New PhD studentships for October 2021 start

We are starting to recruit PhD researchers for Oct 21 start. Join a world-class, interdisciplinary and highly supportive research environment where you can learn about new cell biology projects and advanced microscopy techniques.

Some of the current PhD researchers and alumni of the Applied Biophotonics Group, pictured after their clean sweep of the awards that the 2019 Postgraduate Research Symposium.
  1. iCASE project: Cardiovascular effects of COVID-19 ‘cytokine storm’

This is a 3.5-year MRC DiMeN iCASE studentship based primarily in the Dept of Molecular Biology & Biotechnology, of the University of Sheffield. To read more and to apply, go to the project page on FindAPhD.

We are seeking enthusiastic candidates for an iCASE PhD project investigating the molecular pathology of COVID-19. The primary aim is to develop a detailed and urgent understanding of the so-called ‘cytokine storm’ which follows persistent SARS-CoV-2 infection and leads to the life-threatening cardiovascular disease. We anticipate that our findings will directly unlock life-saving treatments for patients with either COVID-19 or future variants.

>80% of patients hospitalised due to COVID-10 so far have severe vascular diseases and thrombosis, whilst >40% of autopsies detect myocardial hypertrophy.  It is thought that specific cytokines, along with the continued binding of the virus spike protein to the ACE2 receptor over-stimulates the specific cytokine receptors in both the vascular endothelial cells and the myocardium. We will investigate the hypothesis that these events lead to an over-activation of calcium-calmodulin kinase-II (CaMK-II), derailing it from its primary role in cellular homeostasis. We hypothesise that over-stimulated CaMK-II triggers maladaptive protein phosphorylation, remodelling and/or death of these cells, leading to life-threatening acute cardiovascular diseases.

In this project, we will perform novel super-resolution microscopy on tissue samples of lung vasculature and hearts of COVID-19 patients to identify the fine features of the damage caused to the vascular endothelium and heart cells. We will then establish two novel organoid systems (tissue culture assays) – of capillaries and myocardium – to simulate the COVID-19 cardiovascular disease. Coupled with live-cell imaging, these organoids will be subjected to an in vitro version of the ‘cytokine storm’. Based on the pathology and proteins observed in the patient samples, we will use super-resolution microscopy to track the changes in the location and function of CaMK-II. Finally, we will test the effect of a series of pharmacological blockers of the cytokine receptors and CaMK-II to explore strategies to minimise the effect of the ‘cytokine storm’ on the endothelium and myocardium.

As a DiMeN iCASE scholar, you will be eligible for an enhanced stipend (additional £2,500 pa). The project will be primarily based at the Department of Molecular Biology & Biotechnology in the University of Sheffield; in the state-of-the-art laboratory of Dr Izzy Jayasinghe. You will join an interdisciplinary team of diverse researchers with a primary expertise in microscopy and cellular cardiology. The second supervisor is Prof Nikita Gamper, an expert in cytokines, neuroscience and calcium signalling, based in the University of Leeds. The third supervisor is Dr Jung-uk Shim, an expert in microfluidics and their use for tissue culture and organoids. During the studentship, you will spend a 6-month placement with the Leeds-based industrial partner, Badrilla Ltd, one of the UK’s leading producers of antibodies against calcium handling/regulating proteins and biochemical analytical tools (https://badrilla.com/). The industrial placement will offer you an opportunity to contribute directly to their core R&D, product development, market operations, research outputs and consortia.

This is an excellent opportunity to work alongside a world-class team of researchers, to learn state-of-the-art technologies and to contribute to a timely research project with high national and international importance. The MRC DiMeN DTP which this studentship belongs to has previously supported our very own Tom Sheard. Support Tom received from DiMeN includes a flexible funding award to complete a 1-month sabbatical in the University of Auckland, New Zealand, and a highly productive and enjoyable 1-year placement with his industrial supervisor.

Applications close on Jan 14, 2021.

A new correlative super-resolution protocol to visualise cellular function and structure

Super-resolution microscopies are commonly utilised to obtain nanometre-scale information regarding the structures within a cell. This information is rarely accompanied by any functional detail, a key requirement when observing a cellular system. The most recent publication from the applied biophotonics group describes the development of a correlative super-resolution protocol. This protocol enables the fast second-messenger signalling of primary cell types to be visualised in regard to a cell’s underlying structure.

The protocols focuses on the principle of examining calcium sparks in living cells (main panel, left), and then aligning the image frames to the corresponding DNA-PAINT super-resolution images of the underlying clustering patterns of the Calcium release channels, the ryanodine receptors (magnified inset, right)

Application of the correlative super-resolution protocol has been undertaken across various cell types and disease models throughout Miriam Hurley’s PhD studies. Specifically, the group has applied the protocol to study the movement of calcium (Ca2+) against the nanoscale patterns of the ryanodine receptor, a Ca2+ release channel which gives rise to these Ca2+ signals within wildtype primary cells.

The protocol relies upon the combination of total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals with subsequent DNA-PAINT imaging of the RyRs. The publication outlines a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets to demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels which are locally activated during the genesis of cytoplasmic Ca2+ signals.

This work has formed the basis of Miriam’s PhD studies, funded by the Leeds Anniversary Research Scholarship and the Royal Society Research Grant (first research grant awarded to PI, Izzy Jayasinghe).

Access the full article at: https://doi.org/10.1016/j.ymeth.2020.10.005.

Preprint outlining the utility of peptide ligands against RyR2 as potential super-resolution imaging probes

Our latest pre-print is now online, where we evaluated two peptide ligands of the cardiac ryanodine receptor (RyR) as super-resolution imaging probes. The research was conducted in collaboration with one of our industrial partners, Badrilla Ltd., during the industrial internship of iCASE PhD student Tom Sheard funded through the DiMeN DTP.

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The peptides used were the scorpion venom toxin imperacalcin, and CPVT-mimicking DPc10. We evaluated the specificity of the fluorescent peptide conjugates for the target RyR2 in fluorescent imaging experiments, in terms of colocalisation with RyR2 immunolabelling, with confocal and expansion microscopy.

We also performed structural work looking at the DPc10 binding site on RyR2 atomic models to learn more about the peptide’s mechanism of disturbance (DPc10 binding to RyR2 causes domain unzipping and destabilises the channel).

Thanks to the host company Badrilla, the DiMeN DTP for funding Tom’s industrial placement, and the other authors for help with the research.

Access the pre-print in bioRxiv at: https://www.biorxiv.org/content/10.1101/2020.06.26.131854v1.

Feedback on all contents of the work is welcomed.

Follow Tom on twitter : @tmdsheard. https://twitter.com/TMDSheard

New book chapter detailing EExM in Methods in Cell Biology

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How we prepared EExM samples

Our new book chapter has been published, detailing the enhanced expansion microscopy technique (EExM). The protocol offers a route to visualising cellular ultrastructures at a resolution of 15 nm in-plane (and ~ 35 nm axially) with a hydrogel-based molecular-scale imprint of the fluorescence pattern.

We detail how we used EExM to explore structural and biochemical remodelling in cardiomyocytes. The chapter includes a step-by-step protocol, reagent list and troubleshooting tips.

Access the paper published in Methods in Cell Biology at: https://sciencedirect.com/science/article/pii/S0091679X20301084.

Any difficulties accessing the article please contact the Tom Sheard via email at: bs11ts[at]leeds.ac.uk

This is the first book chapter for PhD student Tom Sheard (@tmdsheard on Twitter).

 

Optical microscopy bottlenecks survey

bottleneck-910050_1280We have launched a brief survey examining the needs in the Life Sciences around the use or potential utility of optical (fluorescence) microscopies. This takes ~ 6 minutes to complete and gives you an opportunity to stay in touch or engaged directly with the new developments of the newly launched UKRI Future Leader Fellowship within the Applied Biophotonics Group. Whether you are an experienced microscopist or never had the opportunity to use optical imaging, can such microscopies add value to your research or day-to-day work? If so, what holds you back? If you work within any area of Life Sciences, we would like to hear about your experiences. Please take the survey and let us know.