Thoughts about product inhibition of amylase.

From the two preliminary growth experiments it seems that the strain of L. crispatus I am using is able to breakdown glycogen and use it for growth. If this observation holds (I am currently repeating it a third time, running the starch assay again and running the supernatants on HPLC to measure lactate production), it poses lots of questions for what this means in practice. Although the results have not been robust, it looks like starch-degrading activity is only present when L. crispatus was grown on glycogen and not when the cultures were grown on glucose. This immediately makes me think of carbon catabolite repression: the mechanism where bacteria in the presence of glucose shut down the expression of enzymes metabolizing other, less preferential, carbon sources.
My colleague Jurgen Haanstra posed an alternative hypothesis in the hallway yesterday. This doesn’t necessarily have to be regulation at the transcriptional level. This can also just be caused by product inhibition on the enzyme level! (thanks Jurgen, I really appreciate it). A few minutes later he sent me this paper from 1986 (cause that’s how he rolls):  Glucose feedback inhibition of amylase activity in Aspergillus sp. and release of this inhibition when cocultured with Saccharomyces cerevisiae.

Here, they were unable to measure amylase activity in the supernatants of this fungus, but when they dialysed the enzymes (i.e. replacing the liquid, while retaining the enzyme) amylase activity was back. If this phenomenon is also going on with the glycogen-degrading enzymes of L. crispatus this could not only explain why I am not seeing activity in glucose grown wells, but also why in the starch assay untill now only about half of the starch was degraded. If glucose accumulates, enzyme activity will seize. Pretty basic biochemistry actually. (Hundred years ago someone could have been doing the experiments I’m doing now, nonetheless it is pretty exciting. And as far as I am aware, pretty novel too.)
So, I am first going to simply add some glucose to the starch assay and also other sugars such as galactose and maltose. Maltose is also a breakdown product of glycogen/starch metabolism, so perhaps this will also provide some inhibition. Will keep you updated!

First growth experiments with L. crispatus (warning, unreviewed/unvalidated data) UPDATED

UPDATE 25/9/2017 I have a third biological replicate for L. crispatus growth that confirmed previous findings. I added a graph with average +/- standard deviations.

So, I have been growing the isolates that I ordered (see previous post) from DSM with mixed success.

-The bad news is that the Gardnerella vaginalis -80°C glycerol stock seems to be in bad shape. I inoculated twice successfully from this stock on NYCIII growth media (both liquid and agar plates), but after this it took multiple days for the culture to grow and lately they haven’t grown at all. I suspect this has to do with the aerobic condition in which they are stored. I will retry with plates and media that I will preincubate in anaerobic (N2+CO2) conditions, but there will be some influx of oxygen while inoculating, and at this point I am not sure that it is feasible to handle an study G. vaginalis outside of an anaerobic chamber. Other methods are still optional, such as using closed infusion flasks.

-The good news is that both Lactobacillus crispatus and Lactobacillus iners stocks seem to be growing well. I am using NYCIII medium for both, Lactobacillus crispatus grows within ~16 hours anaerobically, while Lactobacillus iners requires ~48 hours.

I present my first experiences with growing L. crispatus on glycogen.

Some background

The goal of this endeavor is to test whether vaginal bacteria can grow on glycogen, and are able to convert glycogen to lactic acid. Glycogen is an important carbohydrate present in the vagina, shown by old [1] and new research [2]. It is not yet understood how lactobacilli acidify the vagina of reproductive age women, and glycogen metabolism could be an important mechanism. Previously, Spear et al [3] have shown the presence of glycogen degrading enzymes in the vagina. The lactobacilli that were tested in this study showed no glycogen degrading capacity . Spear et al, asserts that the host excretes amylases (glycogen degrading enzymes) in order to assist acidification by Lactobacillus. However, the researchers did not use the most often encountered vaginal lactobacilli: Lactobacillus iners and Lactobacillus crispatus. Here I show initial growth experiments of L. crispatus on glycogen and amylase assay of the supernatants. I have gathered some evidence that Lactobacillus crispatus DSM 20584 strain can utilize glycogen as a source for growth.

Methods and results

I have used NYCIII medium supplemented with either 0.5% glycogen or nothing (water, negative control) or 0.5% glucose as a positive control. To this end I have pipetted 100 uL of a 5% glucose solution or 5% glycogen solution or water and 900 uL of a 1.1x NYCIII medium where I have left out the glucose (see protocols). I have inoculated this with 100 uL of a preculture of L. crispatus DSM 20584 grown for >24 hours at 37°C anaerobically, without shaking. . For every condition, I used three wells as technical replicates. I also have empty controls without cells. Glycogen makes the solution a bit hazy so I wanted to make sure that any increase in optical density is not due to the glycogen itself but this was not the case. I have performed this experiment on two different occasions.
I mixed the culture by pipetting with a 1 mL pipet and diluted the culture 10x with water in a flatbottom 96-well plate to measure the cell density in the plate reader at OD 600. It looked like this


The data look like this: I’ll upload this to a platform once I have a bigger data file to share.

Updated graph with mean +/- standard deviations, unpaired t-test comparing OD600 on glycogen compared to water shows p-value of below .0005.


The optical density is increased when glycogen is added to the media, comparable to the increase by glucose. This is an indication that this particular strain can use glycogen for growth. Next, I wanted to see if I could detect the enzymes that L. crispatus uses to break down glycogen for uptake and metabolism.

To this end, I spun down the cells for 20 minutes at 4°C and maximum speed (4754 rcf, 4499 rpm) and transferred the supernatants to plates I stored at -20°C. I wanted to test whether L. crispatus excreted any glycogen degrading enzymes in the supernatant. I added 50 uL of supernatant to 150 uL of a 1% starch solution in “amylase buffer” (100 mM Na-acetate+5mM CaCl2, pH 5.5). Starch is not the same as glycogen, the polymer has a different structure. The backbone of starch consists of the polymers amylose and amylopectin, which also consist mostly of glucose units but which are branched differently. Looking at Google images for these three polymers gives a good idea

I hope and expect that the enzymes that degrade most of the bonds between glucose units of glycogen will breakdown the bonds in amylose and amylopectin too. Starch has the advantage that it can be easily detected using iodine, an experiment that many of us already did in elementary school. I incubated this for ~24 hours at 37°C. I also checked after one and two hours but did not observe any breakdown of starch. I didn’t use any amylase control yet, only a reference calibration curve for starch. After incubation I added 10 uL, to 290 uL of iodine working solution and measured the absorption at 600 nm. I made a calibration curve using dilutions of the 1% starch concentration.

This experiment is not robust at all, since three times I measured three different things. So I will not share protocol and data yet. During the first measurement this calibration curve was not completely straight, not sure why, but the controls (starch with the supernatants of the empty control) showed an average of 7.6 gram/L starch, which is the concentration you expect. (150 uL of 10 gr/L starch + 50 uL of starchless liquid). However, in the wells with the L. crispatus supernatants something curious is going on. It seems that the amylase activity is found in the wells grown on glycogen whereas the wells grown with no carbon source (water) show the same concentration of starch as the controls (no breakdown). The glucose wells show some increase in starch. I am not sure how this happened, perhaps it has to do with pH, lactate presence, or even some growth in the wells. The wells did not look turbid, but growth can not be entirely ruled out.

The second time I did not freeze/thaw the supernatants but used fresh after spinning down the culture. I saw starch breakdown in the glycogen wells, indicating that L. crispatus excretes some soluble glycogen degrading enzyme in the supernatants (amylase?) whereas the H2O and glucose well did not show signs of starch breakdown. A third time I performed this experiment again with frozen sups I did not see any breakdown of starch.

So, I have to optimize this method, run controls with diluted amylase. I have to find out what’s going on with the increase in OD and finetune the protocol to get reproducible results. Perhaps it will turn out that this method is not robust enough, and I will have to resort to another way of measuring amylase activity.

On my long grocery wishlist with experiments:

  • Improve amylase detection.
  • Perform a HPLC on the supernatants to determine metabolite concentrations and gather more evidence of glycogen metabolism.
  • Use HPLC to measure amylase products (glucose, maltose, trisaccharides etc).
    Continue with Lactobacillus iners and Gardnerella vaginalis to study its glycogen degrading abilities.
  • I would like to partially purify the amylases to perform proteomics, we have applied for a “Hotel”-grant at ZonMW to perform this analysis together with Winclove probiotics at Radboud UMC.
  • Lastly, we want to extend the analysis to other strains of crispatus that were isolated by Remco Kort and his student Jorne Swanenburg in order to find the gene and enzyme involved in this amylase activity. Happy to announce that the biomedical/filosophy student Noa Fuks has offered her help to track down the amylase, while teaching herself some bioinformatics. Good luck Noa!

So, it’s a long grocery wishlist of experiments. If you have any comments, suggestions for alternative amylase activity assay, ideas, theories, criticism, please drop me a line.

1. Cruickshank, R., The conversion of the glycogen of the vagina into lactic acid. Journal of Pathology and Bacteriology, 1934. 29.
2. Mirmonsef, P., et al., Free glycogen in vaginal fluids is associated with Lactobacillus colonization and low vaginal pH. PloS one, 2014. 9(7): p. e102467.
3. Spear, G.T., et al., Human alpha-amylase present in lower-genital-tract mucosal fluid processes glycogen to support vaginal colonization by Lactobacillus. The Journal of infectious diseases, 2014. 210(7): p. 1019-1028.

NYCIII medium

This medium is used for growth of fastidious anaerobes like Gardnerella vaginalis and Lactobacillus iners. In my work I use it also for growth of other lactobacilli such as Lactobacillus crispatus although they will also grow on regular MRS medium. The reason for this is that in most cases I want to compare growth of these different bacteria, and want to keep as many other parameters in the experiment constant.

I adjusted this recipe from the ATCC protocol:

For 500 mL NYCIII medium:

  • HEPES (CellGro) 1,2 gram
  • Proteose Peptone No. 3 7.5 gram
  • Yeast Extract 1.9 gram
  • NaCl 2.5 gram
  • Glucose 2.5 gram (I use monohydrated glucose which means that I need 10% more = 2.75 gram)
  • water 450 mL

I cannot find a way to insert tabs in the wp editor, so this is it for now.

Compared to the ATCC recipe I use less HEPES, and do not adjust the pH. The pH is always around 6.7 before autoclaving and addition of the Horse Serum. In case I want to poor plates, I add 5 grams of agar (1%). I autoclave this mixture at 121°C for 20 minutes. After autoclaving and cooling down I add 50 mL of heat inactivated horse serum.

NYC III medium without glucose, 1.1x

I use this medium in case I want to test carbon sources other than glucose. I use the same recipe as above except that I leave out glucose and only add 400 mL of water. In order to supplement with alternative carbon sources I add 10% of the final volume of a carbohydrate solution (such as glycogen or glucose dissolved in water), with water as the control. The concentration of the carbon source is 10x higher concentration than the final concentration.
So for a 1 mL culture in NYCIII medium with 0,5% of a carbon source I add:
900 uL NYCIII medium without glucose 1.1x
100 uL of a 5% glucose of glycogen solution in water or water as a control.

Principles of Open Kitchen Science

These are the principles or guidelines for Open Kitchen Science. Open Kitchen Science has much in common with Open Notebook Science .As I learn more about Open Science, get comments on these principles and develop my ideas I might update them, in which case I will show the changes made and dates at the bottom of the post.

The general aim  is to increase scientific efficiency by sharing as much information as possible with other scientists and the general public.

  • Every finding in Open Kitchen Science is made public. Even if there is no greater understanding of the mechanism yet or if the finding is a so-called ‘negative results’. Any unreviewed results will be accompanied by a disclaimer that no peer review has been applied yet. All results will be accompanied by a a comment section.
  • Several standards of quality control are applied to ensure that experiments results are correct and reproducible. The gold standard of scientific quality is replication of results by an independent laboratory. This will ensure that all findings are robust and general. In Open Kitchen Science the aim is to replicate other people’s findings or get your own findings replicated. To this end, as much detail about methods, strains and protocols is shared.
  • The silver standard of quality control is traditional peer review. Once greater insight is obtained in mechanism or otherwise conclusions can be drawn about experimental results an open peer review process will be organized. Alternatively, the manuscript could be submitted to traditional academic literature for peer review process but only with Open Access policy.
  • Experimental setup is preferably published before the experiments are executed (as is common in larger clinical trials where RCT’s are preregistered).
  • Any methods developed and used will be made public once they are tested and ready to use.
  • Any other communication on this project, such as posters, slides and talks will be made public.
  • The language used in Open Kitchen Science will be as simple as possible and will prevent the use of unnecessary jargon. The aim is that an informed, educated and interested member of the general public can follow ongoing experiments.
  • This project will use a personal platform www.reblab.org to communicate the ongoing work, but will also provide updates on social media. The project will send out a newsletter to people in the field with updates on progress. In general, platforms are suitable for Open Kitchen Science when they are non-profit and do not require transfer of copyright or any other ownership.

Ordering and stocking strains

Getting started! I have ordered, received and stocked 3 strains at the DSM strain library. Unfortunately they do not have as many different options for vaginal bacteria, compared to all the isolates from the Human Microbiome Project for instances, and the strains I ordered were not all isolated from the vagina. I hope that they will display the same characteristics and can be used as a reference and to assist in setting up my methods. In the future I hope to with more vaginal isolates. I have

To grow the strains I used MRS medium (liquid and plates) for L. crispatus and NYCIII medium for G. vaginalis and L. iners. Unfortunately I do not have access to an anaerobic chamber so I have been using an anaerobic jar. After closing the jar I use 3x: pulling a vacuum (minimally 0.8 bar) and filling with N2+CO2 gas. I grew plates and tubes over the weekend (72 hours) at 37 degrees and stocked them using 0.5 mL 60% glycerol + 1 mL of culture and stored them at -80 degrees. I am not sure how much the oxygen will affect the bacteria during handling and storage, especially Gardnerella is sensitive to oxygen. I will check later if the stocks lead to good growth. I will upload protocols for the media, the stocking and the anaerobic jar soon.

Talk to Open Science conference

The Dutch government has been very active to push the Open Science agenda, both on a national and European level. Their newest initiative is the “National Plan Open Science”, which has been cosigned by several different national science organisations (funding, educations, academia, library, SURFnet etc). You can find it here.

On Monday May 29th, scientists who are interested in Open Science gathered. It was a very fruitful and energetic meeting. I noticed that there is a real Open Science community in the Netherlands, but it is rather patchy, scattered over many different fields. This means that when you are interested in Open Science, the chances are not very high that your closest colleagues share your interest, which complicates it. However, my experience sofar has been very positive (see talk below) and many people in my field were enthusiastic or at least curious to hear more. I was honored to speak at the conference, find my talk below.  It contains several of my ideas about peer review (the silver standard), but also scooping/replicating (the gold standard). There is overlap with previous talks.

(Update 15/06/2017: I have made some grammar and spelling changes and gave intermediate headings for structure. I also uploaded it to FigShare.)

I want to start off by thanking and congratulating our secretary Sander Dekker and his team for his achievements in pushing the open science agenda both here as well as on the European front. I realize that there are many interests you could have been pushing and you chose the topic of open science thereby accelerating the enormous change the scientific communities has to go through. In this talk I would like to give you an insight on my own plans and ideas about open science.

Ladies and gentlemen,

Last year, when I left my postdoc project in the USA, was the first time since in my adult life that I was outside of academia. No longer did I have an .edu account that provided access to literature, no students around, no . I hated every part of it. And I realized that when you leave academia, you’re really out, There is no fluent transition, you’re in or you’re out. You don’t know how high the walls of the academic bubble really are, until you found yourself outside.

I studied here in Delft and in Leiden, did a PhD in Amsterdam and then I continued as a postdoc at Washington University in St Louis Missouri, where I worked on vaginal microbes, lactobacilli but also other bugs. I also write a weekly column in NRC Handelsblad and I worked on a book. That is finally here. It’s called Ode aan de E-nummers, it’s on a hobby of mine, a side project, food technology, and I spend the past weeks promoting it.

The funny thing is that all the written output I create in those different jobs, both as a writer for a newspaper, as a writer of a book and as a scientist, you have to pay in order to read my stuff. The big difference is that when you purchase one of my articles for the newspaper NRC Handelsblad you indirectly pay me, the author. When you purchase one of the articles from a scientific journal, you don’t pay me, you don’t pay the people who reviewed it, you pay Elsevier, or Springer or another publisher. This is problematic, even fraud if you ask me. Furthermore, the public is higher educated than ever. More people are going to colleges where they are exposed to basic scientific ideas, having to read and even write research articles. The saddest outcome of this system is when you end up having to send journal articles to the family of severely sick patients because you are the only access they have. I believe the open access train is well underway, partially thanks to the open science iniative pushed by the Netherlands.

My book is doing well, I can make a living as a writer and entrepreneur. Still my heart breaks with the idea to no be involved with science any longer. The big question for me was how I was going to continue after having left my postdoc.

The push factor in science

There are pull and a push factors in science. The pull-factor is clear. I love science. I love the excitement of understanding how the chemistry of life works. This is the best job in the world. But there are also push factors. I love how free I am as a freelance writer, to just say whatever the hell I want, to communicate freely, on my own, just push the send button, without first having to consult 7 co-authors, and three anonymous peers. As a scientist you’re not free like that. Publishing in the right journals is essential for success. In the academic monetary system, high impact peer-reviewed publication are currency. And those papers need to be shiny, solid filled with good news. Scientists are therefore keeping their cards to their chest. Often you hear people say: “you don’t want to give full openness, because what if somebody takes your research and uses it?”. Then I ask: “isn’t that the whole purpose of science?”

There are some steps taken to open up in academia, but the biggest advances you see on the education front, not the scientific front. In higher education people are slowly breaking down the walls of academia. I was able to follow a Massive Online Open Course on Computer science and bio-informatics from home. I believe real progress is being made on that aspect. On the other side of academia, the research part, we’re far behind. We are still waiting for the digital revolution in scientific communication. The only way you’re able to follow science when you are outside academia, is through peer reviewed articles in journals. That means that from outside the bubble, you can only follow your field with a few years of delay, because that is how long it generally takes for research to make it into a paper.

Luckily papers are not the only way we communicate. In biology, we also show parts of ongoing projects at conferences. There you see rows and rows of carefully crafted A0-sized posters full of information, some people are taking pictures with their phone, because you never see digital versions of those posters, they’re never published. they are only meant for the handful of lucky ones who happened to be present at that conference. Why don’t we publish them? Is it because publishing one of those posters online, on social media or a blog or Researchgate doesn’t result in an impactful publication, with an impact factor? That would sad, right?

I follow many scientists on Twitter. I know what their children look like, I know what music they love or where they go on vacation, but I rarely here what they are doing in the lab. Sometimes they ask each other questions, mostly around scientific trivia or funny side projects in their lab: what is this bug? Why does it look weird? Or they ask: what is the best software to study this problem? Rarely do they say: look what I found. Here is some fresh knowledge, straight from the lab. It is very disappointing how much openness social media has brought to science. It is still a very closed system. Rigid, institutionalized and behind very high walls. That should change.

Open Kitchen Science

So I decided to go back to science, but on my own terms. I want to work from the deep conviction that the purpose of science is to increase our collective knowledge and by doing that make the world a better place. If I manage to increase our general knowledge, or if my work facilitates others to do the same, I achieve my goal. So therefore I chose to share as much possible.

Not only open access, not only sharing raw data and materials. But “real-time open science”. Compare it to vloggers. Those people that share hour-long footage on the regular, banal activities like how they put on their make-up or drive their car or cook their food, eat their food. We have an abbreviation for it: TMI, too much information. That is exactly how I want to do science. I want to publish ALL my findings. The whole process. Every method, everything that works or doesn’t work. Every assay I am trying to develop. Every question I have, every experiment, no, every thought of an experiment and just write it down and put it online, by myself, without having to consult with anyone. I want my work to be open and free for everyone. Any social media or blog is fine, I am now working on crafting my own WordPress-blog, I don’t want it to be too shiny, I don’t care about the form. I care about the content. It just needs has a proper comment section and a clear indication what results have been reviewed, or what results have independently been validated, reproduced.

I am calling this project RebLab, and the type of science I call radical transparent science or OpenKitchenScience. It is not entirely new, a few scientists are choosing to work this way, some call it Open Notebook Science. One great example is labscribbles, where you can follow the work on the Huntingtin protein by dr. Rachel Harding in real time.

Peer review

The biggest science rule I am breaking by doing this is by not having everything peer reviewed. Don’t get me wrong: I believe peer review is very important to maintain a high standard of scientific quality. At the same time I believe we’re overdoing it. Peer review is starting to hold us back. It places a huge hurdle in between scientists because it means everyone is only communicating with a delay. Peer review is probably also the most important reason why results that don’t make it to a paper are never shared,. We are sharing shiny positive nice stories with each other, while so much of our knowledge, ugly practical things, negative results, stays in the lab. Why are we not sharing that? Why don’t we throw it on a blog, send around a newsletter, throw it on Facebook for all I care. Communication in science takes too much effort, time and money, and an important reason is because we want to have everything peer reviewed.

Reproducibility crisis

We are currently facing a reproducibility crisis. Our current system of science, of increasing our knowledge is not working properly. More than one third of the high impact prestigious cancer studies were found to be irreproducible. It was the pharmaceutical industry that pointed to this problem. “Hey guys”, they said “I don’t know what you’re doing. These might be superfancyschmanzy Cell Nature Science results but they are not working for us. They don’t hold up when we are trying to do the same things as you tried”. In social psychology it was apparently even worse and over half of the studies cannot be independently reproduced.

Dutch funding organization NWO is now spending 3 million euros specifically on replication studies. Great! But here is a question: aren’t we already replicating each other’s work all the time? We call it different and we’re normally not very excited when it happens, we call it scooping. In my view peer review is the silver standard. Getting scooped is the gold standard.

Here is an assumption: in the cancer field where one third of those studies could not be replicated, they already knew. This is a highly competitive field. There must be at least 20 labs worldwide working on that same problem. There must have been already awareness in the field that these same results were not found across the board. Perhaps, in a scientific world where the default is to share, and the gold standard is to reproduce and not to review, we would have already known.

First steps towards Open Kitchen Science

In February I went to Prof. Remco Kort, both working at TNO and the Free University of Amsterdam, and I proposed to him to do a project on carbohydrate metabolism of vaginal microbes. I basically want to know what the lactic acid bacteria that colonize the human vagina, what carbohydrates they metabolize. What material they use to convert into lactic acid and acidify the vagina. That’s my research question. That question has been haunting me for a few years now. Luckily that question also has his interest.

And then I said: “it’s not a normal research project. It has to be radically transparent, it has to be open kitchen science”. And he said: “sure”.

Sure? I expected to run into a plethora of problems. This was radical right? Suddenly I doubted the rebellious nature of this endeavor. Hello? I want to publish science on a blog, I want to publish my posters unreviewed. Where are all those walls I need to break down. He said: “fine”.

Then I asked a few scientific journals, the ASM (American Society for Microbiology) journals that we, microbiologists often publish in. I asked: “what if I prepublish my experiments, would you still consider it for your journal?” This is important because any collaborations or students who want to work with me, might still need papers for their CV. And at some point I do want to have stuff reviewed by colleagues. And you know what the journals said? “Fine”.

Then we went to the safety officer to ask if we could do open kitchen science and she said “fine”. Then we went to the communication department to ask if we could do Open Kitchen Science and they said “great”. And then we went to the head of the department to ask if we could do Open Kitchen Science, and he said “awesome”.

It went surprisingly smooth. No big hurdles yet. My blog is not up and running yet, but I hope to make some advances in the lab this summer. What I learned sofar is that it really looks like this whole system of holding your cards close to your chest, this system is mostly cultural. I learned, that there are no rules that stop you from sharing. We humans are herd animals, and we do what the other people do. Scientists are no exception to that rule. We have strong culture and social norms about how you behave in this academic setting. It’s both good news and bad news. A lot of the hurdles for openness are in our minds, the barriers are mostly imagined but they are holding us back. I believe we should change this culture because it is not good enough. It should be standard procedure to overshare. After all, there is no such thing as Too Much Information in science.

Negative Results Week

As a last remark: I am very excited about the National Plan for Open Science. I feel that there is currently true momentum here in the Netherlands but also elsewhere in the scientific community to change the way we communicate in science. I also understand not everyone in the academic world is going to radically open up and throw everything they know and have online, like I plan to do. I believe people need a bit of a nudge to dip their toes in the open science waters and see how it feels. To help them I want to propose to, as part of the Dutch Open Science community, to invite other scientists to engage in a challenge. I believe we should organize a negative results week. we invite scientists to share a short paper, unreviewed, on some platform, that is unowned, independent, Wikipedia-like, open source, with a negative result. Once. Just to see if they like it. If you are interested in organizing something like this, with me, let me know.

Thank your for your attention.

About the human vagina and its microbial inhabitants

During the reproductive years -between puberty and menopause- the bacterial communities colonizing the vagina show a remarkable lack of biodiversity. In contrast to the microbiota of the mouth, the skin or the gut, most vaginal environments have a dominant presence of one of four different closely related lactic acid bacteria: Lactobacillus crispatus, iners, jensenii or gasseri.  Another distinctive aspect of the human vagina is its acidity. Lactic acid, commonly assumed to be produced by these bacteria , lower the human vaginal pH to levels of around 4, considerably lower than that of some apes and monkeys that are our closest relatives. A subset of women have reduced levels of Lactobacillus and a higher pH. Women with this dysbiosis, called “Bacterial Vaginosis” often have no symptoms, but in some cases it leads to abnormal odor and secretions. There are more serious health effects : lower levels of acidity and Lactobacillus are associated with an increased risk of acquiring or spreading sexually transmitted disease. Pregnant women with bacterial vaginosis are at increased risk of preterm birth, possibly due to infection by vaginal bacteria.

We understand very little about the bacterial composition of the human vagina. For instance, it is unclear why humans are the only apes with this high acidity and dominance of Lactobacillus whereas these characteristics are absent in other primates. Why is the human vagina such a good host for these specific bacteria? Furthermore, we don’t know how these lactobacilli acidify the environment, what (food) sources do they use? And what can cause the microbiota to switch to this Bacterial Vaginosis state?

The answer might be found in another characteristic that is distinctive of the human vagina and that is the prevalence of glycogen. Glycogen is a long polymer (chain) of single glucose units that is normally used to store energy for instance in the liver. The human vaginal epithelial layers during a woman’s reproductive years are also rich in glycogen. Its accumulation is regulated by estrogen and it can be used as an energy and carbon source for bacteria to grow and metabolize, but only if they have the enzymes to cut it up in smaller polymers or its single sugars.

Can vaginal bacteria utilize glycogen to colonize the vagina and to acidify the environment? That is one of the main question I aim to answer.

Open Kitchen Science – talk to OMHE conference april 2017

This is a talk I gave to a conference on Higher Education april 2017 where I spoke extensively about my ambitions for Open Science.

I am 32 years old. Last year, when I left my postdoc project in the USA, was the first time since in my adult life that I was outside of academia. No longer did I have an .edu account that provided access to literature. For the first time there was no research anywhere closeby, there were no working groups, literature discussion, or practicum, I was not surrounded by students, I was not surrounded by that exciting vibrant atmosphere, thick with curiosity, and I hated it.

I hated every part of it. And I realized that when you leave academia, you’re really out, There is no fluent transition, you’re in or you’re out. You don’t know how high the walls of the academic bubble really are, until I found myself outside.

I am a microbiologist. I studied here in Delft and in Leiden, did a PhD in Amsterdam and then I continued as a postdoc at Washington University in St Louis Missouri, where I worked on vaginal microbes, lactobacilli but also other bugs. But I am also a writer, a communication specialist as I like to call it myself. I write a weekly column in NRC Handelsblad. Last year when we came back from the USA and I worked on a book. That is finally here. It’s called Ode aan de E-nummers, it’s on a hobby of mine, a side project, food technology, and I will spend the coming weeks promoting it.

The big question is, what to do now. Am I going back to science? There is a pull and a push factor. The pull-factor is clear. I love science. I love the excitement of understanding how the chemistry of life works. This is the best job in the world.

But there are also push factors. I love how free I am as a freelance writer, to just say whatever the hell I want, to communicate freely, on my own, just push the send button, without first having to consult 7 co-authors, and three anonymous peers. I just love pressing the send button. As a scientist you’re not free like that. Publishing in the right journals is essential for success. In the academic monetary system, high impact peer-reviewed publication are currency. Scientists are therefore keeping their cards to their chest. Often you hear people say: You don’t want to give full openness, because what if somebody takes your research and uses it, and then I ask: isn’t that the whole purpose of science?

In higher education you are slowly breaking down the walls of academia. I was able to follow a Massive Online Open Course on Computer science and bio-informatics from home. I believe real progress is being made on that aspect. But on the other side of academia, the research part, we’re lacking behind. We are still waiting for the digital revolution in academic publishing. The only way you’re able to follow science when you’re outside academia, is through peer reviewed articles in journals, and then only when they’re open access. That means that when you’re outside the bubble, you can only follow your field with a few years of delay, because that’s how long it generally takes for research to make it into a paper. Now there are other ways that scientists communicate with each other of course.

When you go to conferences and see rows and rows of carefully crafted posters but you never see those posters online, they’re never published. they are only meant for the handful of lucky ones who happened to be present at that conference. Why don’t we publish them? Publishing one of those posters online, on social media or a blog or researchgate doesn’t result in an impactful publication, with an impact factor.

It’s funny I follow many scientists on Twitter. I know what their children look like, I know what music they love or where they go on vacation, but I rarely here what they are doing in the lab. When they go to a conference I see pictures of the beaches, I see puctures of them drinking beer with their peers, but I don’t see their slides, I don’t see a video of the talk, I don’t see the poster. Sometimes they ask each other questions, mostly around scientific trivia or funny side projects in their lab: what is this bug? Why does it look weird? Or they ask: what is the best software to study this problem? Rarely do they see: look what I found. Here is some fresh knowledge, straight from the lab. It is very disappointing how much openness social media has brought to science. It is still a very closed system. It’s rigid, it’s institutionalized and it’s all done behind very high walls, invisible to the outside world.

So I want to go back to science, but only when I am free to do open science. Not only open access, not only sharing raw data, and materials. But real open science. Compare it to vloggers. You know those people that share hour-long footage on the regular, banal activities like how they put on their make-up or drive their car or cook their food, even eat their food. That is exactly how I want to do science. I want to publish ALL my findings. The whole process. Every method, everything that works or doesn’t work. Every assay I am trying to develop. Every question I have, Every experiment, no, every thought of an experiment and just write it down and put it online, by myself, without having to consult with anyone. I want my work to be open and free for everyone. Any social media or blog is fine, I don’t care about the form. I care about the content. It just needs has a proper comment section.

I am calling this project RebLab, and the type of science I call radical transparent science or OpenKitchenScience.

Now don’t get me wrong. I believe peer review is very important to maintain a high standard of scientific quality. At the same time I think we’re overdoing it. It’s starting to hold us back. I don’t believe it is necessary to apply this gold standard to every single piece of data or result that we share. I believe we should be publishing micropublications. For instance: Look these bacteria indeed are not able metabolize this important carbohydrate. Negative result, important result. After two weeks of trying I finally learned how to dissolve this stupid reagent called blue amylose. This stuff should be shared. Maybe it fits in a tweet, maybe in a small update on my labs website, maybe on a blog, or on researchgate, accompanied by a video, or a picture paragraph of text. We learned that certain strains of Gardnerella vaginalis only produces L-lactate, not D-lactate. We’re probably never doing anything with this, it is just knowledge floating around in the lab, but it belongs to the public. It doesn’t needs peer review, it needs a proper comment section for open discussion like every social medium has nowadays.

Only when you are ready to draft a bigger story, where you link this L-lactate to a lactate dehydrogenase, and a gene and transform it and study it and want to claim to understand the mechanism en conclude bigger things about it, there is no doubt that at that moment you should apply rigorous peer review invite independent researchers to comment on your stuff, anonymously or not.
But the experiments that are leading up to that paper, I want to post them online. Liveblog my research if you will. Like vloggers, overshare.

So. In February I went to Prof Remco Kort, both TNO and VU, and I proposed to him to do a project on carbohydrate metabolism of vaginal microbes. I basically want to know what the lactic acid bacteria that colonize the human vagina, what they eat, what material to use to convert into lactic acid and acidify the vagina. That’s my research question. That question has been haunting me for a few years now. And that question also has his interest.
And then I said but it’s not a normal research project. It has to be radically transparent, it has to be open kitchen science. And he said: sure.
Sure? I expected to run into a plethora of problems. This was radical right? Suddenly I doubted the rebellious nature of this endeavor. Hello? I want to publish science on a blog, I want to publish my posters unreviewed. Where are all those walls I need to break down. He said: fine.

Then I asked a few scientific journals, the ASM journals that we, microbiologists normally publish in. I asked what if I prepublish my experiments, would you still consider it for your journal. This is important because any collaborations or students who want to work with me, might still need papers for their CV. And at some point I do want to have stuff reviewed by colleagues. And you know what the journals said? Fine.

Then we went to the safety officer to ask if we could do open kitchen science, and she said fine. And then we went to the communication department to ask if we could do openkitchen science and they said great. And then we went to the head of the department to ask if we could do openkitchen science, and he said: “great”.

It went surprisingly smooth. No big hurdles yet. It really looks like this whole system of holding your cards close to your chest, where the default is to not share, that this system is mostly cultural. I learned, that there are no rules that stop you from sharing. So why didn’t we just write on Facebook or on a blog or on Researchgate that this strain of G vaginalis only produces L-lactate. Because that’s just not the way we do things. We humans are herd animals, and we do what the other people do. Scientists are no exception to that rule. We have strong culture and social norms about how you behave in this academic setting. That in my view is the most important reason why we don’t open up more.

A lot of the hurdles of openness, of communication if you will, are in our minds, the barriers are mostly imagined, they are in our head, and they are holding us back. I am very determined to fight this standard and to change this culture because it is not good enough. Especially if you are a publicly funded scientist. I believe we should open up are labs to the outside world. It should be standard procedure to overshare. After all, there is no such thing as Too Much Information in science.

Thank your for your attention.

First steps

Hello everyone, first post of my “open kitchen science” research initiative. I am very excited to announce that I have started working at the Free University of Amsterdam, within the Host-Microbiome group of Prof. Remco Kort at the Microbiology department. I will keep a regular update on the progress here. Currently I am still finding my way around in the labs, looking for chemicals, getting the right strains and looking what equipment is available. I will also start filling the website with background information on metabolism of vaginal microbes and the question I want to answer with my experiments.