encourage increased participation of Native American youth in STEM fields. As of 2021, there are less than 1% of Native Americans that comprise the STEM workforce; only 2% of the U.S. population is Native American. She advocated for more resources offered to Native Americans interested in STEM and increased knowledge of her ancestors’ history. Mary attended the 2004 opening of the Smithsonian National Museum of the American Indian in Washington, D.C., wearing a traditional Cherokee dress. Upon her death in 2008, Mary endowed the museum with $400,000. Mary had told the Los Altos Town Crier, “The museum will tell the true story of the Indian – not just the story of the past, but an ongoing story”. Mary had dreams that reached the stars, and she never stopped chasing them. She trailblazed a path for those who followed in her footsteps to find a way, no matter what barriers were present.

Written by Alysia Scott.