Alice has been awarded an UKRI Future Leaders Fellowship

Alice has been awarded a prestigious UKRI Future Leaders Fellowship in Round six of the scheme. Her fellowship “Unravelling the Invisible Complexities of the Genome” will support her ambitious aims of developing a new open biophysical pipeline, combining Atomic Force Microscopy and automated image analysis to determine how complex DNA structures within the genome affect essential cellular interactions. You can read the press release from Sheffield here.

Experience of a Masters Student

Following on from our pervious blog post about how Eddie transitioned from working at AFM probe manufacturer NuNano to doing a PhD in the Pyne lab, this blog post looks at the experience of Billy Davies in the lab before he made the reverse journey from Sheffield to Bristol.

Billy completed a project masters project in the Pyne lab in 2021 as part of his MSc in Molecular Medicine at the University of Sheffield and now works at NuNano in Bristol as a probe consultant.

Billy Davies is now a Probe Consultant for NuNano

What was your project about and what did it involve?

My project studied how DNA topology affected on the binding of DNA Gyrase and its subunits. Gyrase is an essential enzyme involved in relaxing over-twisted DNA, and detangling DNA loops. We wanted to see if different conformations and sites where DNA strands crossed over affected the binding preference of Gyrase. Using AFM, we imaged a variety of DNA substrates, some overwound DNA and others linked rings, with and without Gyrase and its subunits, so we could see if there were preferences for some conformations over others.

What did you learn from your time in the lab?

My time in the Pyne Lab can be summed up by the fact that I came in having heard the technique in passing and left an AFM believer, taking my first job at NuNano (an AFM probe manufacturer). Everyone in the group is so enthusiastic about AFM it was hard not to end up finding it fascinating, even if its completely different to what I had studied in the past, and I really felt like I grew as a scientist by working with them. Despite the restrictions, the amount of effort they made to get me trained and on the microscope was amazing and I’m incredibly grateful to have had the opportunity to learn from leading AFMers such as Alice.

Summarise your experience in the lab

The best thing about being there was how much effort they made to make you feel like a member of the group, and that your project (even if it was one of many) was important for the lab as a whole. Alice was always interested in what we were doing, and listened to any of my ideas or plans even if they weren’t actually very good, and gave us just the right amount of support vs independence.

Billy on the AFM at NuNano

Selling probes to pursuing a PhD- Eddie's move, one year on

This blog post originally appeared on the NuNano website where Eddie sat down (virtually) with his former colleagues to discuss his move from selling AFM probes in Bristol to starting a PhD using AFM in Sheffield. You can see the original post here.

After nearly 2 years working with NuNano as our Sales Coordinator, engaging with lots of scientists using AFM, Eddie Rollins found the lure of academic research too strong to resist.

Eddie made his return to university last November to undertake a PhD with Dr Alice Pyne at the University of Sheffield and we caught up with him earlier this month to find out how he was getting on, one year into his studies.

Eddie Rollins at work in the nanocharacterisation lab in the Royce Discovery Centre.

Great to catch up with you again Eddie! Can you tell us a bit about your PhD?

Sure. I’m trying to understand how small-scale changes in the structure of DNA affects how it interacts with proteins.

These interactions are biologically essential for all forms of life so are important targets for antibiotic and anti-cancer drugs. Understanding how they work could help develop and improve treatments for a wide range of diseases.

AFM is an essential tool to help understand these interactions since it allows us to see individual molecules of DNA and structural changes such as bends and twists as well as directly visualizing the DNA and protein binding.  

In what way are you using AFM? To what extent did your background of working with NuNano help at all?

AFM is a large part of my PhD. I use AFM to image individual molecules of DNA as well as their interactions with proteins. I take advantage of AFMs ability to operate in liquid to get high resolution images of these interactions in a near native state which allows me to see how the local shape of the DNA is affected.

Working with NuNano was a huge help when I started doing AFM as I’d already learnt a lot about the technique from the NuNano team, as well as through discussions with customers about their work in a wide variety of fields.

Additionally, as the probe is such an core part of the microscope, having a good grasp of the different aspects of probe design made a big difference not only in understanding probe choice in my own lab but also in evaluating AFM experiments in the literature.

What was it like moving from working with NuNano to working back in academia again?

Doing a PhD is a long game, the deadline is 4 years away at the start, so you have to create your own structure and your own intermediate targets.

While at NuNano I had a constant stream of calls and meetings with customers on top of the regular meetings and deadlines that come with being part of a growing company. This was probably the biggest adjustment.

Whilst I’ve enjoyed the freedom and space to do research and learn I do sometimes miss the faster-paced world of commerce and chasing sales!

How was it moving to a new area and starting a new job whilst we were still very much in the full throes of the Covid pandemic last autumn?

Moving during a pandemic was pretty difficult. Not only the practical side of it was hard but it’s taken a while to get to know the city and people in it. Thankfully Sheffield is a very green city with easy access to the Peak District so I manged to discover a lot of lovely parks and explore the Peaks on my bike.

The PhD itself also didn’t start as I had imagined as I wasn’t able to get into the lab for four months - though that did give me a lot of time to read and attend the glut of virtual conferences going on at the time.

Although it was a bit frustrating at the time it meant that when I did get into the lab I had a lot better idea of what I was doing and was able to hit the ground running and get some good data, so it worked out well really.

As an insider to academia now, what are your thoughts on the need and value of an AFM Community?

AFM is a powerful technique with potential applications in numerous areas from nanotechnology to diagnostics. This breadth of application has the potential to fragment the field so having a strong and generous AFM community is essential.

Sharing developments and best practice between people and labs working in different areas through such a community is one way to ensure that all forms and applications of the technique can benefit from these improvements.

I believe an AFM community that involves the commercial instrument and probe manufacturers is key to seeing the applications developed in academic labs applied more broadly in non-specialist setting like the clinic.

What’s brilliant is that since starting my PhD I’ve seen the AFM community come together around the question on data analysis and seen projects emerge to share software between groups (see www.github.com/AFM-SPM). I can see this being a particular area of collaboration allowing progress like we’ve seen in the cryo-EM field.

What has been the biggest lesson for you in the past year?

I think one of the biggest lessons I’ve learnt this year is that doing AFM provides fantastic opportunities to collaborate with a variety of researchers.

Being able to directly view nano-scale structures can clarify mechanisms that otherwise can only be visualised abstractly as cartoons so combining AFM with other approaches can reveal things invisible to each technique in isolation.

This is true of all scientific instruments and methods however AFM’s fairly unique set of advantages and disadvantages makes it a particularly helpful partner for illuminating processes at the nanoscale. 

Eddie and James Vicary (NuNano MD) at low-key COVID send-off for Eddie after 18 months working for NuNano.

Paper published showing the helix of ‘dancing DNA’

Alice and Kavit alongside collaborators have published a paper in Nature Commmunications this week, ‘Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides’.

Videos accompanying the paper, derived from high resolution AFM images of DNA and molecular dynamics (MD) simulations, show for the first time how small circles of DNA adopt dance-like movements.

The footage is based on the highest resolution images of a single molecule of DNA ever captured. They show in unprecedented detail how the stresses and strains that are placed on DNA when it is crammed inside cells can change its shape.

Previously scientists were only able to see DNA by using microscopes that are limited to taking static images. But now the Yorkshire team has combined advanced atomic force microscopy with supercomputer simulations to create videos of twisted molecules of DNA.

Alice said:

 “Seeing is believing, but with something as small as DNA, seeing the helical structure of the entire DNA molecule was extremely challenging. 

The videos we have developed enable us to observe DNA twisting in a level of detail that has never been seen before.”

The images are so detailed it is possible to see the iconic double helical structure of DNA, but when combined with the simulations, the researchers were able to see the position of every single atom in the DNA and how it twists and writhes. 

Every human cell contains two metres of DNA. In order for this DNA to fit inside our cells, it has evolved to twist, turn and coil. That means that loopy DNA is everywhere in the genome, forming twisted structures which show more dynamic behaviour than their relaxed counterparts.

The team looked at DNA minicircles, which are special because the molecule is joined at both ends to form a loop. This loop enabled the researchers to give the DNA minicircles an extra added twist, making the DNA dance more vigorously. 

When the researchers imaged relaxed DNA, without any twists, they saw that it did very little. However, when they gave the DNA an added twist, it suddenly became far more dynamic and could be seen to adopt some very exotic shapes. These exotic dance-moves were found to be the key to finding binding partners for the DNA, as when they adopt a wider range of shapes, then a greater variety of other molecules find it attractive.  

Previous research from Stanford, which detected DNA minicircles in cells, suggests they are potential indicators of health and ageing and may act as early markers for disease.

As the DNA minicircles can twist and bend, they can also become very compact. Being able to study DNA in such detail could accelerate the development of new gene therapies by utilising how twisted and compacted DNA circles can squeeze their way into cells. 

One of our collaborators who produces the minicircles used in this study said:

“Dr. Pyne and her co-worker’s new AFM structures of our supercoiled minicircles are extremely exciting because they show, with remarkable detail, how wrinkled, bubbled, kinked, denatured, and strangely shaped they are which we hope to be able to control someday.”

You can read the paper here:

Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides

Paper on DNA supercoiling published in Nature Communications

Alice and Kavit along with collaborators from around the UK have published on, ‘Base-pair resolution analysis of the effect of supercoiling on DNA flexibility and major groove recognition by triplex-forming oligonucleotides’

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Isabel and Alice publish paper on a new way to detect antimicrobial resistance

A new, quicker way of detecting antibiotic resistance in bacteria has been developed by a team of scientists from the EPSRC funded interdisciplinary research collaboration, i-sense.

Isabel Bennett and Alice Pyne along with collaborators at UCL have developed a new technique that uses nanotechnology to detect antibiotic resistance in approximately 45 minutes.

The standard method for detecting resistance is a relatively slow process that typically takes between 12 and 24 hours. The ability to reduce this time could significantly help the ongoing battle against antibiotic-resistant bacteria - a problem which is predicted to cause 10 million deaths per year and cost the global economy $100 trillion by 2050.

Speeding up the time it takes to identify antibiotic-resistant bacteria could improve our ability to prescribe antibiotics correctly and reduce the misuse of antibiotic treatments - a key step in the fight against antibiotic resistance.

The new method was developed by Isabel at UCL in collaboration with Alice and Prof. Rachel McKendry, also from UCL, and uses a new Atomic Force Microscopy (AFM) detection system. 

Our method allowed us to quickly differentiate between resistant and sensitive phenotypes in multiple strains of E. coli, a bacteria implicated in a number of challenging infections including UTIs.

Dr Isabel Bennett

UCL

This method uses a nanomechanical cantilever sensor together with a laser to detect single bacterial cells as they pass through the laser’s focus, which provides a simple readout of antibiotic resistance by detecting growth (resistant) or death (sensitive) of the bacteria.

By placing a reflective surface - a small stiff cantilever - in a filtered growth medium in a petri dish and reflecting a laser off it onto a photodiode detector, it is possible to detect bacteria as they pass through the path of the laser, therefore altering the signal at the detector.

Following the addition of the antibiotic to the petri dish, the study has shown that it is possible to detect whether fewer bacteria interfere with the laser beam, thereby indicating cell death in the antibiotic-sensitive bacteria.

The new technique developed by Isabel builds on an AFM method from a previous study, however this method doesn’t require the bacteria to be immobilised - making the new detection system much faster.

Isabel said: “Our method allowed us to quickly differentiate between resistant and sensitive phenotypes in multiple strains of E. coli, a bacteria implicated in a number of challenging infections including UTIs.”

Alice added: “We were able to show that our faster method was able to reproduce values from gold standard measurements, such as MIC’s in a fraction of the time.”

The study - Cantilever Sensors for Rapid Optical Antimicrobial Sensitivity Testing - was conducted by Dr Isabel Bennett as part of her PhD supervised by Dr Alice Pyne and Professor Rachel McKendry.

The research by the all-female team of scientists is published in the journal ACS Sensors. The journal has published an interview with Dr Bennett following the paper being selected as an ACS editors choice.

Read the paper here

Paper published in ACS Sensors

Isabel and Alice along with Prof. Rachel McKendry have published their paper, ‘Cantilever Sensors for Rapid Optical Antimicrobial Sensitivity Testing’ in ACS Sensors find the paper here: https://pubs.acs.org/doi/10.1021/acssensors.0c01216

A new chapter - Alice moves to the University of Sheffield to take up a lectureship in the Department of Materials Science

After almost a decade, Alice is leaving UCL to take up a position at the University of Sheffield in the Department of Materials Science and Engineering.

I am really excited to start a new chapter in my research, continuing on with all of my current collaborators, but also to find new collaborators, both in Engineering and Materials Science, and throughout the University of Sheffield. This should be made a little easier by the growing DNA presence at the University of Sheffield, which I encountered at #NAF2019, and whom I hope to meet more of through SInFoNiA.

I can’t wait to get started, and to meet my new colleagues. I’d also like to thank everyone at UCL who’s contributed to my journey there, its been an incredible place to learn and work. I look forward to visiting, I will have an honorary position there, so will be back often!

 

The Robert Hadfield Building, University of Sheffield

Paper Published in Nature Communications: Capturing the Membrane Attack Complex on Camera

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