Quantum dots for Immunofluorescence

Guest post by Dave Mason; reblogged from rapha-z-lab

In modern cell biology and light microscopy, immunofluorescence is a workhorse experiment. The same way antibodies can recognise foreign pathogens in an animal, so the specificity of antibodies can be used to label specific targets within the cell. When antibodies are bound to a fluorophore of your choice, and in combination with light microscopy, this makes for a versatile platform for research and diagnostics.

Most small-dye based fluorophores that are used in combination with antibodies suffer from a limitation; hit them with enough light and you irreversibly damage the fluorochrome, rendering the dye ‘invisible’ or photobleached. This property is the basis of several biophysical techniques such as Fluorescence Recovery After Photobleaching (FRAP) but for routine imaging it is largely an unwanted property.

Over 20 years ago, a new class of fluorescent conjugate was introduced in the form of Quantum Dots (QDots); semiconductor nanocrystals that promised increased brightness, a broad excitation and narrow emission band (good when using multi-channel imaging) and most importantly: no photobleaching. They were hailed as a game changer: “When the methods are worked out, they’ll be used instantly” (ref). With the expectation that they would “…soon be a standard biological tool” (ref).

So what happened? Check the published literature or walk into any imaging lab today and you’ll find antibodies conjugated to all manner of small dyes from FITC and rhodamine to Cyanine and Alexa dyes. Rarely will you find QDot-conjugated antibodies used despite them being commercially available. Why would people shun a technology that seemingly provides so many advantages?

Based on some strange observations, when trying to use QDot-conjugated antibodies, Jen Francis, investigated this phenomenon more closely, systematically labelling different cellular targets with Quantum dots and traditional small molecule dyes.

Francis_et_alFig3_GM

Figure 3 from doi:10.3762/bjnano.8.125 shows Tubulin simultaneously labelled with small fluorescent dye (A) and QDots (B). Overlay shows Qdot in green and A488 in Magenta. See paper for more details. 

The work published in the Beilstein Journal of Nanotechnology (doi: 10.3762/bjnano.8.125) demonstrates a surprising finding. Some targets in the cell such as tubulin (the ‘gold standard’ for QDot labelling) label just as well with the QDot as with the dye (see above). Others however, including nuclear and some focal adhesion targets would only label with the organic dye.

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The important question of course is: why the difference in labelling when using Quantum Dots or dyes? This is discussed in more detail in the paper but one explanation the evidence supports is that it is the size of the QDots that hinder their ability to access targets in the nucleus or large protein complexes. This explanation further highlights how little we really know about the 3D structure of protein complexes in the cell and the effect of fixation and permeabilisation upon them. Why for example, can tubulin be labelled with QDots but F-actin cannot, despite them both being abundant filamentous cytosolic structures? At this point we can’t say.

So why is this study important? Publication bias (the preferential publication of ‘positive’ results) has largely hidden the complications of using QDots for immunofluorescence. We and others have spent time and money, trying to optimise and troubleshoot experiments that upon closer study, have no chance of working. We therefore hope that by undertaking and publishing this study, other researchers can be better informed and understand when (or whether) it might be appropriate to use Quantum Dots before embarking on a project.

This paper was published in the Beilstein Journal of Nanotechnology, an Open Access, peer-reviewed journal funded entirely by the Beilstein-Institut.

 

Breaking up is hard to do: New insights into cell signalling revealed

Press release originally issued by the University of Liverpool on June 23, 2017:

A rewrite of biology classroom textbooks could soon be on the cards as scientists at the Universities of Liverpool and Washington reveal important new insights into how cells communicate with each other. The research is published today in the prestigious journal Science.

Cell signalling refers to the mechanisms cells use to communicate with each other. In humans, signalling normally regulates cell growth and repair and therefore contributes to diverse basic processes that control tissue physiology and brain function. However, abnormal cell signalling contributes to many diseases, including diabetes, cancer and neurodegeneration. For this reason, the proteins that control disease signaling are important targets for many types of clinically-approved drugs.

In the new study, the researchers focused on understanding how proteins assemble into higher order signalling complexes, which control aspects of cell communication and cell fate such as the decision to live or die, using the ‘textbook’ cyclic AMP (cAMP) signalling pathway.

In the late 1960s, cAMP was shown to activate an enzyme complex termed Protein Kinase A (PKA), which can exist in both ‘active’ and ‘inactive’ forms depending upon the type of complex assembled.  It had long been thought that cAMP levels were sensed in cells by a release of the active kinase component from the larger PKA complex, rather like ice cubes breaking apart after being added to a drink.

Using contemporary scientific strategies, including electron microscopy, mass spectrometry, chemical genetics and real-time imaging, the researchers found that instead of being broken apart by physiological levels of cAMP, the signalling complexes remain intact, directly delivering the appropriate message into the correct part of the cell interior.

The study was carried out by Dr Dominic Byrne and Dr Matthias Vonderach in the laboratories of Dr Patrick Eyers and Professor Claire Eyers in the Department of Biochemistry at the University’s Institute of Integrative Biology, in collaboration with Dr Donelson Smith and Professor John Scott and colleagues at the University of Washington.

Dr Patrick Eyers explained: “We believe the finding that PKA protein complexes respond to the second messenger cAMP in a different way than we had assumed for nearly half a century, might bring about other changes in how we understand cell communication, especially the type of signaling we study that involves protein modification (phosphorylation) by protein kinases.

“It might also prove important for a better biochemical understanding of how medicines affect PKA signaling complexes, allowing us to develop drugs with fewer side-effects.”

The next challenge for the team will be to try to explain how the larger PKA signalling complex functions in cells, and how it is regulated by various factors.

The research received funding support from the UK Biotechnology and Biological Sciences Research Council, North West Cancer Research and the Howard Hughes Medical Institute.

The paper ‘Local protein kinase A action proceeds through intact holoenzymes’ is published in Science [DOI: 10.1126/science.aaj1669]

Learning to Communicate – a Johnston Post-Doctoral Development Fund report

This is a guest post by Andrew Holmes, Postdoctoral Research Associate in the Mammalian Behaviour & Evolution group of the Institute of Integrative Biology.

 

The Johnston Post-Doctoral Development Fund enabled me to attend a Royal Society residential course in in communication and media skills in June 2017. The course was hosted at the Kavli Royal Society International Centre at Chicheley Hall in Buckinghamshire, a Grade I listed 18th century mansion set in 80 acres of beautiful grounds that has been used in films such as Pride and Prejudice and The Meaning of Life. Hidden amongst the trees near the house lurk two large fiberglass pterodactyls from an earlier Royal Society event, now abandoned and eerily weather-beaten.

Andrew1.pngImage: Chicheley Hall and gardens (left); Pterodactyls amongst the trees (right).

The course was run by Dr Jon Copley, an Associate Professor at the University of Southampton and former reporter and editor at New Scientist, and Geoff Marsh, a freelance multimedia producer and science writer for publications including Nature. It was great to ask them about their own experiences in science communication, in particular Dr Copley was able to provide insight into his experiences working with the BBC on nature documentaries.

In the first half of the course we discussed and practiced how to write short popular science articles, using the ‘inverted triangle’ approach to present what was most important in a concise and engaging starting paragraph and then going into more details as the article continued. This approach is great for communicating to non-specialist audiences as well as in the lay summary sections of grant proposals.

We also covered writing press releases, long-form science writing and using social media. I have recently started my own website (https://thescikuproject.com) using scientific haiku to explore research findings. I have very little experience of using social media and the course has given me the confidence to start using it to promote my own website and research.

Andrew2Image: Andrew Holmes (left); Chicheley Hall Gardens (centre); a resident of Chicheley Hall (right).

The second half of the course covered the media and science, discussing the differences in function, requirement and audience expectations between media types – radio, tv, print and online reporting. By learning how the media works and the requirements of journalists we were able to understand how to interact with the media and retain more guidance of how our work is reported.

We also practiced being interviewed: a ‘soft’ radio interview; a ‘hard’ radio interview with probing questions about the ethical and societal issues associated with our work; and a TV interview via a remote link. Discussing our work in different contexts and having an awareness of the practical requirements of media production has helped me feel more confident about interacting with the media and promoting my work to the public in general.

I felt the course was excellently run and covered some very interesting and useful topics that as scientists we aren’t often trained to consider. By learning how better to present my research to a variety of audiences and through a number of formats I feel much better prepared to use my communication skills to help improve the impact of my research and promote my science to the world outside of academia.

I thank the Johnston Post-Doctoral Development Fund committee for this opportunity and hope they feel that it was justified – I certainly feel that I gained a lot from it.

 

 

Visit to Cannon Sharples Primary School

On Tuesday 16th of May, Marie Phelan of the Technology Directorate (NMR Metabolomics) and two interns from the Ngee Ann Polytechnic in Singapore visited Cannon Sharples Year 6 pupils. The primary school in Wigan was holding a careers week, so as part of their #raising aspirations initiative Marie was invited to talk about higher education and scientific careers. In addition, interns Shina Teo and Xin Hui Er on their 4-month placement with the University of Liverpool spoke to the pupils about college life in Singapore and their experiences at the University. The 38 pupils in attendance gathered into groups to figure out what specialist skills various careers required and to play the celebrity education quiz.

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Marie talking about her career to year 6 pupils at Cannon Sharples Primary School in Wigan.

Interns Shina Teo and Xin Hui Er     Pupils pick skills for specific careers

The story behind the paper

We recently published a paper on genomic surveillance of a diarrhoeal pathogen Shigella sonnei across Latin America which represented the culmination of over five years of collaboration, as well as training and development in the region

Graphical abstract

In collaboration with the Wellcome Trust Sanger Institute, the Pan American Health Organisation and PulseNet Latin America and Caribbean (PNLAC), we whole genome sequenced over 400 Shigella sonnei collected from nine countries over two decades. Shigella are the most important bacterial cause of moderate-to-severe childhood diarrhoeal disease in low to middle income nations, and countries in Latin America still experience endemic disease and explosive outbreaks. By sharing information on common pathogen subtypes through public health networks, like PNLAC, pathogens can be traced epidemiologically to facilitate early identification and intervention in disease outbreaks. Whole genome sequencing is transforming surveillance of bacterial pathogens, as it provides the highest resolution of pathogens subtypes and can also be used to explore other genetic factors of interest, like antimicrobial resistance. However, its cost precludes routine use in some areas, which are unfortunately some of those regions where the most Shigella disease is seen.

In this study, we sequenced approximately 50 isolates from nine countries in Latin America and use whole genome phylogenetics to reveal those sublineages that were responsible for most of the disease in the region. We identified a novel global lineage of Shigella sonnei, and by correlating the geography of where isolates came from to their evolutionary relationships, we could see international transmission of some sublineages and what the distribution of different sublineages was across the continent. Visit the microreact page to play with the data yourself.

We were also able to identify key determinants of antimicrobial resistance in the pathogens and how they were distributed among the different sublineages, providing key information for managing this important disease in the region.

In addition to constructing this invaluable regional framework for ongoing surveillance, this project helped build capacity for whole genome sequencing surveillance in the region. Over the course of the collaboration, the World Health Organisation sponsored the establishment of whole genome sequencing facility at the reference laboratory for PNLAC, ANLIS in Buenos Aires, Argentina (see photo). In the paper, we show how locally-generated sequencing data from this facility can be integrated into the regional surveillance framework to determine whether outbreaks were due to locally-circulating lineages or resulted from the importation of new sublineages.

In addition to laboratory capacity building, the collaboration involved training an ANLIS researcher (Josefina Campos – see photo – who now runs the genomics facility there) in bioinformatics, and conducting training courses (in conjunction with Wellcome Trust Advanced Courses) for medical, veterinary and public health professionals in the region, including courses in Argentina, Uruguay and Costa Rica (see picture).

There are 29 authors on our paper and every one of them worked hard on, and cared deeply about, the outcome of the study as well as the training programs and capacity building surrounding it. Every paper has a story behind it, and this one, like so many others, is so much more than it appears.

Photo: Top ANLIS in Buenos Aires, Argentina. Bottom (from right to left) ANLIS collaborator Josefina Campos and co-corresponding author Nicholas Thomson (WTSI) outside the Malbran (ANLIS) Institute; Genomics for Epidemiology and Surveillance of Bacterial Pathogens course instructors and participants held in February 2015 in San Jose, Costa Rica; co-corresponding author Kate Baker with bust of Carlos Gregorio Malbran, the ANLIS institute’s namesake.

Lay summary on Latin America.png

IIB visit from Alzheimer’s Society research grant monitors

IIB received a visit on Thursday 15th June from a group of public volunteers who act as lay reviewers of research grant applications and also monitor ongoing research funded by the Alzheimer’s Society. They were hosted by Prof Jerry Turnbull who is currently undertaking preclinical research funded by Alzheimer’s Society on candidate heparin-based drugs aimed at lowering amyloid levels. It is hoped that these might provide a safe early treatment to tackle an underlying cause of the disease, since current treatments only tackle disease symptoms. The research monitors were taken on a tour of the lab and updated on progress with the ongoing project. This was followed by lunch and lively discussions with Jerry Turnbull, Ed Yates and Jill Madine and their lab members.

Alz Soc visit 15 June no2

Grasslands at Ness Family Science Fair

Raj_Ness_Science_Fair_Mar_2017

Raj Whitlock and PhD student Di Yang took part in the Ness Family Science Fair for British Science Week in March. They manned a stall with model grassland communities, and talked with members of the public about the importance of plants, how plants work, grasslands, their conservation and threats to plants from climate change. Their installation at the science fair also included a grassland-inspired arts area, which became a popular relaxation point for parents and kids in the hustle and bustle of the event (which was attended by ~1500 people).