Adventures in Lentivirusland

I've already written a bit about my troubles regarding the packaging of my lentiviral particles. At the end of my last lentiviral post I had figured out that the packaging of my CherryPicker vector was delayed and optimal collecting time was around 60 hours and not 48 hours. Additionally, I found that the amount of virus-associated p24 (lentivirus capsid) was highest in my target cell medium making harvesting and subsequent transduction quite easy. The problem was, however, that the amount of virus-associated p24 capsid only tells you how much virus you have in your supernatant. It does not indicate the amount of infectious virus in your sample and that's pretty much what you care about when you're harvesting lentiviral supernatant. 

Before, when I was using GFP as a reporter gene I would estimate the lentiviral titers by flow cytometry (FACS). I would transduce Huh7 cells in a serial dilution and then analyze the percentage of green cells in a FACS machine and then calculate transduction units per milliliter. Since I don't currently have access to a FACS machine to detect mCherry I thought that I could maybe make use of the TCID50 method to estimate the titer.

I diluted my newly collected lentiviral supernatant into a 96 well plate (similar as shown in the picture) and evaluated what wells still had fluorescent mCherry signal. Then I estimated the titer using the Spearman & Kaerber algorithm. I also estimated the titer of some GFP viruses I had along with my previous CherryPicker stocks so I had something to compare to. 

Not only does this method estimate the titer of my lentiviral stocks to be quite similar to what I had already estimated with either p24 ELISA and/or FACS but it also showed that the CherryPicker viruses that I collected after 60 hours had a very high titer. Higher than all of the previous CherryPicker stocks and even the GFP stock. 

So it seems that I have solved my lentiviral crisis. At least, this lentiviral crisis. I currently have some other ones I might share later. 

Soon I will also post a little bit about my musical guilty pleasure. 

Until next time!

Hüldi

A wonderful assortment of Christmas cookies

Dear friends

Quite some time has passed since my last post and thus, with delay, I wish you all a very happy and wonderful New Year - and I hope you will keep following our blog with curiosity and interest. I was pretty busy just before Christmas, as I not only had to finish important experiments in the lab and last sentences for my review; but we also had to move our lab and the flat to a new city. I was very touched by the great and emotional moments during our goodbye party - I certainly will remember all these funny chats while pipetting or splitting cells, the BBQs on the terrace with its amazing view over the city and I'm very grateful for all the experience and knowledge I acquired while working with so many different people - thanks for that!

After moving my belongings to the new flat and having unpacked all those boxes, just one week was left till Christmas and I was enjoying the time baking Christmas cookies for my family and friends. Christmas cookies have a long tradition in our family, and every year they are prepared in the same fashion concerning ingredients and shape and with the same accurateness that i learned from my mother.
This year it was my responsibility to prepare the cookies, as everyone was still working and as busy as one is around that time of the year! I have to admit, it was a lot of work to bake ten different types of cookies all by myself - but i really enjoyed the time to relax after busy weeks in the lab.

A resume of my homemade cookies:

Now, to the essential part of the blog: my different type of cookies - i will shortly describe each of them, so you can get inspired for next Christmas and if so, just contact me and i will be happy to share recipes and experience with you;-). One of my favorite cookie is the "spetzbueb", which is made of a very thin dough mainly containing butter, eggs and flour and whatever jam you like. I prefer, as the original recipe suggests, Raspberry Jam. If you manage to cut the cookies as thin as possible, they will melt in your mouth;-)

In the upper right corner of the plate you find a cookie based on egg white (like meringue), dates and almonds. This is the first time I ever made them and they were the favorites of many people - the foamy egg white makes them rather light for a cookie and the pieces of dates give them a very exquisite taste. Just bake them until the are slightly colored - otherwise they become dry and crumbly very fast. As many people became fond of them I will recycle the recipe also during the year for birthdays or other events, I am thinking to prepare an alternative with dried apricots or with figs - how does that sound to you?

On any Christmas-Cookie plate, cinnamon-stars should always be present. If you don't bake them too long they are still nicely humid and the taste of cinnamon will inspire you and get you in a very "Christmassy" mood.  You can also modify the icing by adding cinnamon which will intensify the flavor of the cookie a lot. I advise to not cut the stars to small, as they not only become dry much faster, but you also need much more time to ice them;-)

In the lower right corner you will see dried apricots coated with dark swiss chocolate. They are very easy and fast to prepare - but nevertheless taste lovely! They always remind me of that party night where we prepared a chocolate fondue and dipped all kinds of fruits into dark chocolate - for me just the perfect combination.

The brown cookies called "Basler Brunsli" are said to originally come from Basel and are a combination of dark chocolate and almonds, with a drop of cherry liquor. You like them or you don't - either way, they will be present on my Cookies plate every year - you can't meet the taste of everyone i guess;)

In line with the pistachio cake i posted some months ago, I intended to implement this idea into a Cookie and was happy to see that in its newest issue, Betty Bossy had the same idea and she added small pieces of orange peel ;-). I was very curious to see how that cookie will turn out, as its the first one to be so intensely colored. In my opinion, the green hearts looked very beautiful and enhanced the overall impression of the plate. However, the particular taste of pistachio was missing somehow - although I liked the flavor of oranges. Next year I will improve the recipe by adding the particular "pistachio dough" I used to prepare the biscuit of the cake.

I do hope you got inspired by the variety of cookies I presented, and that you are already looking forward preparing them for next Christmas...I am sure your family and friends will very much appreciate your effort.

Yours, Eva

Say goodbye to 2013!

Happy sciency new year to all our readers! We are currently divided between two countries but our blogging will return strong and amazing in January. Next year we also promise to do more science (see below). 

Tonight is also the perfect time to listen to this This Week in Virology podcast. In the last episode of 2013 they cover all the cool things that happened in virology this year.

Happy new year!

-Hüldi

Lab moving and Christmas

We've been extremely bad bloggers lately. But we have an excuse. Sort of. We have been relocating our lab and ourselves from St.Gallen to Bern. Moving is very time consuming and exhausting but thankfully it's all over now. Although we still have to organize our stuff at the lab in Bern. 

Before we moved we had a really nice goodbye party in St.Gallen with good food and way too much alcohol. Side note: Bring popcorn to every party you go to. People love popcorn.

 

We were also thrilled when we found out that our last lunch at the hospital in St.Gallen was this:

Now everyone is home for the holidays, eating and sleeping. Reloading for a new exciting year at a new lab!

Eva has also baked some Christmas cookies she will show you. I will also post an update on my lentiviral adventures soon.

-Hüldi

The great concert month of 2013: November

In November I attended four concerts, one every week. Two of these were attended with my fabulous co-blogger Eva. The schedule looked like this:

November 5th: Thirty Seconds to Mars, Hallenstadion, Zürich.

November 12th: Emiliana Torrini, Club Xtra, Zürich.

November 18th: Placebo, Hallenstadion, Zürich.

November 28th: Sigurros, Oslo Spektrum, Oslo.

Thirty Seconds to Mars:

Going to see 30STM was a spur of the moment thing since I had only discovered the band about two weeks before the concert. I have pretty much been obsessed with them since the beginning of November. Now, I'd be lying if I said that a lot of that obsession didn't have anything to do with the pretty frontman Jared Leto. However, his brother the drummer, is also quite awesome. Everytime they showed him playing on the jumbo screen you couldn't look away. He has such a dynamic energy when he plays that it kinda made me want to learn to play the drums. The concert was also extremely good. Jared is very interactive with the crowd, although sometimes a bit too much, since I come to a concert to hear the band. Not fans singing their songs. A lot of people were brought up on stage and they released giant balloons for the crowd to play with. I was so happy after the show that I actually bought a band T-shirt. I almost never do that. Especially after such a short relationship.

This is my current favourite song by Thirty seconds to Mars; Kings and Queens from the album This is War. Their new album is called Love Lust Faith Dreams, available at iTunes here.

    

Emiliana Torrini:

I've been a fan of hers for years! Actually, I can't quite remember if I have been to a concert with her before. That's not so good. Blame it on my educational alzheimers. The mood at this concert was fantastic. She is a great musician. She plays mostly slow and somewhat sad songs but she is truly one of my favourites. There was no extravagant light show, just her and the band. She also attempted to speak German so I could actually not understand much of what she said. But she made people laugh. She also played almost all of my favourite songs from her new album and the older ones I've fallen in love with. I actually wanted to buy a T-shirt at this concert too but I forgot to withdraw some cash. In Switzerland you must always have cash. Otherwise you miss opportunities like this.

This is my all time favourite Emiliana Torrini song; Today Has Been Okay from the Album Fisherman's Woman. The new album is called Tookah and is amazing. I highly reccomend it. Buy it on iTunes now! 

  

Placebo: 

I've always liked their music quite a lot even if I was never really a die-hard fan. As a result, I didn't know many of their new songs but they also played some of the good older ones that I do like. The only downside was that I was quite flu-ish during the concert. Towards the end I could no longer speak. The music was good and the mood excellent. However, there was this one guy that kept shouting "PLACEBO" at unappropriate times. But he probably just wanted to make sure that the band didn't forget who they were. What I kind of missed at this concert was the interaction with the fans. 30STM overdid it a little bit but Placebo hardly even acknowledged the crowd. But overall it was a good show. Although, I would have preferred not to be sick.

This is one of my favourite Placebo songs (even if it's a cover); Running up That Hill originally from the album Sleeping With Gosts. Their new album is called Loud Like Love, available on iTunes here.

   

Sigurros: 

Been a die-hard fan of theirs for years! But I always missed their concerts. When they played in Bergen I lived in Iceland, when they played in Iceland (for free!) I lived in Bergen. When they played at Iceland Airwaves I lived in Switzerland. When they played in Switzerland tickets sold out before I could get my hands on some. An so on and so forth. So when they were playing in Oslo on my Birthday I took it as a sign. That was my concert. And I was going. I went. It was awesome. Here I also bought a T-shirt. Even if I had no cash. Scandinavia is card friendly.

This is my favourite song from their new album, Kveikur. They started the concert with this song. It was perfect.  

I'm pretty sure that I will have a hard time topping the great concert month of 2013. But I will try my very best.

-Hüldi

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.