Coronavirus – data mining

WARNING. I am not a medical doctor nor an epidemiologist. The analysis I am sharing here is only for the data geeks around that are curious. Please follow advice of your national authorities and health system.
NOTE. This post was updated on 15/3.

The data | The data repository for the 2019 Novel Coronavirus Visual Dashboard operated by the Johns Hopkins University Center for Systems Science and Engineering is available on GitHub.  To adjust mortality rates by local demographics, I have downloaded the population pyramid data from www.populationpyramid.net. There are several estimates for age-dependent mortality. I was able to find only the following pre-print for mortality in Hubei compared to the rest of China. The dataset analysed was small, let me know if you find something better.

The software | In spare time, I prepared a bit of Matlab code that can import the JH data and does just two simple things: compare trends between different countries and compare age-adjusted moralities. The code is available on GitHub. Keep in mind, sorry to repeat, this is just for the curious geeks.

Trends

I summarize a few countries I checked. As I am Anglo-Italian, and with Italy and UK having adopted very different strategies to fight conoravirus, I developed this code to check trends between UK, Italy and Hubei. It is interesting that these three territories have similar population sizes but, until now, experienced the epidemics in different ways. Hubei got off-guard because it is the origin of the epidemics. Italy, together with South Korea and Iran got off-guard because they thought the coronavirus was somehow under control. UK might have done fewer mistakes so far and controlled the spread of the virus better and it has decided not to further contain the epidemics against WHO advice. Let’s see.

I synched the curved to a number of confirmed cases equal to 400. By chance, this is about the number of cases since we have data from Hubei, the day when Hubei went into lockdown, and a similar number when UK decided not to contain the virus.

Italy, just before and just after introduced first a local lockdown and then a national lockdown. Italy and Hubei seem to be on a similar trajectory of confirmed cases. For the UK is too early to say. We should keep in mind that confirmed cases depend on the methodologies of testing. Hubei’s and Italy’s health systems got overwhelmed, therefore it is possible at a certain point might have struggled to test the general population. The UK has decided to stop screening the general populations. Therefore, the reported deaths might be more realistic as numbers. At the time of writing, the JH dataset is one day behind, but we know that the UK is now in line with the other curves, and Italy is overshooting Hubei’s trajectory. Mortality rates are heavily affected by the reporting of confirmed cases. We will know the actual mortality rates only after epidemiologist will be able to do their statistical work retrospectively. More on this at the end of this post.

What about other countries? South Korea is interesting as they did not go into lock down but they also had a major outbreak. They were able to contain it by tracking those infected.

Assuming that Korea counted all covid-related deaths, their strategy was rewarded with a successful containment and lower deaths than other regions. Spain seems to be the EU country that will struggle next, let’ see…

Unfortunately, it seems that Spain is on the same trajectory compared to Italy and Hubei. But remember, Hubei succeed to contain the outbreak, which gives hopes. This and the experience in Korea is why WHO is still recommending to attempt containing the virus.

The same is true for France.

What about Germany?

For confirmed cases, Germany looks like on a similar trajectory. However, unless I did a mistake, the mortality rate seems very low. There are reports on the news that Germany considers as covid-related deaths only those patients who did not have other important related pathologies.

This, of course, would bias completely the curves we presented, but the situation in Germany might be not different form other countries. We’ll understand this in the future. Now a few comments on mortality rates. Initially, many of us were puzzled by the differences in mortalities between countries. There are several factors that influence these statistics: i) confirmed cases are underestimated in different ways in different countries because of testing capacity or policy. ii) covid-related deaths seem to be accounted similarly in many countries, except for Germany. iii) different countries have different demographics and iv) when a health system is strained both mortality might increase and confirmed cases decrease. All this considered, I just thought to give a reference for demographic adjustments.

I used mortality figures in Hubei and rest of China as to estimate the worst and best case scenario for an overwhelmed and a coping health system. The red and blue curves are these values adjusted by demographic differences in each country.

Then it seems that the high mortality in Italy is just demographics. Pay attention that these are cumulative statistics and, therefore, even if the situation improves massively like in Hubei, the mortality remains high because historically it was high. Thus, so far it looks like that only in Hubei and Italy the outbreaks arrived to the point to fully overwhelm the health systems. However, check the drift of the Italian curve, that is what might (hopefully not) happen in other countries that are on similar trajectory.

Keep in mind, I am no expert. I think however that there are two possibilities that explain this, and probably they both coexist. First, when ICU is overwhelmed, we rescue fewer people. Second, when a country is overwhelmed, there might be also fewer testing. So, plenty of limitations in this data (mortality rate data are no great, I am no expert, and several factors might explain the trends)… but at least there is some pattern that might indicate what is happening.

To conclude. Every country can still do what Hubei did. Not my work, but WHO’s. We need to protect the most vulnerable waiting for the vaccines and drugs that WILL come. Take care and find ways to keep positive and help people around you!

Coronavirus – getting ready in a lab

I am publishing here the recommendations I circulated to my colleagues, as this might help others to formulate their strategies or me to receive suggestions on how to improve. At the bottom of the post I also share my opinion about the situation, just to explain why we are taking action. Disclosure: I am no expert in this area, therefore I analysed data just to form my own opinion and to organize our work. Please check institutional guidance and reports.

Dear all,

      While we can still hope that no major disruption will occur, it is increasingly likely that the epidemic will not stop any longer. What is concerning, from a logistics point of view, is that this might last for several months as the responses of the public authorities will focus, rightly, in slowing down the epidemics. I would like to invite you to observe some basic rules, but also reflect on issues you might have not considered:

  1. The most important thing is to address the upcoming months with a scientific mind and no panic. Please follow the indications provided by the NHS, WHO, and the University.  While I do not doubt that all of us already wash their hands! Please do so also when you come in from outside, something we might usually not do.
  2. I am happy for you to work from home when you can. Most of you will have to carry out experiments, but I am happy for you to cluster reading and to write in specific days and to work from home. Would you need access to your computers from home, just organize this with IT but the Unit will provide appropriate IT arrangements soon.
  3. Some experiments could be rather expensive. Please let me know when this is happening so we could plan them properly. I would like not to delay important work though, so we might take some risks (on funds not on safety) but we could manage these risks proactively. For example ensuring that very expensive steps are executed in the shortest period of time and with sufficient people being aware of the experiment. For example, we have several commercial and in house developed cell lines that have not been archived yet.
  4. I can foresee two situations where we need to help each other. First, the case where a single individual will self isolate and they need help to store, throw materials, or shut down a microscope. I think Slack will suffice, but we should have also a ‘buddy system’. For example, if I started an experiment on a microscope that would last two days, I can inform someone else who would have the expertise to safely terminate the experiment.
  5. The second case is a bit more extreme but not really unlikely as other university campuses around the world have been closed. The Unit will soon provide specific guidance. Please think about which element would be critical, for example we will have to shut lasers down, air compressors to avoid them running out of oil etc.
  6. Do consider if you travel, even just within the UK, you might get stuck somewhere. Please check the University policies that are updated daily. I will not recommend specific actions related to personal trips, except to comply with public health guidelines and to think about the possible consequences to get stuck at home in one or another town.
  7. There is no indication – at the moment – that we will experience disruption to the supply chain. However, this might happen. Have a thought if we will run out of some consumables in a couple of weeks and perhaps order now.
  8. Also, very important. There are people coming from areas that are quarantined. Unless you are sure they are breaching rules, be supportive and do not make too many jokes. Some people are more sensitive than others.

To conclude, please do not allow the situation to make you anxious or too worried. For the general population, the main issue in not health but arranging life around likely restrictions of movement to permit the NHS to cope with the extra workload. For us, provided we will put first safety of ourselves, colleagues and family, we have opportunity to keep reasonably productive even in this situation simply organizing.

Feel free to propose ideas or to have a chat with me in private if you have any specific concern.

My opinion on the situation and on what is happening

I am growing of the opinion that the Italian situation is happening only earlier than in other European countries, not that is a special situation. Spain, France and Germany might be already on that path (10-14 days of delay compared to others). UK is probably an extra week late, meaning that by the beginning of April, or earlier, we will experience similar disruptions we are observing in Italy (hopefully not). Also, I had a look at mortality rates. Once taken into account the demographic and that in Italy we are experiencing a situation similar to Hubei (health systems overwhelmed) and not to the rest of China (managed containment of the disease), the stats of Italy do not seem odd to me any longer.


At this point, all other European countries will experience the same unless they enact strong preventive measures. To me it seems governments in Europe and USA have preferred to shield economy first rather than people, or they are simply incompetent, to then get caught off-guard and inflicting to the economy the same level of damage they would have got intervening earlier.
We can organize, minimize disruptions and deaths. Not eliminate them but we can do better we are doing. If only politicians would exert leadership, at local and national level, and – of course – people would comply with the indications…


Last word of caution. People might be complacent also thinking they have the best health systems. This is not the issue, the UK system will be as easily overwhelmed as the Italian one. In fact, there are fewer ICU beds in the UK than in most EU countries, including Italy. The point is to slow down the spread of the disease to keep our health system working within certain operational margins.

Bottom line. Am I writing to get your more worried or anxious? NO. The large majority of us will have minor health issues. However, the public health policies that will be necessary to minimize the negative impact on the NHS will cause major disruptions. Therefore, organize not by panic buying, but thinking ahead… how to work, look after family, etc, etc, when restrictions will be imposed.


Last thing. There is a tendency to minimize the situation as people dying is elderly affected by other pathologies. In Germany, it seems that they do not even consider those patients as CoV-related. The large majority of those people could have lived a much longer life, they are not (all) terminal patients. Moreover, with patients piling up in dedicated wards and ICU, everyone risks more because they will not receive adequate treatment, irrespective if they have been infected or not.


So… the apocalypse is not coming, but just the time to work together to get pass this.

Tackling cancer heterogeneity by live single-cell ‘systems biology’.

NOTE: This assay is the introduction to my research vision I wrote five years ago but that did not make into the programme grant we wrote. I think this is still current and, as it is unlikely I will publish this text, I am releasing it in the public domain with very little editing. I should note, particularly, that some paragraph remains unreferenced.

The genome, biochemical networks and phenotypes | Somatic mutations and gene copy number variations (CNVs) accumulate over time, stochastically altering the abundance and the functions of gene products. At first glance, efforts to identify and characterize somatic mutations provided a comparatively simple model: a few hundreds genes (proto-oncogenes and tumour suppressor genes) are often mutated contributing mechanistically to tumourigenesis (driver mutations). Some driver mutations are very frequent, but driver mutations that are less frequent in a cancer type overall dominate in number within an individual tumour, presumably conferring a more subtle growth advantage than others taken individually [1,2]. Also CNVs are very common in cancer. Sometimes, a clear role of CNVs in tumourigenesis can be established; most of the times, however, the effects of CNVs are difficult to predict or characterize because of the very different possible dependencies between phenotype and concentration of a gene products (e.g., haplo-insufficiency, quasi-sufficiency, triplo-sufficiency, etc.) [3,4]. Concentration effects and “subtle driver mutations” complicate the interpretation of genomic studies and may be best described by a continuum model for tumorogenesis where the all-or-none effects of individual genomic alterations are the frequent exception rather than the rule [4,5]. Notwithstanding the invaluable insights that genomics studies have provided and will continue to provide in our understanding of cancer, diagnostics and therapy, the role of these genomic alterations in tumorogenesis will be better understood in the context of the alteration of molecular networks underlying the respective cancer-associated phenotypes [1,6].

Few phenotypes are selected by mutation, those that enable cancer evolution [7] by increasing clonal heterogeneity (by genetic mutation, aneuploidy or epigenetic instability) and that permit growing in a hostile environment (avoidance of immunosurvaillance, metabolic deregulation and stromal hijacking). Moreover, cell survival, cell fate determination and, later in cancer evolution, cell migration are the key phenotypes that make of cancer the devastating diseases it is. Genomic alterations select for these phenotypes by influencing a comparatively small number of biochemical networks. Indeed, cancer-associated somatic mutations cluster in pathways controlling cell-cycle or cell-death, RAS/PI3K/MAPK, TGFβ, APC, STAT, NOTCH, WNT, HH and mTor [1,4,7]. Unsurprisingly, somatic evolution of cancer reshapes a comparatively small number of biochemical pathways that control cellular and tissue homeostasis to offset, often in a subtle manner, the net proliferative rate of cells. The study of these pathways is no less daunting than the understanding of complex genomic alterations. However, biochemical networks have evolved to exhibit robustness in the presence of intrinsic noise present in biological systems (e.g. stochastic variations in transcription or cytokines concentrations). Robustness of biochemical pathways permit to stably encode for cellular functions and cellular states. It is therefore conceivable that the myriads of possible genomic alterations and individual gene-products simply concur to generate a discrete set of biochemical states corresponding to cancer-associated phenotypes.

Other “big data disciplines” (e.g., transcriptomics, proteomics and metabolomics) have provided the opportunity to study the working mechanisms of biological systems alongside genomics. Some groups have suggested that integrative biology [8,9], the effort to integrate data from these various disciplines, may permit avoiding the biases and inherent flows of individual –omics techniques and, at the same time, may deliver a new approach to the study of human disease. This approach is summarized by the term “network medicine” highlighting that molecular networks altered in disease can be both the target for future therapeutic strategies and the possible source of novel biomarkers. A biochemical network, common to many different cell types or even species, exhibit a different “network utilization” in different physiological and pathological contexts. Mutations can therefore offset the utilization of molecular networks and their dynamics. On the one hand, better understanding of how networks encode functional states and cellular decisions under physiological conditions and how these are altered in disease will offer more and better targeted therapeutic opportunities. On the other hand, defining cancer-associated network utilizations and engineering tools (probes and instrumentation) to reveal them will provide fundamental insights to optimize patient stratification for improved theranostics and prognostics.

Heterogeneity, causality and phenotypes | Phenotypic heterogeneity, including genetic and epigenetic polymorphism, and polyphenism, is at the basis of both unicellular and complex lifeforms. These three levels of phenotypic heterogeneity are recapitulated in cancer and constitute often insurmountable obstacles to effective therapeutic intervention. Intra-tumour heterogeneity, either within the primary tumour, within a metastasis or between different metastatic foci is indeed the primary cause for the emergence of drug resistance and tumour relapse. The genetic basis for phenotypic heterogeneity within a tumour is rather established. However, other non-genetic factors can be regarded as equally important.

For instance, upon treatment, a fraction of tumour often exhibit drug resistance. In part, this can be caused by pre-existing tumour cell clones carrying mutations that, by chance, will confer resistance to any given drug. Alternatively, this may be caused by tumour initiating cancer cells, stem-like cells that are usually quiescent, less vulnerable to treatment and that can regenerate the tumour upon termination of the therapy. Moreover, non-Darwinian mechanisms for the emergence of drug resistance have been proposed as well, whereby cells trigger a transient drug-resistant phenotype that, in time, can be then converted to a stable inheritable state by subsequent somatic evolution.  Fractional killing may also be explained by non-genetic heterogeneity. For instance, Spencer et al. have shown that in a clonal population of cells, TRAIL elicits a heterogeneous phenotypic response with cells undergoing apoptosis at different times or surviving indefinitely. The authors elegantly demonstrate that this phenomenon is caused by stochastic variations in the abundances of the many proteins involved in the apoptotic molecular network.

Genomics, transcriptomics, proteomics and metabolomics allow the characterization of tens of thousands of biomolecules at the same time. Furthermore, the increasing sensitivity of these techniques provides – or may provide in the future –  single cell “–omics” characterization. However, the invasiveness of these techniques will limit their applications to the study of individual time points. Thus, causality can be established only by inference. Techniques capable to provide low invasiveness and biochemical information on living cells are thus extremely useful to complement models derived by ‑omics techniques and to provide a tool for testing hypothesis derived from analysis of big data.

It is thus evident that time-lapse imaging of individual living cells with biochemical information is strategic for the understanding of the heterogeneous response of biological systems and to establish causality between biochemical events and cellular decisions. At the same time, genetic heterogeneity within a tumour and between tumours induces differences in network utilizations with significant consequences for prognosis and treatment. Also in this context, biochemical imaging techniques are necessary to understand the phenotypic heterogeneity of a tumour and, at the same time, may be useful to define network-based biomarkers.

The next generation of Systems Biology | Several groups have identified the need to integrate fluorescence microscopy in the systems level study of the cell and organisms [10-15]. The term “Systems Microscopy” has been suggested for the description of microscopy tools applied to this field [14]. In order to strategically complement other approaches, Systems Microscopy has to deliver single cell resolution, temporal characterization of living cells and high quality quantitative data and has to be applied to the most appropriate biological context (e.g., for epithelial cancers, adherent 2D, 3D, organotypic cultures or in vivo rather than in suspension or cellular homogenates) [10]. Whereas –omic techniques can sample the biological space over the fullness of biochemical moieties (genes, RNAs, proteins, metabolites) albeit with poor sampling of individual cellular behaviours and spatio-temporal organization, Systems Microscopy samples the fullness of the spatio-temporal organization of molecular networks but reports about a limited number of gene products or biochemical events [10]. Therefore, Systems Microscopy elegantly complements big data studies.   

We envisage two (not mutually exclusive) approaches to Systems Microscopy: high throughput screening platforms and single cell biochemical multiplexing. High Content Screening (HCS, also known as imaging cytometry or high throughput imaging) is the current tool of choice for Systems Microscopy. Relying on robotics, automation and unsupervised or semi-supervised data analysis, HCS enables the screening of large numbers of cellular perturbations (e.g., siRNA or compound libraries) with commercial instrumentation making the correlation of these perturbations with morphological estimators and fluorescent markers possible. Several groups have also highlighted the importance of integrating quantitative biophysical imaging techniques such as Fluorescence Correlation Spectroscopy (FCS) and Foerster Resonance Energy Transfer (FRET) in Systems Microscopy in order to deliver data of high quality. Despite this, HCS has been integrated with these techniques only in a few academic-based efforts [16-19]. HCS expands the sampling of biological space of imaging technologies to deliver another set of “big data” but with single cell resolution.

We are pursuing a different approach to Systems Microscopy that maps in space and time an increasing number of fluorescent markers within the living cell. Fluorescence is not amenable to the simultaneous detection of many fluorescent molecules because of the broad excitation and emission spectra of common fluorophores. Therefore, we are determined to develop new techniques (bioprobes and instruments) that exploit all properties of light (photon arrival times, colour and polarization) efficiently to maximize the biochemical resolving power of microscopy. We aim to monitor nodes of molecular networks (e.g., quantifying the dynamic phosphorylation of several substrates) in living cells in response to stimuli, discerning between physiological and pathological (oncogene-driven) network behaviour (topology). The integration of Optogenetics tools (e.g., light-inducible oncogenic signalling) enables perturbational analysis of biochemical networks and facilitates the execution of complex biochemical imaging assays fully automated with no requirement for sample manipulation other than by light. Therefore, these techniques will be strategic for the study of biological networks at low throughput with high quality data; thanks to this all-optical approach, they may also be integrated with HCS increasing the quality and quantity of information and decreasing steps in chemical manipulations of the samples (e.g., addition of doxycycline to stimulate the expression of a gene)

[1] Vogelstein et al. (2013) “Cancer Genome Landscapes” Science
[2] Wood et al. (2007) “The genomic landscapes of human breast and colorectal cancers” Science
[3] Solimini et al. (2012) “Recurrent hemizygous deletions in cancers may optimize proliferative potential.” Science
[4] Davoli et al. (2013) “Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns to Shape the Cancer Genome” Cell
[5] Berger et al. (2011) “A continuum model for tumour suppression.” Nature
[6] Jorgensen&Linding (2010) “Simplistic pathways or complex networks?” Current Opinions in Genetics and Development
[7] Hanahan&Weinberg (2010) “Hallmarks of Cancer: The Next Generation” Cell
[8] Erler&Linding (2010) “Network-based drug and biomarkers” J Pathology
[9] Barbasi et al (2011) “Network Medicine: A Network-based Approach to Human Disease” Nat Rev Genet
[10] Megason et al. (2007) “Imaging in Systems Biology” Cell
[11] Verveer&Bastiaens (2008) “Quantitative microscopy and systems biology” Histochem Cell Biol
[12] Ankers et al. (2008) “Spatio-temporal protein dynamics in single living cells” Current Opinion in Biotechnology
[13] Pepperkok&Ellenberg (2006) “High-throughput fluorescence microscopy for systems biology” NAt Rev Mol Cell Biol
[14] Lock&Stromblad (2010) “Systems microscopy: An emerging strategy for the life sciences” Exp Cell Res
[15] Conrad&Gerlich (2009) “Automated microscopy for high-content
RNAi screening” J Cell Biol
[16] Esposito et al. (2007) “Unsupervised fluorescence lifetime imaging microscopy for high content and high throughput screening” Mol Cell Proteomics
[17] Mathews et al. (2008) “A high-content screening platform utilizing polarization anisotropy and FLIM microscopy” SPIE BIOS
[18] Talbot et al. (2008) “High speed unsupervised fluorescence lifetime imaging confocal multiwell plate reader for high content analysis” J Biophotonics
[19] Barber et al. (2013) “The Gray Institute ‘open’ high‐content, fluorescence lifetime microscopes” J Microsc

Our superpower is you (first draft)

This is a preliminary draft for a leaflet aimed at outreach events. Following the work we have done on Women in STEM at the last year Cambridge Science Festival, this year we’ll add a second leaflet to be more inclusive. This will be a word search puzzle that we will release into the public domain for your peruse. The idea is to have a list of people to include different genders, ethnicities, sexual orientations and disabilities. While my team is working on the graphics and the production of the leaflet, I prepared the first draft of the name list. As I believe it can be certainly improved  I wished to ask for suggestions and criticism.  

The message: “There is not much in common among these inspirational people, no colour, no gender, no physical ability. Hard working and smart, nothing else defines a scientist. A scientist is someone like you.”

Alan Turing (1912-1954),  Mathematician, a founder of Theory of Computation

Florence Nightingale (1820-1910), Social reformer and statistician, the founder of modern nursing. nurse

Neil Divine (1939-1994), Astrophysicist, a major contributor to the modern theory of
star formation.

Lynn Ann Conway (1938), Computer scientist and electrical engineer. Pioneer in electronics and computing.

George Washington Carver (1860-1943), Agricultural scientist and inventor. Promoted alternative crops to cotton and methods to prevent soil depletion.

Ernest Everett Just (1883–1941), Biologist, a pioneer in the studies of fertilization and early development,

Stephen Hawking (1942-2018), Theoretical physicist and cosmologist, a pioneer in the modern theory of cosmology and black holes.

Edwin Krebs (1918-2009), Biochemist. Pioneering work on post-translational modification of proteins and cell regulation.

Albert Einstein (1879-1955),  Theoretical physicist, founder of the theory of relativity, one of the two pillars of modern physics.

Charles Darwin (1809-1882), Naturalist, geologist and biologist, a pioneer in the science of evolution.

Rita Levi-Montalcini (1909-2012), Neurobiologist, pioneering discoveries in neurophysiology.

Chien-Shiung Wu (1912-1997), Experimental physicist who made significant contributions in the field of nuclear physics.

The design of the leaflet will be based on a diverse group of superheroes, with the message “Our superpower is you” meaning that science main resource is one: people.

 

 

This is ATLAS.ONE (a high speed high resolution biochemical imaging platform)

Project ATLAS

In 2018, we decided to invest capital funds provided by the MRC and the MRC-DBT with the aim to make our technologies more accessible to the biomedical researcher laying down also the possibility to deliver advanced biophysical assays at high throughput [REF1] with a focus on 3D cultures. Why ATLAS? I often code-name internal projects, possibly with evocative names that might capture the pathos of the project. As these are capital investments to strengthen specific areas that will be essential for our long-term applications, I named this investment ATLAS, as the Titan that was condemned to hold up the heavens on its shoulders. In our case, I am building the base for two microscopes that will support my research projects, and support those of the others working at the MRC CU, in the longer period.

This is ATLAS.ONE

Here, I will briefly introduce ATLAS.ONE, the first of the two microscopes we have started to develop. The aim is to develop high spatiotemporal and biochemical resolution with a imaging platform that could be readily accessible by a non-expert user. We are testing the solid-state FLIM (Fluorescence Lifetime Imaging Microscopy) camera PCO.FLIM by PCO for video-rate biochemical read-out of genetically encoded probes or protein-protein interactions.  This is the commercial incarnation of part of my PhD work [REF2REF3] so brilliantly developed and delivered by PCO (no commercial conflict). After considering different possibilities to gain some resolution to better discriminate cellular compartments, we decided to integrate this platform with a simple SIM (structured illumination microscopy) setup based on LCoS spatial light modulators. Biochemical perturbations will be implemented with a CellASICS microfluidic platform. This is a capital investment and we will first focus on methodological advancements, however, we will deploy this platform to characterize genetic and non-genetic heterogeneity in cancer cell lines. While we will look for external funding, we’ll start working on KRAS-dependent signalling pathways and metabolic pathways.

20190110_095745

Who is involved?

You are welcome to get in touch if you wished to coordinate developments or to use this platform – once established – either with own resources or common grant applications. Currently, ATLAS.ONE is supported by the MRC and the MRC-DBT for capital funds and the following people for development and applications.

Andrew Trinh and Alessandro Esposito (MRC Cancer Unit), developing the system and applications together with Christian Frezza and Annie Howitt (MRC Cancer Unit), developing single-cell metabolic assays.

Guy Hagen, University of Colorado, to collaboratively develop SIM

Gerhard Holst, PCO to advise on camera integration.

 

SOP – Ti:Sapphire / Leica SP5 alignment

This SOP is published only for a social media discussion. The author does not take any responsibility for the utilization of this procedure. The system discussed here is a customized two-photon microscope, based on a Coherent Chameleon Vision 2 and Leica SP5. The optical path is fully enclosed and the SOP is written for maintenance. 

Basic rules

1.       Align laser with a lid room (smaller iris, smaller damage to the eye)

2.       Never align eyes with the height/direction of the laser beam

3.       Use the most appropriate personal protective equipment such as goggles and a white lab coat

4.       Perform laser alignments with the least number of people present in the room. Ideally, alignment is a 1 person job and a colleague is aware you are performing this task, within core hours

5.       Use devices such cards, cameras and viewers to visualize the laser beam

6.       Take short breaks every ~45 minutes of work. Do not continue alignment if too tired. Alignment of optics can be a stressful and lengthy procedure; try to identify the right moment to take a long break to relax

7.       Alignment is carried out only by authorized users

PPE for Ti:Sapphire laser

          VC5 IR card viewer from Thorlabs. WARNING: card viewers reflect part of the laser beam. Therefore, they must be used with caution, strictly using protective goggles, directing reflection away from the eyes

          Hand-held IR viewer from Newport. WARNING: hand-held IR viewers limit dexterity and must be used always with protective goggles.

          LG9 Amber lenses from Thorlabs. OD5+ on the 720-1090nm range; OD7+ on the 750-1064nm range. WARNING: goggles never fully protect from direct high power laser beam.

List of authorized users

Alessandro Esposito (MRC Cancer Cell Unit)

Coherent’s field engineer can align the laser under their own responsibility. Coherent’s field engineer can align the beam path until after the Pulse-Picker. The rest of the optical path must enclosed at any time or isolated with a beam stop.

Leica’s field engineer can align the complete beam path under their own responsibility with the exception of the Pulse-Picker. Alterations of the beam path have been discussed with Leica representatives.

Standard Operating Procedure

Room preparation

1.       Show warning at the door

2.       Lock the door

3.       Switch on the system as needed (shutters ON)

4.       Wear PPE as appropriate

5.       Open the beam path as needed (keep lens tube arriving to the scan-head until the last moment)

6.       If a large section of the beam path is opened, always block the laser beam with the beam stop after the optical element that is aligned in order to avoid the laser beam being reflected in dangerous directions (eye, skin, fire hazard) when misaligned

Beam alignment

7.       Always activate laser shutter when the beam is not undergoing alignment

8.       Apply #6 every time a section of the laser beam is aligned

9.       Start laser alignment, proceed with pairs of mirrors from the position closer to the laser up to the scan-head, trying to operate the laser beam within a central part of the mirrors

10.   Always ascertain that all optomechanics is stably connected to the optical table and that no optical device can fall, tilt, flip…

11.   Re-aligned section should be covered (at least temporarily) while progressing towards the scan-head

12.   When arrived at the EOM, remove the device (Leica’s shutter and half-plate may be removed as well). WARNING: the entrance window of the EOM is located within a brass cavity. Upwards reflections of the laser are possible.

13.   Using irises, make sure the laser beam is parallel to the table

14.   Reposition the EOM, coarsely aligned to the laser beam. WARNING: after the EOM there is a periscope. Use a beam-stop before the periscope, beam reflection towards undesired direction is otherwise possible.

15.   With a power meter, measure power of the laser before the EOM. Relocate the power meter after the EOM and iteratively maximize power through the power meter with the EOM in “high” state.

16.   Coarsely align the periscope if necessary, then reintroduce Leica’s shutter and half-plate if previously removed. BE SURE the periscope is locked to the optical table in a stable manner.

17.   Remove lens tube and MFP cover.

18.   Install Leica’s alignment tool on the scan-head

19.   Iteratively align the front iris of the alignment tool and the back aperture of the alignment tool.

20.   WARNING. During the iterative alignment of scan-head, PPE is usually hindering an already lengthy procedure. Avoid removing PPE. Check actions to be taken.

21.   When the two apertures are aligned, start scanning trying to see fluorescence from a bright sample on the screen. Keep adjusting alignment and MFP screws until alignment is completed.

Preparing the room to normal operation

22.   Close the optical path. Before securing all covers and panels, check that the alignment is still ok.

23.   Secure all safety panels

24.   With the laser ON, shutter OFF and during scanning, verify with the IR viewer that no beam is exciting the enclosed laser path.

25.   Remove safety warning on the door and operate equipment as normal.

Embrace your public speaking anxiety

About a decade ago, I went to a PI during a retreat to ask a question. Nervously, but politely, he asked me to be left alone as he was rather anxious for a talk he was about to deliver.  A few hours later, a PhD student at the time, I was freaking out for my own talk, but it was comforting, in a way, to see that an established scientist I highly regarded and I had considered rather self-confident was in a similar state-of-mind.

Comforting? Why not scary? Would you never get rid of public speaking anxiety? I am no anxiety coach and, for that, browse around. However, I wished to share my own experience as it might be useful for students. I now noticed I am that ‘senior’ scientist at that retreat (or something similar) and that junior colleagues might misunderstand my confident speaking in public as evidence of no-stress, no-shyness, a gift from birth. So, even though your solution might be a different one, here I tell you which was mine.

Be prepared! Be prepared? (take 1)

Trivial, isn’t it? I am not going to give practical suggestions here, except set yourself comfortable deadlines. With experience, you will be able to work on a talk until a few minutes before delivery, but earlier in carrier, you have to prepare all your material far in advance. However, even very experienced academics and businessmen when facing more unique scenarios work hard to prep a meeting and give this enough time and resources.

Be prepared! Be prepared? (take 2)

Perhaps, the most difficult thing you might find, it is to commit to a deadline, after which you have to be ready. But, here the challenging bit, even if you feel still unprepared (and some people may never be able to shred off that feeling) or if you are actually unprepared because you miscalculated something, you have anyway to commit to the next difficult bit, be mentally and physically prepared, something you might be completing neglecting. Deadlines are deadlines and the starting time of your talk is unmovable. Therefore, start to mature a process and to understand how long you need to be ready before a talk. Some people is a natural and need no or little preparation. Other people need time: never underestimate how long time you need. Most of my following comments are about this stage of preparation. The bottom line, when the deadline strikes, be sure you are ready and if you are not, do not allow doubts to undermine the next phase of preparation.

Commit physically: water and energy

During a stressful moment, your physiology will be heavily altered and you might lose control. So, think how not to. Personally, before a talk I try to drink lots of water to ensure I will be properly hydrated, and I also make sure I have water available during the talk. Once I didn’t, and I was not well. I coughed though all my talk and it was not a very ideal situation. Also, be sure you have energy, so a bar of chocolate or a juice, can help. Ah… ok, is this obvious?… pay attention – water in > water out. As basic as it seems, be sure you went to the toilet at the latest opportunity before the event. You do not want to be dehydrated, but even not to be distracted by your bladder while on stage.

Mind you that this is even more true when you have very long days, such as more articulated interviews or conference commitments.

Commit physically: oxygen

Breathing, for me, is the next most important issue. You might find yourself in need of oxygen after a few slides and attempting to do the world record in apnoea while speaking in public. You could pass through an entire 20 minutes presentation incapable to breath properly, increasing your level of anxiety at each slide. You are in front of an audience, it could be two people at an interview, or a thousand people in a theatre, if not a million in TV. However, giving a good breath permitting your lungs to be completely emptied and filled with fresh air takes a few seconds. This can be easily concealed in a transition between two slides, or during a question. And… if you cannot conceal it… do it anyway, 5 seconds spent silently breathing properly will be immediately forgotten by your audience, but a poorly delivered 20 minutes talk will be remembered.

Once again, get ready for it. First of all reflect on your breathing habits, far away from a talk. If you give enough thoughts about the issue, whenever you will struggle, a mental trigger will snap and make you aware of the occurring issue for you to take action. More importantly, if issues in breathing are recurrent for you, just do exercises in the 5 minutes preceding your talk. Breath in deeply and breath out slowly. This will decrease your anxiety and will prepare your breathing for the talk. You can do it while seating in the audience or even while speaking with others.

Commit physically: avoid distractions

Personally, I have a routine. Before a talk, I remove everything from my pockets, or even the badge, anything superfluous. After a few talks delivered with my pockets inside-out dangling from my trousers, I also double-check that I am generally presentable! So, on stage or seating in front of a panel, I have no distractions from the badge hitting the microphone, the phone vibrating, the keys stuck in my thigh. Well, the phone: switch it off well in advance of your talk and dump everything in you bag.

Commit mentally: have fun

Those were a few suggestions, and more or different tricks will work for you, to ensure your physical state will be ready to support the potential stress you might experience while speaking in public. Of course, your state of mind will play an equally important role. Perhaps, I should advise to not care, to convince yourself that the event you are preparing does not matter. This is probably key, more in general, to achieve the resilience necessary in the academic world. For me that does not work very well, as I tend to be heavily invested in everything I do. So, what it works for me is to repeat myself I need to have fun speaking about science, my work, or the work of others – otherwise is really not worth. A bit of self-couching targeted to focus your mood towards excitement, how great can be to speak or debate science.

I did receive my dose of criticisms in my career, but let me tell you which is one of the best compliment I ever got. Do you remember the talk I was freaking out during my PhD? Well, after my talk, which might not have been even an excellent one, I overheard the head of a department advising two junior PIs to speak with the energy and enthusiasm I was speaking with. I guess you should remind yourself of how exciting the work you do is and if you disagree with this, change job or lie to yourself for a couple of hours.

Commit mentally: focus

You would not run the athletics world final 100m, physically unprepared and with no excitement. You would also not run it thinking about random stuff or worrying not to win it. Watch athletes on their blocks, the intensity of their eyes, the deep focus they concentrate on the start gun and those few seconds after. Focusing might take a fraction of a second if you were a natural or simply experienced. Also, keep your focus during the talk, try to nurture that unconscious little voice that can warn you everytime you are going off-track.

The top right-hand corner syndrome (TRiHCS) is a risky issue in our business. TRiHCS happen when your mind wonders off, but you keep speaking. TRiHCS happen when you zone out and speak for 2 minutes about an irrelevant detail being fixated on a corner of a room, while you are not engaging with the audience and perhaps even with the main topic of the talk. If you get TRiHCSed, your timing and narrative will derail. But, do not worry, if you notice it in time, you can easily recover.

OK, ok… TRiHCS? I just made this up, but I promise you, it is something that does happen!

Look after yourself…

Pay attention to yourself. It is easy to get anxiety compromise your health in the long term, or your performance in the short term. In an ideal world, you can sleep, eat, drink, meditate as a Yogi. In the real world, assaulted by too many things to do, it is likely you will experience periods of stress and long hours. However, you will have to know your limits and try to stay far from the edge and arrive to an event in good physical and mental conditions. Your institution and funders will offer you a provision of well-being courses, advice and activities. However, your institution and funders will implicitly ask you to neglect completely their own advice and deliver huge returns for them at any cost (for you). Like for any job, the day will come that you cannot run any longer over the edge. Then, manage anxiety, either it is just for public speaking, or for anything else… embrace it, as in ‘do not ignore it’, ‘do not fight it’ as it fights back, but manage it and if you can’t, ask for help.

Look after yourself… plan your cool-off stage

I did some crazy things aiming to present data still warm from the microscope (yes, it is a thing if you use high power lasers), consciously cutting sleeping times down (within reason) and working over the edge. Even if you do not, but public speaking really takes a toll on you, look after yourself after the main event. You need to consider two phases. One, which might be short or very long, depending on the event, is the immediate aftermath. I used to be a runner, and I used to give everything until the end of the race, which made it very likely for me to fall on the ground exhausted after the line… but you learn to immediately stand-up, walk, then do a run at slow space and hydrate.

Somehow, after a peak of stress you need to do something similar, often quietly and in public. This may have to happen in a few seconds before taking further questions. So, regain mental and physical composure, re-gather your focus and energy, again consider drinking water or a juice.  You will need this, particularly, in a day-long event full of meetings. It can really take just one minute, but if you do not do it, you might crash and underperform in the aftermath of a public speaking event. Do not underestimate the task you will have to follow after the main event and the energy you will need for them.

Then, at last, all is over. Really look after yourself because if the event you prepared took really a lot of energy from you, there might be consequences. You will discover what is best for you, if to completely relax and instruct yourself, or to simply take it easy for a few hours or a few days.

Conclusions

Keep in mind that what I have written here it is not an expert-opinion, but a personal experience. My suggestion to embrace your public speaking anxiety comes from trying to advise junior colleagues and realizing I did not wish to give the same suggestion a GP once gave to me: ‘you should avoid stress’. This is the wrong suggestion, in my opinion, as most of us, certainly in the ultra-competitive academic world, will have to manage plenty of stressful situation. Thus, the keyword is ‘manage’ not ‘avoid’, be the master or mistress of your stress-responses and, yes, avoid only those things that might push you too far beyond what you can manage. So, embrace your public anxiety speaking, mould your response to it in time and you will eventually grow out of it, or if not, at least you will manage.

Of course, whatever I described here is not something I usually think about, even during big talks. I made an effort to catalogue the various ‘tricks’ I – sometimes unconsciously -matured in 15 years of presenting scientific work in public. But recently, I had noticed that – either as a natural predisposition or by training – delivering a talk is more than just speaking in public. It is a process that requires physical and psychological strengths, like an actor preparing for a play or an athlete for a race. Scientists, noticing it or not, need to nurture these strengths, even not for their audience, but at least for looking after their health.

[TALK] Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Friday 14/09 at 14.30 | Dr. Simon Cook (Signalling Laboratory, The Babraham Institute) will present the following talk, at the Clifford Allbutt Lecture Theatre, Clifford Allbutt Building (former LMB building). All welcome to attend.

 Goldilocks and the two ERKs; signalling in the ‘sweet spot’ underpins resistance to ERK pathway inhibitors

Simon Cook, Signalling Laboratory, The Babraham Institute

Tumour cells with BRAF or RAS mutations are ‘addicted’ to ERK1/2 signalling for proliferation and RAFi and/or MEKi are now approved for use in the clinic.  However, despite some striking clinical responses, resistance emerges within 9-12 months resulting in disease progression. Acquired resistance to MEKi often occurs through amplification of BRAFV600E or KRASG13D which act to reinstate ERK1/2 signalling.

Here we show that BRAFV600E amplification and MEKi resistance are fully reversible following drug withdrawal.  Resistant cells with BRAFV600E amplification become addicted to MEKi to clamp ERK1/2 signalling at a level optimal for cell survival and proliferation (2-3% of total ERK1/2 active, quantified by mass spectrometry).  This is seen in cell culture and in vivo where growth of resistant cells with BRAFV600E amplification as tumour xenografts is inhibited in mice that do not receive MEKi.  ERK1/2 hyperactivation (~20% active) following MEKi withdrawal drives expression of the cyclin-dependent kinase inhibitor (CDKI) p57KIP2, which promotes G1 cell cycle arrest and senescence, or expression of NOXA and cell death; these ‘terminal’ responses select against those cells with amplified BRAFV600E.  ERK1/2-dependent p57KIP2 expression is required for loss of BRAFV600E amplification and determines the rate of reversal of MEKi resistance.  Thus, BRAFV600E amplification confers a fitness deficit during drug withdrawal, providing a rationale for intermittent dosing (‘drug holidays’) to forestall resistance.

Remarkably, MEKi resistance driven by KRASG13D amplification is not reversible. ERK1/2 reactivation in the context of amplified KRASG13D does not inhibit proliferation but drives a ZEB1-dependent epithelial-to-mesenchymal transition that increases cell motility and promotes resistance to chemotherapy agents, arguing strongly against the use of ‘drug holidays’ in cases of resistance to MEKi driven by KRASG13D amplification.