Artsy Science

Almost a year ago our lab published a paper in mBio about the newly discovered coronavirus MERS-CoV (although it was called HCoV-EMC back then). In short, our conclusions were that the MERS coronavirus grows very well in human airway epithelial cells cultured at Air-Liquid Interface (ALI) and as other human coronaviruses it evades host innate immune recognition. Additionally, we showed that treatment with recombinant human interferon can reduce the replication of MERS-CoV in our culture system. 

We also submitted a suggestion for a cover image once the paper was accepted for publishing. We wanted to have a little fun with it and make it a bit psychedelic and interesting. The image is made from a confocal Z-stack of human airway epithelial cell culture infected with human coronavirus 229E, a common cold virus. The stack was reconstructed in 3D using Imaris software. The same software was used to change the colors but the image itself was assembled in CorelDraw.

Upper left corner: Nuclei (blue), Cilia (red), viral RNA (green).

The paper also has some psychedelic images of ALI cultures infected with SARS and MERS CoVs. Those who are interested can find the paper here. 

The Science News on Monday

Today in "News on Monday" : 

Please tell us what is the difference between an 
invertebrate cell & a vertebrate cell !   

Published: 2011, PlosPathogens                       

Virus: Nidovirus, Coronavirus, Nam Dinh virus (NDiV), Vietnam

Host species: Insects, mosquito

Aim of the study: 

To compare the genome organization and main enzymatic activities of the first insect-born nidovirus with known representatives of the Nidovirales

Gen /protein of interest: 

nsp14 Exonuclease (ExoN) , nsp15 Endoribonuclease (EndoU)

Main discovery in 2 sentences: 

1) The acquisition of an ExoN is a prerequisite for the evolution of large RNA-genomes

2) The EndoU is not a nidovirus-specific marker, as it is not present in insect-born nidoviruses

Implications for the field: 

1) Studies on CoV-MHV and SARS with defective ExoN demonstrated that while replication kinetics and viability of the mutants were only mildly affected, the mutation rate and frequency was significantly elevated throughout the genome. Furthermore, in vitro studies revealed that the 3'-to-5' ExoN is able to excise single nucleotide at the 3' end of RNAs, thus implicating a role as a proofreading enzyme, which was until then only know for the DNA-world. The discovery of the insect-nidovirus with a intermediate genome size of 20.2 kb and its possession of an ExoN is a major step in CoV-research, as it marks the bottom line of a "large-Nidovirus" possessing an ExoN. With its discovery, the gap to the next known RNA-virus lacking an Exon could be reduced to only 0.8 kb. To conclude, ExoN confers very high replication fidelity which is needed for the maintenance of a very large and complex genome size. 

                       

Secondly, it was believed that the endoribonuclease (a nuclease that cleaves cellular or viral RNA) was unique to Nidoviruses and was thus proposed to be a nidovirus-wide marker. However, the discovery that the newly identified insect-virus NDiV and also Roniviruses (invertebrate-virus, replicates in crustaceans) dont possess an EndoU-activity contradicts this hypothesis. As Roniviruses belong to the large nidoviruses, it can be considered unlikely that the EndoU is involved in RNA-proofreading and replication. 

The biochemical characteristics of the EndoU-activity of Coronaviruses or arteriviruses are very well discribed, however its function  during virus-replication and its involvement in innate immune evasions is unknown.

Remaining questions:

1) Is it possible to further reduce the gap between RNA-viruses with and without ExoN-activity?

2) Is the ExoN involved in innate immune evasion?

3) why do invertebrate-Nidoviruses (irrespectively of their genome-size) not encode an EndoU?

4) what is the difference during virus replication in a vertebrate and in a vertebrate cell?

Plus factor of the paper: 

The discovery of a first insect-born Nidovirus is a very important step in this field, as it opens the possibility to compare viruses from the same order that have a different host range. Furthermore, the genome-wide comparison between known vertebrate nidoviruses and NDiV was executed in very detail, as all important enzymes were discussed separately. The discovery of Nidoviruses without an EndoU is very crucial for further work on EndoU activity and this will surely give very interesting insights into the replication of viruses with different host range.

Drawback of the paper:

Generally, the paper is well written, but quite long and especially the discussion is very extensive and could have been reduced without any loss of content. Furthermore, more in depth implications of the present work were missing.

Lentiviral packaging for Christmas

A large part of my PhD project revolves around creating and using various lentiviral particles for gene transfer into mammalian cells. The principle of lentiviral packaging and transduction goes something like this. You transfect your packaging cell line (I use HEK-293T) with plasmids encoding the envelope and capsid virus proteins along with the transcriptase and integrase. That will be sufficient to create your pseudotyped particles. Lastly, you include your transgene or shRNA for expression/knockdown in your target cells post transduction. 

 

www.amsbio.com

The number of plasmids used for transfection can vary but in my project I use four; one with my shRNA (so far only scrambled), MDL (HIV gag-pol), VSV-g (the glycoprotein from the Vesicular Stomatitis Virus for target cell entry) and Rev (HIV rev). Transfection of those four plasmids will result in the packaging of lentiviral particles containing my scrambled shRNA. The vectors I use belong to a 3rd generation lentiviral packaging system. This system is safer than the 2nd generation system but slightly more cumbersome as it requires the transfection of four separate plasmids (more information about packaging plasmids). The resulting lentiviral particels are replication deficient and are only capable of one round of infection and integration.

When I started I was using a roughly 5kb scrambled shRNA plasmid with a GFP reporter gene. Originally I had some problems collecting supernatant with high viral titer. That problem I could easily solve by using younger cells. Generally it's not recommended to use HEK-293T cells after passage 30. Mine had not quite reached that age but it seemed they were no longer up for the daunting task of assembling my particles. Once I changed the cells everything went somewhat smoothly. Or at least as smoothly as things can go in science. I optimized the transduction of my target cells and determined that the methods I was using were compatible with our specialized culture system.

Then we had the brilliant idea to change the shRNA vector. We found this extremely neat little vector called CherryPicker. It is basically an mCherry fluorescent protein coupled to a truncated transferrin receptor. Both the mCherry and the receptor reside on the outside of the cell allowing for cell sorting using either the fluorescent tag or the receptor.
 

 

www.clontech.com

This can come in handy when you don't have access to a FACS sorter. Cell sorting using cell surface receptors and magnetic beads require no advanced equipment and should be quite straight forward. I say "should be" because, currently, my magnetic sorting does not work. Maybe I'll write about that later.

Once I attempted to produce lentiviral particles using the CherryPicker vector instead of the GFP one the titers went way down. I made three different stocks and titrated them on Huh7 cells using the protocol by Bard, Salmon and Trono from 2010 (Pubmed). This titration method had worked great for the GFP particles but only one of my three CherryPicker stocks had a measurable titer and that one was definitely not something to be proud of. Then I thought that maybe the ratio of my packaging/shRNA mix weren't optimal for the CherryPicker. That vector is over 11kb, so almost twice the size as the GFP-shRNA vector. Maybe I needed to add more? Or less? 

No. My current ratio (which is roughly 1:1:1:1 for all plasmids) turned out to be the the best one for the CherryPicker. However, the amount of the HIV p24 capsid protein (measured using p24 ELISA) for CherryPicker particles was only about 2/3 of the amount of p24 for GFP particles produced in the same way, with the same ratio. Then I started thinking that maybe the packaging of CherryPicker particles was delayed, since the shRNA vector is quite large. Maybe it was slowing down the process?

So I set up an experiment where I collected particle containing supernatant at 24, 36, 48, 60 and 72 hours post transfection (I even chronicled my excitement for you). I did this both for the traditional HEK-293T cell culture medium (DMEM+10%FBS) and also for the medium I use on my target cells (hence the two tubes). Serum can have an adverse affect on some cell types so it would be optimal to be able to collect the particles in either serum free medium or your target cell medium. Traditionally, harvesting of lentiviral particles in done around 48h post infection and that's what I've been doing so far. However, for the CherryPicker I found that there is a big jump in the amount of p24 capsid between hours 48 and 60 while the amount remained quite stable between hours 60 and 72. That indicated that the packaging of my CherryPicker particles in indeed delayed compared to the GFP vector and the amount of p24 is also higher when they are collected in my target cell medium! 

All in all, if it actually turns out to hold true when repeated, I may have solved my lentiviral packaging crisis. Just in time for Christmas. 

-Hüldi

A pistachio-heart

For a special person... for your "someone"

Heart-shaped cupcakes

who could resist ??

 

Flügelt ein kleiner blauer
Falter vom Wind geweht,

Ein perlmutterner Schauer,
Glitzert, flimmert, vergeht.

So mit Augenblicksblinken,
So im Vorüberwehn
Sah ich das Glück mir winken,
Glitzern, flimmern, vergehn.

Hermann Hesse

This poem is about a tiny, blue butterfly that lightly drifts in the sky - it sparkles, it twinkles and then it disappears - and everything left is a short moment of pure happiness.

I guess the take-home message from that poem is, that every so short moment of happiness and satisfaction should be cherished, as you never know how long it will last... These tasty cupcakes will sweeten every so worthwhile moment and will show to your special someone(s) how much they mean to you. 

Yours, Eva

Pistachio-Raspberry Dream

An amazing cake for a good friend

Personally, i really like birthdays - not the part where you should or have to find a present for a person, but it is a great opportunity to surprise a friend with a homemade dessert. And in my view, is there anything more personal than a homemade cake that you create with joy and love...

However, sometimes I find it quite challenging to find a nice recipe and to imagine how the cake will taste and if your friend will like it... so one idea of this blog is to exchange ideas of successful and tasty desserts...

I came across this splendid cake when I was searching for a dessert that will be the star-dessert for a party at work... and i found this cake on a blog of an amazing french lady..she is not only a very creative baker, but she also takes beautiful pictures of her cakes! So don't hesitate to get lost on her blog: http://pucebleue-jenreprendraibienunbout.blogspot.ch/

So, lets have a closer look at the actual cake...it consists of three different layers, each with a very unique flavor.. At the bottom, you find a pistachio-dough...fluffy and not dry at all, with a very deep and profound aroma of roasted pistachio and almond...my favorite part of the cake i have to admit...second, a layer of raspberry jam is the very fruity, but not too sweet continuation...finally, the top layer consists of refreshing quark and lime..you can adjust the level of acidity according to your own preferences...and if you are not a fan of lime or lemon at all - why not using as an alternative a fluffy, white-chocholate mousse?;-) 

 
I will show in my next post, how you can modify this recipe to

make people happy who are not really in favor of milky products....

 Yours, Eva    
you will find the recipe below:



Ps. If you have any questions concerning the recipe or if you have suggestions /alternatives to the proposed three layers - don't hesitate to ask or leave a comment - i always appreciate any kind of input;-)

Science and Food!

We wanted to add a bit more detailed post about our intentions for this blog. As I already mentioned in my first post the name Thirty seconds to PhD is partly stolen from the band Thirty seconds to Mars. We discovered the band, loved it and wound up seeing them in concert all in the span of 10 days. However, the seed was actually seeded at bit sooner, during an alcohol-less dinner at a PhD student conference in Bern. We were discussing how there are a lot of bloggers out there that blog about clothes, hair extensions and staying at expensive hotels. Obviously, there is nothing wrong with that but we felt that we have something to add to that since our life experiences are somewhat different. I mean, who has time for hair extensions when lab is calling. Our main source of blog material will most likely come from our work in the lab, describing our victories and struggles. We thought we might include some protocols we have optimized along with general posts about lab work and troubleshooting. Also, we sometimes attempt to find answers to some of our problems online but to no avail. Therefore, we were hoping to connect with other PhD students through this blog and swap war stories and knowledge. 

All this lab stuff might get a little dry in the long run so we thought we might include some food. Because, who doesn't like food? I myself cannot really cook but I have figured out some easy recipes that might come in handy for others who are also culinary challenged. So I might possibly share some of them with you. That is if I ever get around to cleaning my kitchen. My better half, however, is superb at baking so she will include recipes and photos of her artwork. I know everyone is looking forward to the fancy cake she'll make next week. 

Hopefully, we will also find our way to more concerts so we can share some more cool balloon photos with you. I also thought we could gather and post a list over scientifically oriented TV shows and movies for us geeks. 

If anyone has something they want to say about science or food, please contact us and become a guest blogger! If you're not up for that kind of a commitment don't hesitate to comment on our posts.

 

Let's blog the hell out of science and food together!

How to write a scientific review?

Task of the next weeks: how to write a scientific review?

I found a nice guideline that might help you once you are in a situation like this:

http://ueberfachliche-kompetenzen.ethz.ch/dopraedi/pdfs/Mayer/guidelines_review_article.pdf

further i recommend: prepare a well-structured overview before you get started. Divide your review article into small paragraphs and make a list of topics and papers you want to include. Then think about how to connect them and how to make a coherent story out of it.

in addition, before you get started (also when writing a paper): get your literature into a library, in order to be able to cite the articles in your paper. I highly recommend mendeley. You can add pdf-files to your references and access the library at several computers (at work and at home) and also online. Was a great help for me so far.

You guys know what's not fun...?

...to collect supernatant late on a Saturday night. Especially when it takes longer to walk to and from work than it takes to collect your samples. Yes. Those are two tubes. Two whole tubes. 

I'm having some trouble with my lentiviral particle production. I'll blog more about it once I finish this experiment and might perhaps know what's what.

Friends.

During a fast paced and stressful study it's important to have good friends. These are two of my friends; Sexual Harassment Panda and BiPolar Bear.

They're good buddies to have. 

Traditional Swiss Butterzopf

Not to miss on a sunday morning - a fresh butter Zopf...

A breakfast on Sunday morning without a butterzopf is like xmas without presents - something is missing. The tricky thing with homemade zopf is though, once you discover how easy it is to prepare and how amazingly it tastes (as I add slightly more butter) - you will find it difficult to go back to the zopf from the store. I personally prefer the Zopf with some butter and sweet jam or honey, others are more excited about a salty breakfast and eat it in combination with cheese, egg or cold meat. Another advantage of the homemade zopf is, that you can easily modify its shape - may it be a star for xmas, a heart for easter or birthdays...

Yours, Eva

Balloons...

Here's a photo from yesterday's concert featuring some balloons.

Because it's awesome. 

Yeah. 

New blog!

Yes, I know. I do this a lot.
But I am serious this time... sort of.

Me and my co-PhD student Eveline will be documenting our exciting lives in the world of science. The name of this blog (and the idea of it) came to us yesterday during a Thirty seconds to Mars concert. We felt it correctly described the feeling of working towards a PhD, it's so close but somehow so unattainable.

So get ready for pictures of late night titrations and kinetics. Photos of beer and bad weather. Also, Eveline bakes. So maybe there will be cakes.

And really, what else do you need?

-Hüldi