PhD life: sharing science

The past few weeks I have not written much on this website. That does not mean I have not been doing any outreach activities. In contrast, I have done several and for a widely varying public. The first took place in a greenhouse in the middle of The Hague, the second in front of a group of Master students and the third at a symposium organized by NWO. An interesting mix of places and people and all new experiences for me.

International Fascination of Plants Day
On April 18th I went to The Hague, a city in the West of the Netherlands that is the seat of our parliament. For that day (and two following days), plant scientists and Dutch breeding companies had erected a greenhouse on the Square in front of the parliament buildings in honor of the ‘International Fascination of Plants Day’. Of course there were lots of plants, but also information panels about plant science and agriculture and even a small laboratory in which people could isolate RNA from small tomato plants.

Fascination of Pants Day in the Hague
Fascination of Pants Day in The Hague

Journalists and people from the parliament were specifically invited for breakfast and lunch, but the greenhouse was open for the general public the rest of the day. During the day I talked to many different people about plant science. Among them were the owners of a small flower breeding company, but apart from them I talked mostly with ‘common folk’, who came across the greenhouse by accident and decided to step inside – or were persuaded by me to do so. I very much enjoyed telling people about plants, making them aware that plants are actually pretty cool things and, whenever possible, very briefly introducing them to the kind of research that is going on at the moment.

Giving my first lecture
After talking to the general public in April I stood in front of a bunch of Master students on June 6th. These were students following a Master’s program in life sciences, but not necessarily anything at all to do with plant sciences. I was not there to give an regular lecture – the two lectures of the day, about RNA and RNA sequencing, were covered by two colleagues. Instead, I got to do the nice part: I got to present the part of my research that revolves around RNA sequencing.

The group was attentive, I got several questions during my talk, several more afterwards and when we called the session to an end a few more students came up to me to ask more questions. The questions ranged from questions about the techniques I used and the results I gathered to the application of my research. It showed that the students had paid attention ánd were interested in the material. First lecture for a group of university students: check!

Invited speaker at a symposium
Just this week, on June 20th came another first for me: I was an invited speaker at a symposium. The conference was organized by the NWO for people who work on a project that is supported by either the NWO’s Agri&Food or ‘Horticulture and starting materials’ grants. I received my personal grant from the latter and was therefore invited. Or well, in fact it was my supervisor who was asked to give a talk, but since the request was about the project I had written and am carrying out, he sent the request on to me. From 10 am to 2 pm the day was divided into three session for talks and a poster / lunch session.  Each presentation session had three parallel programs: two with four talks each and one in which public-private partnerships –between a university or research institute and a company – were discussed.

NWO symposium 'Co-creation? Naturally!'
NWO symposium ‘Co-creation? Naturally!’

I already enjoyed the first two sessions immensely. The topics varied widely, which worked well because talks were only seven minutes long and were geared to a non-expert audience. I heard about wasps being trained to find mites – which cause plant disease – more quickly in rose greenhouses, for example, and about how to model sustainable intensification in agricultural settings. Cool! My own talk was in the third session and was well very received. Like with the Master students, I got lots of questions, also after the end of the session. I even got my first speaker present: a bottle of wine. It made my day – even though I do not like wine at all.


Background: plant science

I do not need to convince anyone of the importance of studying cancer treatment, heart disease prevention or the mechanisms of HIV/AIDS. The importance of plant science, on the other hand, is not that clear to everyone. This is a shame, because plant science has the potential to save many lives ánd to decrease the negative impact of humans on our planet.

Food shortage and hunger
In 2015 about 795 million people in the world did not have access to enough food to meet the minimum daily dietary energy requirements [1]. This is a huge number compared to the amount of people being diagnosed with cancer, 14 million in 2012 [2], or living with HIV / AIDS, 36.7 million in 2015 [3]. Hunger is caused by many factors, among them economic and political ones.  Environmental factors also explain a large part of food shortage in certain areas. Plant diseases cause 10 to 40% crop loss – depending on who you ask – each year [4,5], drought and extreme temperatures result in additional losses. Increasing temperatures due to climate change and stricter rules for the application of pesticides and fertilizers will increase the severity of these problems in the years to come. And remember, without food, there will be no people to get diseases for which we need to find a cure…


Increasing food availability through plant science
Plant science encompasses many subfields that together cover a wide variety of topics, including but not limited to: plant disease, plant response to abiotic factors such as light, temperature or nutrient concentrations, plant development, plant interactions with microbes in the surroundings and plant ecology. All of these subfields look at plants from different angles and not all of them have clear follow-ups that will increase food production. However, any extra knowledge that we gather on plants will help us understand them better. Ultimately, this will increase our ability to grow them optimally in the field and thus to increase food production.

A science success story
The discovery of the so-called submergence-tolerant (Sub1) rice varieties is a clear success story showing how data gathered in a laboratory can lead to increased food production. Rice is an important food source for many people, especially in Asia and Africa. While rice is a relatively flood tolerant crop, modern rice varieties do not do well when they are completely under water (submerged) or in low floods over long periods. While several local rice land races are tolerant to flooding, they have other traits that prevent farmers from using them, such as low yields or poor grain quality.

Discovery of a gene that makes rice plants flood tolerant
Researchers were interested in those local varieties, though: they wanted to discover which genes lead to the increased tolerance. They studied this by crossing food tolerant and flood intolerant rice varieties, like in the paper I described earlier on the discovery of another gene, HCR1, that was found to possibly have changed in plants in response to different aboveground water environments to increase plant fitness. Like in the previously described paper, a part in the genome was found to correlate with the phenotype of interest, in this case the increased tolerance to flooding. This part of the genome was named submergence-tolerant (SUB) 1.

Making varieties used in the field flood resistant
After the discovery of the part of the genome that makes local land races flood resistant, people wanted to use this knowledge to increase flood tolerance of varieties used by farmers in the field. In 2003, the International Rice Research Institute (IRRI) started a breeding program in which they crossed land races with SUB1 in their genome on the one hand with varieties used in the field on the other hand. Offspring with the full genome of the agriculture variety with only the addition of the SUB1 part from the land races were selected and distributed among farmers in, among other countries, Bangladesh and India. Large field trials were conducted and showed no difference between Sub1 rice and the original varieties apart from increased tolerance to submergence. This makes sense, considering that SUB1 is only expressed in submergence conditions. As visible in the photo below, Sub1 rice has a much greater crop yield then the commonly used varieties in flooded fields.

The original modern variety on the left, the variety with the SUB1 part of the genome from a local land race on the right
The original modern variety on the left, the variety with the SUB1 part of the genome from a local land race on the right. Photo source

Sub1 rice is a great success. Many parties, such as the Bill & Melinda Gates Foundation, are contributing financially to further distribution and research. For more information, visit the research organization’s  website.

Changing my blog’s focus
I am a plant scientist and very enthusiastic and optimistic about the ways that plant science can contribute to our society. With sufficient funds and talented people I think it is possible to create more success stories like the Sub1 rice variety. In the past months I have realized that only few people are aware of this important possible contribution of plant science to society. Therefore, I have decided to shift the focus of my ‘journal club’ and ‘background’ stories to be mostly plant related. In this way, I hope to open more people’s eyes to the possibilities of plant science. Who knows, maybe one day this increased awareness will result in something like a ‘plant research to feed the world fund’ in addition to the ‘world cancer research fund’ and the many other disease-related funds out there.

1: The State of Food Insecurity in the World 2015, by the Food and Agricultural Associations of the United Nations.
2:Cancer, by the World Health Organisation.
3: Global health observatory data: HIV / AIDS, by the World Health Organisation.
4: Combating plant diseases is key for sustainable crops, by Science Daily (2011)
5: Almost 40 per cent of worldwide crops lost to disease, by The Crop Site, 2012

Information on the Sub1 research and application is from:
Ismail AM, Singh US, Singh S, Dar MH, Mackill DJ (2013) The contributin of submergence-tolerant (Sub1) rice varieties to food security in flood-prone rainfed lowland areas in Asia. Field Crops Research 152: 83-93.

PhD life: conversing at a conference

I did not spend April 24th and 25th listening to a colleague’s presentation at lab meeting, doing lab work, meeting with my student or analyzing data in my office. Instead, I was in Gif-sur-Yvette, in France, attending a two-day symposium on interactions between beneficial microbes and plants.

Conferences: one of the perks of being a researcher
Attending conferences definitely ranks high on the list of ‘awesome things you get to do as a researcher’. With a little luck, they take place outside of your home country, allowing you to see some more of the world while ‘working’. While my first international conference was in Maastricht, the Netherlands – also fun, by the way, I gave my American collaborator a ride on the back of my bike, a first for her -, I attended a conference in Portland, Oregon, USA last year. This year, my boyfriend and I got to combine my conference attendance with a weekend in Paris. Not at all bad, right? And that is not even the best part of attending a conference. The best part is the enormous enthusiasm boost that comes with it.

A rather small, specific conference
Conferences can vary widely in the number of people attending, the broadness of the topic, the location and the budget. In comparison with the 500+ people attending at the previous two conference I attended, this conference with its 120 attendees felt relatively small. It still had the same international vibe to it, though, with people from quite a few different countries attending, including France, Belgium, Saudi Arabia, the Netherlands, Germany, England, China and the US. Also, the mix of ‘levels’ was good: there were PhD students and postdocs, but also a lot of principal investigators. The 18 presentations spread out over 1.5 days were all given by principal investigators. Apart from these talks, there were 40 PhDs and postdocs presenting posters during the coffee and tea breaks. A busy schedule, but interesting because the topic – beneficial microbes and plants – is quite specific and close to my own project.

New data during presentations
Listening to presentations at conferences can be exciting. First, you are among the first to see new data – although most 30 minute talks are not only about new data: they generally cover results gathered over many years that together form a nice coherent story. Second, apart from the results, talks regularly also go into new methods used to get those results, thus giving new ideas for own research projects. Together this makes for a head spinning with new ideas after a couple of talks. (There are of course also talks that are not that interesting or presenters that do not present all too well and yes, whiling away the time of those presentations on my phone is very attractive…)

Talking science next to posters
Apart from presentations almost all conferences also have sessions for poster presentations. Before I started my PhD I saw posters as a high school thing: cutting and pasting stuff on a big sheet of paper and putting that paper up somewhere for no one to ever really read. In science, posters turned out to be much more than that. In fact, poster presentations are actually very nice. For one, it is the easiest way to talk science with people you do not know since there is a clear topic and excuse for a talk on hand: the poster. Second, people interested in your topic will actually read, or ask about, everything on your poster: they are genuinely interested in the whole thing. Third, it gives easy, quick glimpses of the research performed by other people and you can cherry pick which you want to know more about. Fourth, a discussion about someone’s work in front of a poster is often much more spontaneous and fun than a presentation from a stage. All together this makes poster sessions a lot of fun. In fact, twice I and someone else were still in the middle of an entertaining discussion about science in front of a poster when we were pretty much forced to break up because the next presentation session was about to start.

Getting to know fellow scientists during dinner
Apart from the talks and the posters there was one more component to the program: the dinner. To keep the attendance costs as low as possible, the food and location were not at all fancy. We did have a good time though: being surrounded by people from Belgium, France, Hungary and China made for enough conversation topics. All together the talks, poster sessions and dinner ensured the typical conference-induced enthusiasm boost about science in general and my own project by the end of it.

Now I need to get back to work and ensure that I have new things to tell when I visit the next conference next year…

PhD life: company visits

A few weeks ago, three of my colleagues and I felt as if we were back in school and going on a field trip: we went to Warmenhuizen and Enkhuizen for the day! Both these cities are located in the North of the Netherlands in an area known as ‘the seed valley’. We went there to visit two of the many companies that give the region its nickname, Bejo Zaden and Incotec.

Bejo Zaden and Incotec’s contribution to our research
The primary reason for our visit was to discuss how these two companies can contribute to our current research projects. As discussed before, almost all positions in academia are paid for by grants. In the field of plant research, most of the available grants require that part of the costs are covered by companies. The companies can contribute by giving money, or by making ‘in kind’ contributions. This could be sponsoring a machine to analyse samples, supplying research materials (microbes or seeds, for example) or even doing an experiment. The latter often comes in handy in plant research. Field trials, that is, experiments with hundreds of plants performed outdoors, and also large greenhouse experiments are generally hard to perform at a university because of space and manpower limitations, but are relatively straightforward to perform at a company. Since both Bejo Zaden and Incotec signed up to sponsor one of our projects, we went there to discuss the way in which they might be able to make their contribution. While we were there, we also got to see what these companies look like.

Bejo Zaden
We arrived at Bejo Zaden around 10 am and were greeted by three employees that all perform research at the company. First, they introduced their company and all of us introduced ourselves and our research topics to each other. After learning a bit more about research at a company and explaining our research interests, we went into the factory.

It was amazing to see the scale of the company and to realize how much is earned in this business. If I remember right, 1 gram of high quality tomato seeds costs about 50 cents. And we saw tons of these seeds… To get these high quality seeds, there are many hurdles to be taken. First, the incoming seed batches – Bejo has breeders all over the world – are checked for seeds of weeds by eye or by machines that remove the weeds by air pulses. Then, the germination percentage, the percentage of seeds that produces a plant, needs to be determined. If this is low, seeds can be sorted according to weight and size, again in huge machines. One or more of these samples might have a higher germination percentage than the total batch. Once a seed batch passes these tests, it undergoes rigorous disease tests. If there are fungi or bacteria present in or on the seeds, the whole batch needs to be disinfected and checked again – or thrown out. Two of our guides work in the department where they are trying to find quicker and easier methods to detect disease. Once (part of) the seeds are deemed of sufficient quality, the seeds are often coated with a colored layer, just like the chocolate in M&Ms is covered with colored sugar. In the case of the seeds, the coat can contain fungicides to protect the germinating plant from fungi, or fertilizers or growth promoters. Sometimes a thicker coat of about 3 mm is made to simply increase ease of sowing. While tiny, light seeds will be blown away by wind and seeds of different shapes will be hard to distribute evenly, a thicker coat around seeds results in seeds that are easily evenly distributed. Finally, the seeds are packaged, divided over pallets and ready to be shipped all over the world.

During the tour we unfortunately did not get to see the labs and greenhouses where most of the research is performed. This is probably partly due to the work having to stay secret. For the same reason they could not tell us whether they are already trying to use beneficial bacteria or fungi in their seed coating – which is what our research project will hopefully ultimately contribute to. They do not want other companies to know what they are up to. After the tour, we talked about what the company might be able to do for us. These options include providing us with seeds of their crops and performing disease assays. The details will need to be discussed later. A quick lunch later, we heading off to our next stop: Incotec.

Incotec does not produce seeds. Instead, they focus entirely on the seed coating mentioned above. They try to find new methods to do the coating and new coating components. This latter part is all the more important because the laws restricting fungicides, fertilizers and pesticides are becoming stricter. Thus, new methods need to be found to protect the seed during storage and after sowing. Among promising methods are the addition of beneficial bacteria and fungi that we work with in the lab as replacements for pesticides and fertilizers. Apart from developing new methods, Incotec also performs seed coating for other companies. In fact, Bejo Zaden is one of the bigger clients of Incotec. Once again, after having talked with a few employees we got a tour through the factory. Here we saw how the coating is performed and how seeds transform from tiny specks into little colored balls.

When we had seen all there was to see, we got back into the car for our 1.5 hour car ride back to Utrecht. It was good to see what companies in our field – which are not only contributors to our projects, but also potential future employers – are doing and to get a sense of the scale of them. It is too bad that it is hard to get a real sense of what working there would be like, since we did not get to see the laboratory part of the companies. However, it is definitely valuable to be forced to think about the application of our research and to look at our results it from a company’s perspective.

Journal club: resistance to wheat disease through recognition of a protein

Introduction: Wheat yellow rust disease
Every year large wheat crop losses are caused by wheat yellow rust disease, a disease on wheat caused by the fungus Puccinia striiformis f. sp. tritici (Pst). This fungus is not able to make all plant species sick: Nicotiana benthamiana, a relative of tobacco and a much studied species, is resistant to it. This plant is resistant because it can recognize the fungus and activate an immune response. Understanding how this plant recognizes the invading fungus and thus becomes resistant will increase our chance of ever breeding a resistant wheat variety that is also resistant to this fungus.

Results: Recognition of a protein is the key to a resistant plant
In their paper, Dagvadorj and colleagues show that N. benthiamiana recognizes a small protein secreted by the fungus. Recognition of this protein results in the expression of genes that lead to the activation of defense responses in the plant. The defense responses result in reduced disease symptoms after pathogen attack. Future studies identifying the receptor on the plant cells that recognizes PstSCR1 will therefore provide material to improve disease resistance against a range of pathogens.

For the enthusiasts: How did the researchers get these results?
This paper follows up on another paper published in 2009 that looked among all the genes of Puccinia striiformis f. sp. tritici for genes that are expressed when the fungus is infecting a plant. They found fifteen genes that encode secreted proteins and end their paper with ‘[t]hese genes are candidates for further studies to determine their functions in wheat-Pst interactions.’

Dagvadorj and colleagues performed just such a study. Their study focuses on one of the fifteen genes, one that codes for PstSCR1, as a possible explanation for the resistance of N. benthiamiana to Pst. They first check whether PstSCR1 mRNA is produced when the fungus infects wheat. This is the case from 72 h to 8 days post infection. Next, they checked the effect of PstSCR1 on plant immunity. To do this, they expressed PstSCR1 on N. benthiama leaves before putting pathogens on the leaves. The infection, as measured by the lesion diameter on the plant or the number of spores produced by the pathogen, is reduced in leaves with PstSCR1 expression. One possible explanation for the reduced infection is activation of the plant immune response. To test this, the researchers checked mRNA levels of genes involved in the plant immune response after infiltration of the leaves by purified PstSCR1. Both defense genes that were checked, NbACRE31 and NbCYP71D20, were indeed activated by PstSCR1. Thus, the detection of this protein results in the activation of the plant immune response, which in turn results in resistance against the pathogen secreting the protein.

Discussed paper
Dagvadorj B, Ozketen AC, Andac A, Duggan C, Bozkurt TO, Akkaya MS (2017) A Puccinia sriiformis f. sp. tritici secreted protein activates plant immunity at the cell surface. Scientific reports 7: 1141
DOI: 10.1038/s41598-017-01100-z

Paper that precedes this one:
Yin C, Chen X, Wang X, Han Q, Kang Z, Hulbert SH (2009) Generation and analysis os expression sequence tags from haustoria of the wheat strpe rust fungus Puccinia striiformis f. sp. Tritici. BMC Genomics  10: 626

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Academia: research funding

As described in a previous blog post, most of the jobs in academia are temporary positions. Only the person in charge of a group, the principal investigator, and sometimes the technicians have permanent positions and thus only their salary is paid for by the institute or university that they are working at. All other positions are funded by grants.

The types of grants
Grants come in many different forms and are awarded by many different institutions. There are grants for visiting a lab in a foreign country for a short period, grants for an entire PhD or postdoc project and grants that supply enough money to pay several PhDs or postdocs. Grants in the Netherlands can be paid by the NWO, the Netherlands Organisation for Scientific Research or by the European Union, such as the Marie Curie grant for a Postdoc abroad or the European Research Council (ERC) grants for researchers aiming to start up their own group. Grants can also be (partly) funded by charities, such as the Hartstichting, which supports cardiovascular disease research, or by companies, such as the many breeding companies in the Netherlands that support plant research. In all cases, scientists judge the applications and decide which of their colleagues get the money. Care is taken that the people in the decision committee are as objective as possible, for example by not letting them judge people they have worked together with. In this way, at least in theory, the grant applications are judged solely on the quality of the researcher and the proposed project.

Who applies for the grants?
The visiting scholar and Marie Curie grants are personal grants, which means that they are written and defended by the person who will do the work. These kind of grants also exist in very small numbers for PhD projects – that is how I got the money for my project. In general, though, most grants are written by the principal investigator of a lab. Once the money is granted, the PI will interview people and hire someone to perform the work described in the grant.

Critique on the system
Without grants, research cannot be performed in the lab: while there is money for the salary of the principal investigator and a technician, there is generally no money for research supplies. Moreover, without PhDs and postdocs there are simply too few hands to get anywhere. Thus, to keep a lab going, principal investigators spend a lot of their time writing and defending grant applications to hire new people. In addition, they spend time to review the grants of other people. This is part of the reason that there is quite a lot of critique on the current funding system. Another critique is that it doesn’t always select the best science. People who have already gotten grants in the past have a higher chance of getting grants again because of those previous grants on their CV. Likewise, people with one or two papers in great journals are much more likely to get grants for a long time afterwards. This leads to vicious circles with those lucky enough to have received grants or a good paper early in their career getting more and more money afterwards. Currently no good alternative has been found yet, though.

Further reading
Since there is much more to be added to this discussion, but I do not want to make this post way too long and do not know everything myself, here are some sources for extra reading:
More about the procedure of deciding which grants get funded (in this case by the National Institutes of Health in the USA)
More about who pays for research, again in the USA, and how that might influence how the research is carried out
More on why it’s sometimes hard to see immediately why certain research, especially fundamental science, is ultimately beneficial for society – and thus to convince the public and politicians why funding is needed
More about the critique on the current EU funding system
More on a recently proposed new funding system in the Netherlands

Academia: the academic career ladder

PhD, postdoc and PI are terms that have two things in common: 1) most people have little to no idea what they mean and 2) they are all names for jobs on the academic career ladder. Okay, and 3) they all start with a ‘p’.

The first step: doing a PhD
I am currently at the first step of the career ladder in academia: doing a PhD. Apart from some shorter PhDs in the medical field, most PhDs take (at least) four years. As discussed in several other articles on this website, doing a PhD is mostly about doing research. Apart from doing research, the tasks of a PhD candidate generally involve some teaching and student supervision tasks. Those latter things do not contribute to the final evaluation of the PhD candidate, though. A PhD project is judged on the PhD thesis that is written in the end. This thesis is a book containing an introductory chapter, two to many research chapters, a discussion and a summary for non-scientists. While it is not strictly required, it is better for future career possibilities when at least some of the chapters of the thesis have been published in a scientific, peer-reviewed journal before handing in the thesis. After approval of the thesis by a committee, it has to be successfully defended in front of a group of scientists before the doctor degree is awarded.

Postdoc time!
The new doctor can now move on to the next step in the academic career ladder: doing a postdoc. A postdoc – short for postdoctoral researcher – is also a temporary position, which generally last two to three years, there are also people doing much longer postdocs. The main responsibility of a postdoc is still doing research. However, more responsibilities can be required. Postdocs might partly supervise PhD students or give lectures, for example. While postdoctoral research can be in the same research field as the PhD was performed in, this is not necessarily the case. In general, people at least go to a different lab, and often even to a different country.

Moving towards a permanent position
After about two postdoc postitions, the next step is a tenure track position. A tenure track is a job for four to five years and often comes with money to pay a PhD. At Dutch universities, tenure trackers are generally placed in an existing lab. This is in contrast to the situation in the US and at Dutch research institutes like the NIOO and the Hubrecht, where people with a tenure track position almost always start a new lab. In the five years of the contract, the  person needs to prove that he / she is ‘worthy’ of a permanent position. In practice, this comes down to publishing enough papers in important enough journals. If these requirements are met, the person will get a permanent position as ‘principal investigator’, or ‘PI’. At research institutes like the NIOO and the Hubrecht, principal investigators are often not connected to a university and thus not Professors. At universities, principal investigators are generally either assistant, associate or full Professors.

Apart from the PhD candidates, Postdocs and principal investigators, most labs in the beta-sciences also have technicians. Technician positions are generally permanent positions, thus ensuring continuity in a lab where most people leave again after a few years. Technicians are part of the support staff: they help out with experimental work and make sure that everything works smoothly by ordering supplies, keeping track of stocks, arranging lab cleanings, etc. From my experience in different labs I learned that good technicians are vital for good research.

Advancing on the ladder
As already mentioned in several of the interviews on this site, permanent positions are hard to come by in academia. The following graph, published in one of the major journals, illustrates this well.

Since 1982, almost 800,000 PhDs were awarded in science and engineering (S&E) fields, whereas only about 100,000 academic faculty positions were created in those fields within the same time frame. The number of S&E PhDs awarded annually has also increased over this time frame, from ~19,000 in 1982 to ~36,000 in 2011. The number of faculty positions created each year, however, has not changed, with roughly 3,000 new positions created annually

Even when someone reaches the highest level of the career ladder, that of principal investigator, that does not mean that he / she can relax and focus on research from then on. In fact, people in those positions in the beta sciences, generally do not carry out research anymore at all. Instead, they spend their time managing the lab, supervising PhD candidates and postdocs and applying for money. Money, more specifically research funding and grant application, is worthy of a post itself, though, so I will go into that in a later post.

Figure and supporting legend from Schillebeeckx et al. (2017) The missing piece to changing the university culture, Nature Biotechnology 31: 938–941. Reprinted by permission from Macmillan Publishers Ltd: Nature biotechnology, copyright 2017.



Journal club: heart development

Main messages
The three major cell types of the heart develop and mature at different time points during heart development.The precise development depends on the localization of a cell within the heart. A mutation in the gene NKX2.5 can disturb the developmental process in mice. This probably explains the heart defect of both newborn people and mice that have this mutation in their DNA.

Background and aim
At birth, our hearts are fully functional pumps, pumping oxygen-poor blood to the lungs and oxygen-rich blood from the lungs to the rest of the body. The blood flows into the heart in the two atria, which can be visualized as chambers. Upon contraction of the walls of these chambers, the blood is pushed into the next chambers: the ventricles. From there, the blood is pumped out of the heart to the rest of the body and lungs. To be able to perform it’s pumping function, the heart contains muscle cells, allowing the heart to contract, endothelial cells, lining the blood vessels and heart chambers like a kind of inside skin, and fibroblasts, making the support framework of the heart, the so-called the ‘extracellular matrix’. This complex structure needs to develop during only nine months of pregnancy. In their paper, DeLaughter and colleagues studied the expression of genes in the heart of embryonic and newborn mice to learn more about how a heart and its different cell types develop. Ultimately, this will help us understand how defects in certain genes result in heart defects in newborn babies.

The researchers determined the expression of genes using RNA sequencing. They determined gene expression at different time points during development: at embryonic day 9.5, 11.5, 14.5 and 18.5, on the day of birth and at 3, 7 and 21 days after birth. Interestingly, they did not study gene expression in the whole heart at once, but in single cells. As explained here, different cell types perform different functions because different genes are expressed in each of them. By studying gene expression in separate cells, the researchers could determine the cell type and maturity of each of the analyzed cells.

Results & discussion
The ratio of the different cell types changes over time
The researchers discovered that the proportion of endothelial cells was stable over development. In contrast, the number of fibroblasts increased from none at all at embryonic day 9.5 to a little less than half of all cells at birth. This increase was at the expense of the cardiomyocytes. The decrease in the percentage of cardiomyocytes is possibly due to the decrease in cell division of these cells: more than half of the cardiomyocytes expressed genes associated with cell division during the start of embryogenesis, but none expressed them 21 days after birth.

Cells in the different heart chambers and different development time points can be distinguished based on different gene expression patterns
Interestingly, gene expression of the cardiomyocytes from the atria was clearly different from that of the cardiomyocytes from the ventricles. Not only that, even the cells from the left and the right ventricle could be distinguished based on gene expression! The difference between the ventricles became less clear over time, indicating that the chambers first establish their own characteristics, after which all of the cardiomyocytes mature.
The samples obtained at different time points were also clearly different from one another. Interestingly, comparing gene expression data from human hearts to this mice data, showed that human gene expression in the heart shortly after birth corresponded to the mice data obtained at 1-3 days after birth.

A mutation causing heart defects in humans delays maturation of the heart cells
Finally, the researchers looked at mice with a defect in one of their genes: gene NKX2.5. Both humans and mice with a defect in this gene, so with a so-called mutation, develop hearts in which the four chambers are not fully separated. Heart cells of mice with this mutation showed gene expression differences compared to mice with healthy hearts. These differences indicated that the mutant hearts had much fewer cardiomyocyte cells that had reached maturity at birth. Interestingly, while NKX2.5 is expressed mostly in the cardiomycytes, the maturation of endothelial cells was also affected.

DeLaughter and his colleagues have given us a better idea how heart cells develop and mature in an embryo. They also showed how this information can be used to understand the cause of heart abnormalities present at birth.

DeLaughter DM, Bick AG, Wakimoto H, McKean D, Gorham JM, Kathiriya IS, Hinson JT, Hornsy J, Gray J, Pu W, Bruneau BG, Seidman JG, Seidman CE (2016) Single-cell resokution of temporatl gene expression during heart development. Developmental cell  39: 480-490.

Disclaimer: blog posts in the category ‘journal club’ are not intended to cover the whole paper discussed. Instead, I discuss the parts that I think are most interesting for a general public. I try my utmost to prevent any mistakes in these blogs, I apologize in advance for any mistakes that I make anyway.

Background: RNA sequencing (method 1)

The goal of the method
RNA sequencing is a method that is used a lot in molecular biology laboratories around the world. The method is used to measure the expression of genes. All cells in a certain organism have the same genes. Differences in the expression of those genes between the cells results in different cells being able to perform different functions: an immune cell can combat pathogens, a muscle cell can contract, a cell in a plant leaf can perform photosynthesis. Therefore, studying gene expression can learn us more about the developmental processes underlying the differentiation of the cells and the formation of multicelllular organisms like us humans. In addition, changes in gene expression allow a cell – and thus also multicellular organisms –  to respond to the environment. Knowing which genes are expressed in response to certain stresses, such as heart failure in humans, or drought in plants, can give us an idea of how cells and organisms deal with those stresses. Ultimately, this can aid in for example developing treatments to prevent heart failure in humans or breeding programs to increase tolerance to drought in crop species.

The method itself
RNA sequencing measures gene expression by measuring the amount of RNA present. As explained here, RNA is the intermediate in the process of using the DNA code as a ‘building plan’ to form a protein. The more RNA from a certain gene is detected, the more active the gene was. Once the order of letters in a string of RNA, in other words the sequence of a piece of RNA, is known, it is easy to determine from which gene it originated. This is easy because the sequence of a piece of RNA corresponds to certain letters of the DNA sequence it was made from: an A in the RNA is a T in the DNA, a G in the RNA is a C in the DNA, etc. Thus, once the sequence of a piece of RNA is known, one can deduce the sequence of the DNA it was made from. With databases on the internet, the gene corresponding to this DNA sequence can be determined.

To measure the amount of RNA present, the RNA is first converted to cDNA (complementary DNA) by the researcher. Thus, synthetic A, T, C and Gs are combined with the mRNA pieces, in addition to ‘reverse transcriptase’: a protein that can bind DNA nucleotides one by one to an mRNA molecule to create a complementary piece of DNA: the cDNA. This is necessary, because the sequencer, the machine that reads the sequences, cannot read RNA. The cDNA samples are then sent off to a sequencing facility, where the sequencer ‘reads’ all the pieces of cDNA. In other words, it reads the order of A, T, C and Gs of all the pieces of the cDNA present. This information is put into a huge file, containing all the sequences, the so-called reads, that the sequencer read. This file is returned back to the researcher, who can use a computer program to find out from which DNA the RNA-turned-into-cDNA pieces originated.


In the above-shown case, the red lines represent the RNA sequences of which the cDNA pieces were read by the sequencer. The black line represents the DNA of the studied organism. With a computer program the reads have been aligned to the genome based on their sequence . Finally, by looking up which parts of the DNA correspond to genes – this information is available online for most organisms -, the researcher knows which genes are expressed. In this case, gene 3 is expressed a lot, whereas gene 4 is not expressed at all.

Use of the method
Generally, the gene expression of not one, but several samples is determined in an RNA sequencing. A healthy heart of a newborn and a healthy heart of an adult, for example, or a diseased heart and a healthy heart. By comparing gene expression in the two (or more) conditions, one can learn about developmental processes and/or stress responses.

RNA sequencing is used in many different fields of biology, ranging from cancer biology to microbiology. On this website several papers are discussed using this method: this paper on light perception in plant roots and this paper on heart development.

Background: DNA and the central dogma

DNA and its messenger RNA
DNA is the genetic code that every organism inherits from its parent(s). If you compare a cell with a factory, the DNA is the entire set of building plans for all the different machines in the factory and any other instructions necessary for the factory to function. Genes are the parts of the DNA that correspond to the building plans. In the case of a cell, the building plans contain the instructions for the formation of proteins and other biomolecules essential to the cell’s survival. To build a machine in a factory, a copy of the correct building plan needs to be taken to a workshop. Similarly, to construct proteins in a cell, a copy of the correct gene needs to be taken to the protein-builders: the ribosomes. This copy is called messenger RNA (or mRNA).

DNA and RNA composition
When observed under a microcope, DNA looks like a twisted ladder. This structure is called a helix. It consists of two parallel strands of chemical compounds called nucleotides, each of which is loosely bound to its partner nucleotide on the parallel strand. DNA consists of four nucleotides: adenine (A), thymine (T), cytosine (C) and guanine (G). A can only loosely bind to T, and vice versa, and C can only bind to G. Thus, if one strand of the DNA helix reads ‘AGATCC’, the parallel strand, read in the same direction, will read ‘TCTAGG’. RNA also consists of nucleotides, but instead of T, it contains uracil (U). In contrast to DNA, RNA is most often single stranded.

The central dogma
When a certain protein is required in a cell, an mRNA copy has to be made from the corresponding gene. To make this copy, the DNA at the site of the gene detaches from the parallel strand. This unwinding of the helix lets RNA nucleotides bind. Once the entire gene is covered by the correct RNA nucleotides, the formed mRNA strand leaves and moves to the ribosomes, that is, the protein builders. There, the mRNA ‘building plan’ is used to make the required protein. DNA –> mRNA –> protein is known as the ‘central dogma’ of molecular biology.

Gene expression determines a cell’s function
Each cell of a multicellular organism, such as a plant, fish and yourself, contains the same DNA. However, cells within the organism can perform very diverse functions. This is possible because different genes are active in different cells. Ultimately, this results in different proteins being made and different cells being able to perform different functions: an immune cell can combat pathogens, a muscle cell can contract, a cell in a plant leaf can perform photosynthesis. Changes in the environment also influence gene activity, allowing a cell or organism to respond to the changes.

Genes are the parts of the DNA that code for proteins. To make proteins, a copy of the DNA, called mRNA, is moved towards the protein-builders, where the new protein is made. Changes in gene expression govern cell development and function and its responses to the environment. In multicellular organisms, gene expression in all the individual cells ultimately determines the appearance and fitness of the total organism.

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