Saturday, October 26, 2013

Prokaryotic Saturday: everything is always more complex of what it seems


Although I skipped last week post, this is the  1st month of PS!  J
Smiling cupcake time:

And for today’s post let me talk about this paper of Sorek and Cossart. You have to pay to get it, but I have it, so if you feel like reading, email me...... did I just say that?.

So, Sorek and Cossart discuss some findings that have been reported by studying transcriptomes of bacteria. They also spend a couple of paragraphs describing the main approaches for transcriptome studies, RNA-Seq and tilling arrays.

First of all, let us clarify what transcriptomes are. From all the definitions I've read, the one that I liked the most (although I don’t remember where I read it, sorry) said that a transcriptome is the dynamic part of a genome. After getting the genome of an organism, the whole research thing doesn't stop there, you have to know where are those genes located, and most importantly what are they doing and how do they interact with themselves and the environment. That information you get it from a transcriptome analysis.

So, for years many eukaryotes have been widely studied, and there are several genomes and transcriptomes available for model eukaryotes. And as usual, bacterias and archaeas were overlooked. Mainly because of that old belief that microorganisms, for being small, are simple and insignificant (well, poor things, what can I say, sometimes that is also believed of short people, or kids). But guess what?!! that is not only wrong, it is WRONG!!!

But that was not the only reason; actually it is more difficult to study transcriptomes of bacteria than those of eukaryotes. Again, more development is there for techniques to work with eukaryotes. In bacteria the major problem is the need to enrich mRNA in the sample. Prokaryotes lack the 3'-end polyA tail, and >95% of the RNA is ribosomal RNA and you need mRNA, the other 5%. But now it is possible to enrich mRNA and Sorek explains a bit of this process in this really cool paper.

 As I told you, the two main approaches that have been used to study transcripts in prokaryotes are RNA-seq and tilling arrays. RNA-seq can be done in several of the platforms available (you know SOLiD, Illumina, etc etc). First you extract your RNA and synthesize cDNA by reverse transcriptase (RT). As bacteria don't have the polyA tail, then some priming step has to be there for RT to work, with oligo(dt), random hexamers or artificial poly adenylation. Then, don’t forget the fact that we need mRNA and it is only a 5% of the sample. So, several methods are there for enrichment, and in Sorek and Cossart paper they are briefly explained. Here they are :
  •  rRNA capture with magnetic beads, so at the end you remove the beads and the sample ends up being mRNA
  • degradation of the 5’P RNAs, so mRNAs in bacteria have an analogous to the cap in eukaryotes, a 5’PPP (triphosphate). So the idea is to get rid of all those RNA species, tRNA and rRNA, that does not have a 5’PPP.
  •  Polyadenylation of mRNA. Here an artificial polyA is added.
  •  Capture the undesirable RNAs with Hfq, a very famous protein originally discovered in E. coli that binds to RNA.

I do not know if there are more enrichment methods. Details about each of these I've told you are presented in the paper, and some other references are suggested in case you want to learn more. Authors also give the name of the kits to do all these protocols, you know, if there are two people in the world doing same thing, there should be a kit for that.

So, once you have your mRNA, you get your cDNA libraries, and again, several methods are there for doing this. And the final output of the RNA-seq are millions of reads (from 20 to 200 bp), that you use to align to a reference genome, and the expression of the genes is measured depending of how many reads are aligned to that particular region in the genome.

And in the other method, tiling arrays, after the cDNA synthesis, the library is hybridized to an array and expression is measured using signal intensities. Enrichment is not necessary here. So both methods require a reference genome, and tilling arrays do not need an enrichment step. Several prokaryotic transcriptomes that have already been completed using these methods include: Listeria monocytogens, Bacillus subtilis, Halobacterium salinarum, Burkholderia canocepacia, Listeria monocytogens, E. coli, Salmonella, Sulpholobus solfataricus

And what all people found, in general, is that prokaryotic transcriptomes are decidedly complex. Gene structures had to be remodeled as new genes were discovered and specially important ncRNA (non coding RNAs that are well know to play key roles in regulation of gene expression) were found. In this paper they mentioned one study (Wurtzel et al), in which they found 162 transcription start sites equivocally annotated. Although I don't know exactly why ORFs are usually predicted upstream of their actual places, this clearly shows how useful transcriptomes are.

Also, several riboswitches structures (you can read more about riboswitches in this previous PS) were discovered. It looks like 2% of bacterial genes are under riboswitch-mechanisms control. The detection of riboswitches is done by analyzing contiguous regions of the 5’UTRs at different conditions, when expression of such regions is interrupted at one growth condition and not in the other, you have spotted a riboswitch. Although this is a paper from 2010, an important comment they did is that there should be more focus on the 3'UTRs, especially in the case of archaea, which were already reported to have 3'UTR with regulatory roles. It'd be nice to find out what has been done in this regard in the last three years.

And now, to almost conclude this post, the most exciting (in my very personal point of view) part of the paper: gene plasticity. In this paper I found the reference of an experiment conducted in 2007, in which Mycoplasma pneumoniae was grown in 173 different conditions (woooth?!! ….... well, this shouldn't surprise us, we know someone who likes experiments of 100+ reactors ;)

So in this study, the found that operons (the bacterial genes) act in an homologous way to eukaryote genes, emulating alternative splicing… although, bacteria were here before eukaryotes, should we say that eukaryotes are emulating bacteria?

Probably, yes. In this study of the oh-so-many-conditions they saw that polycistronic regions can be transcribed as monocistronic when conditions varied, so, from one part with many genes, sometimes only one was expressed. So operons are versatile entities. And it looks like archaea act similar. So, who is copying who?

Another important discoveries were the definition of some ncRNAs in critical processes such as quorum sensing (more about this amazing bacterial signaling processes here) and the antisense transcription of some genes. So, isn't this cool? Although they are small, with small chromosomes and everything, prokaryotes overlap their genes, and it looks like "this is the rule, rather than the exception". 

I would like to keep writing, but this post is already too long. Please, check out the paper and if you have any comments, leave them down below these lines .

Wednesday, October 23, 2013

Want to read minds?

What do you think if, while you’re Reading books you get an ability to read minds?
in Science journal, mentioned that reading can help develop the ability to read minds or rather factions that other people express when conversing with them.
“The skills we use to navigate these ambiguous fictional worlds serve us well in real life” David Kidd (the author of this study) said. But it is not a fact.
So, if you are interested you can read here.

To continue reading, at the bottom you will find several comments with differing points of view on what the author is trying to explain. One of them mentions the book "Thinking Fast and Slow" by Daniel Kahneman and I couldn't avoid to look for it, and then I search in Google (and if you do the same, you will find this link).
In the book I found some interesting that I want to share with us.

And reads:

Now look at the following problem:

17 × 24

You knew immediately that this is a multiplication problem, and probably knew that you could solve it, with paper and pencil, if not without. You also had some vague intuitive knowledge of the range of possible results. You would be quick to recognize that both 12,609 and 123 are implausible. Without spending some time on the problem, however, you would not be certain that the answer is not 568. A precise solution did not come to mind, and you felt that you could choose whether or not to engage in the computation. If you have not done so yet, you should attempt the multiplication problem now, completing at least part of it. You experienced slow thinking as you proceeded through a sequence of steps. You first retrieved from memory the cognitive program for multiplication that you learned in school, then you implemented it. Carrying out the computation was a strain. You felt the burden of holding much material in memory, as you needed to keep track of where you were and of where you were going, while holding on to the intermediate result. The process was mental work: deliberate, effortful, and orderly—a prototype of slow thinking. The computation was not only an event in your mind; your body was also involved. Your muscles tensed up, your blood pressure rose, and your heart rate increased. Someone looking closely at your eyes while you tackled this problem would have seen your pupils dilate. Your pupils contracted back to normal size as soon as you ended your work— when you found the answer (which is 408, by the way) or when you gave up.

Amazing, isn't it?
I don’t know how real is that reading books help to understand people, unless you read this kind of books, but I think it is a good tool to perform our personality and most of all for self-knowledge.

Friday, October 18, 2013


Hola ! les comparto una foto que tomé hace unos domingos afuera de mi casa, me encontraba fotografiando esas flores, y de pronto llegó este invitado a desayunar, fue difícil capturarlo pero al fin se quedó quieto. Espero les guste! y si de paso me ayudan a identificar la especie estaría bien jeje, Saludos!

Thursday, October 17, 2013

Are you on the road ?

Hola a todos!

Por X o Y, hoy debía publicar algo en el blog (no me pregunten por qué jeje), bien, pasé buscando algún tema para compartir con ustedes pero no, no se me ocurría nada, revisé las páginas que usualmente leo en mis ratos libres pero no, no encontraba nada :/ así que decidí solo escribir lo que me viniera a la mente, y husmeando en mis entrañas me puse a pensar, llevo 1 año y 8 meses dentro del lab, nuestro lab, y comencé a procesar varias ideas. Destino ? Casualidad ? Causalidad ? o que había pasado ? jeje (de inmediato visualicé mi situación actual), ahora estoy todas mis tardes dentro de un laboratorio, tratando de encontrarle un porque a las cosas, tratando de superarme día a día, tratando de sacar una buena investigación, trabajo, tesis o como lo quieran llamar, y de pronto me pregunto: Realmente vale la pena ? realmente vale la pena dejar mis ratos libres y ponerme a leer un articulo ? dejar plantados a mis amigos por sacar mi trabajo a tiempo ? valdrá la pena aprender una técnica para que mañana ya esté obsoleta? Valdrá la pena todo lo que estoy haciendo ? ... La respuesta es Si, y creo que muchos de los que leemos este blog coincidirán conmigo, en todo este tiempo que he estado en el lab, siento que he crecido mucho en todo aspecto, desde el académico hasta el personal (importantisimo), he cambiado mi forma de ver la cosas y le he dado una dirección a mi vida (tal vez a veces no lo demuestre jeje), siento que he encontrado el camino que debo seguir para ser lo que quiero ser, y creo que es lo importante, saber qué quieres ser y a donde quieres llegar, tomando en cuenta dónde estas, realmente no se que estaría haciendo en este momento de no haber sido invitado a formar parte de este gran equipo, sinceramente cuando ingresé al lab no sabía lo que me esperaba, pero fué realmente satisfactorio ir descubriendo poco a poco, conocí a muchas personas dignas de admirar, personas que me han inspirado y motivado para seguir por este camino, personas que me han enseñado muchisimas cosas no solo del tipo académico, personas que podría considerar genios, hice grandes amigos también! . Hoy sé a donde quiero llegar, la pregunta es: Estoy haciendo lo que tengo que hacer para lograrlo ? (créanme que no es la primera vez que me hago esta pregunta) la respuesta no la se, tal ves yo diga que si, sin embargo para alguien que ya pasó por donde me encuentro actualmente la respuesta será no, me he dado cuenta que "estar en el lab" no es solo ir y sentarte a tomar café, leer un par de cosas interesantes, medir producción de metano e irte, me he dado cuenta que no es así. Que si me di cuenta tarde? No, solo que tenía otra concepción de las cosas, ahora sé que se trata de estar generando día a día ideas que puedan ayudar a tu trabajo y al de los demás, hablar de temas de interés social, no solo temas científicos, tratar de contribuir con los trabajos de los demás, generar discusión entre los que estemos en el momento, colaborar unos con otros, transmitiendo conocimiento para crecer todos y así hacer crecer al lab y al mismo tiempo generar un cambio, entonces la respuesta es SI ? Aun no lo se. Solo sé que el estar ahí me llena de responsabilidades que debo cumplir, no tendría sentido estar sin hacer nada, quejándome de una y otra cosa sin generar un cambio, si estoy es por algo y para algo que no ? aunque nunca me he quejado de tener mucho trabajo, tal ves lo he mencionado, pero al fin y al cabo estoy haciendo lo que me gusta! y eso me hace sentir bien. 
Somos una generación totalmente nueva y tal ves los old school que me lean ya sabrán las respuestas a muchas de estas preguntas, pero espero y los nuevos tomen lo que les sirva para seguir creciendo, porque, quién tiene las respuestas para todo ? realmente nadie, todos en su momento estuvimos confundidos de lo que realmente queremos, todos tuvimos dudas y miedos pero supongo que es normal, y también es normal que vayan desapareciendo (y aparezcan otras nuevas) conforme continuemos creciendo en los distintos aspectos. Es bueno platicar con alguien que ya pasó por lo mismo, te da otro panorama, piensas las cosas de otra manera, y así debe de ser creo yo, aprender de los demás, es bueno salir y conocer el mundo, darte cuenta que todos estamos conectados sin importar cuan lejos nos encontremos, en fin, pertenecer a este gran equipo es una de las mejores cosas que me han pasado, he aprendido muchisimas cosas, he sufrido, he llorado, conocí al otro isco, he ganado confianza en mi mismo, me he dado cuenta que soy capaz de alcanzar mis metas y eso me hace realmente feliz porque nada se me ha regalado, pero sobre todo, le di un enfoque a mi vida, hoy sé lo que quiero ser y sé como lo voy a lograr. No digo que todo esté trazado, porque he hablado con personas que han cambiado sus planes por una u otra razón, pero al menos tengo la visión puesta en algo y creo que es importante. 
Tal vez piensen que andaba existencial jaja, pero hace unos días una persona  me dijo que sino expresamos nuestra forma de pensar tal vez las otras personas jamás conozcan quienes somos y de qué somos capaces, por eso quise compartir algo de mis pensamientos con ustedes ;) espero sus comentarios, regaños, etc etc ...


Saturday, October 12, 2013

Prokaryotic Saturday: Movies of the microbial marine world… coming soon on your favorite theater

I hope you all are spectacular!

.... I am spectacularly tired. It was a good week, actually was a lot less hectic than the previous week (believe me, A LOT!), but still, I feel tired. So for today I’ll keep this short  :)

I have some bad news, nobody called in the middle of the night from Stockholm this week. I think is still too soon. Maybe in a couple of decades I’ll get that call. For now, from my little space on the internet I congratulate the winners of the Nobel Prize 2013, here they are:

  • Physics: François Englert and Petter W. Higgs. "For the theoretical discovery of a mechanism that contributes to our understanding of the origin of mass of subatomic particles, and which recently was confirmed through the discovery of the predicted fundamental particle, by the ATLAS and CMS experiments at CERN's Large Hadron Collider".
  • Chemistry: Martin Karplus, Michael Levitt and Arieh Warshel. "For the development of multiscale models for complex chemical systems".
  • Pysiology (Medicine): James E. Rothman, Randy W. Schekman and Thomas C. Südhof . "For their discoveries of machinery regulating vesicle traffic, a major transport system in our cells"
  • Literature: Alice Munro "master of the contemporary short story" (I really liked this, "a master", hehe).
  • Peace: Organization for the Prohibition of Chemical Weapons (OPCW). "For its extensive efforts to eliminate chemical weapons".
  • Economy: this one will be announced on Monday 14 (they know how to keep the suspense). 

And you can read more about them here, here and here.

So for today’s PS I was planning to talk about that paper I left last week, Sorek et al., 2010. But then it happened that I came across something else. In the last few days I've been reading several reviews of Dr DeLong (this and this other). And most recently this one named “Microbial Earth: the motion picture”. In this paper DeLong is writing about what would be the difficulties in making a documentary about the marine MICROBIAL life. 

How will you make a documentary about marine microbial life at the level of one of those BBC documentaries about nature? It would be very difficult. As DeLong pointed out, there are many interactions that “microbiologically” speaking you won’t be able to capture. At least for now it’s impossible. In this super short and super-easy-to-read paper, four movies about several microbial dramas are announced… can’t wait to watch them. 

If you want to know more about the impressive work of Dr DeLong, here is his web page, and his wikipedia page. And here is a really cool picture of him working:

Here is a lecture he gave in 2009, I warn you, it's an hour long.

Please, if you got something to say, leave your comments below this post or in twitter @ale_alvarador.
Although if you don’t, I understand, this is not the best PS, I have to do justice to DeLong in a post, later.

All right, if you want to suggest a topic for the upcoming PSs, something you would like to discuss, feel free.  Would it be a good idea to write some PS in spanish?

See you next week!!

Saturday, October 5, 2013

Prokaryotic Saturday: molecular switches and Travis' experiment

You won’t believe how much I love Saturdays. It’s very difficult to be depressed in a Saturday, seriously. Who can be depressed on Saturday? I mean, you can be depressed on Monday, that’s fine, or Wednesday, I understand. But Saturdays are just perfect. See, no matter at what time you sleep or wake up, you won’t be late to anywhere and you can also work whenever you want and it won’t be wrong.  It’s perfect!

And another good thing I have on Saturdays is the Prokaryotic Saturday, yeeey!!! 

For today’s writing I had three papers from where to choose. The options were:

1) Sorek, R., and Cossart, P. 2010. Prokaryotic transcriptomics: a new view on regulation, physiology and pathogenicity. Nat. Rev. Genet. 11 (1): 9-16. (this one is behind a paywall right here, but I have it, so if you would like to read it, email me –hope this is not considered an illegal statement-)
2) Joint, I., Doney, S.C. and Karl, D.M. 2011. Will ocean acidification affect marine microbes? ISME J. 5(1): 1-7(Available online here).
3) Smolke, C. 2005. Molecular switches for cellular sensors. Engineering and Science. 68 (4): 28-37. (Available online here).

So I chose the last one. Anyway, if you would like me to write about the other two, just let me know. I will probably write about Sorek and Cossart paper next week, the other of ocean acidification I’m not really sure, maybe later. 

Now, let me tell you why I chose Smolke’s paper. Reason #1, because I liked it so much, reason #2, because I had a test about this paper and although I read it my test wasn't very good AT ALL... :( aaaaaaah!! This was my face when I saw my result:

 So I want to use this Prokaryotic Saturday to redeem myself (gosh, I'm such a nerd).  

The paper is a little old but it is very enjoyable (something you can’t say about many scientific readings) because Smolke writes this particular paper in a very “friendly manner” (maybe that’s why I didn't take it too seriously-damn!). 

So Smolke’s team designs biosensors using RNA molecules. So basically she is trying to engineer sensors based on the RNA property to associate with proteins. Remember that RNA (and DNA) can form tertiary structures and create binding sites. In the case of RNAs, they can actually act as regulatory elements forming structures called switches, or riboswitches. These riboswitches are in bacteria, they are RNA structures that can fold and bind specifically to certain molecules (or ligands), these can be a protein, a mineral, etc. And as you can see in the picture, the binding between the aptamer (the part of the RNA that binds to the target) and the target molecule modify the structure of the entire RNA molecule, “hiding “ either transcription start sites or ribosome binding sites, in both cases controlling (turning on and off) gene expression. 

Other regulatory RNA elements are the antisense RNAs. These are small RNA pieces that are complementary to mRNA, so when they both, mRNA and antisense RNA bind, translation is blocked. 

Smolke in her paper explains how she takes advantage of these RNA tricks in the design of sensors. She is not giving any methodology, just an idea of how the process works. The first step is to combine the target molecule (the molecule you want your sensor to detect) with RNA, and RNA can be synthesized in the lab from DNA, and you can buy nucleic acids from several companies. Which DNA you use to make your RNA and exactly how do you decide which sequences you use it is not explained here. But once you have your RNAs and your target molecule, you incubate both. Then you will see that some RNAs will bind with the target, some will not. Later you take those RNAs that worked, you amplify and you continue again, with the incubation of the target molecule and the now smaller pool of RNAs. It is, as Smolke defines it, a talent search. 

But the sensors also need to be measurable. So you can add a sequence that codes for a fluorescent protein in a way that if the target molecule binds, the RNA will or will not express the protein. So imagine, you place these sequences in a cell, you add your target molecule and you wait and see. If the cell glows (or “produce” the fluorescence), then your sensor either is on or off, depending on how you designed. And according to Smolke, it is also possible to design biosensors that are able to detect concentrations of the target molecule, this is, it is not only an on and off response, but either how much of the target is in the medium. .. wooow!! 

At some point she explains the experiment of one of her students, Travis, and at the end you can’t conclude something else than “Travis is a really smart guy”. And now you will see why. What Travis did is to create a biosensor that detects a chemical called theophylline, which is found in tea and is similar in its structure to caffeine. He used yeast to insert the sensor. And this is the figure of the results of Travis’ experiment:

In the y-axis we have the expression of GFP (green fluorescent protein), so basically what he did was measure how much the yeast cells were glowing depending on the amount of target molecule (theophylline), x-axis. 

So the blue line is the switch response. And in the beginning the binding wasn't changing, but a dramatic change was observed when the target molecule reached something around 1 mM, meaning that the biosensor needed to have a certain concentration of target so this can actually be “detected”. So Travis’ biosensor worked, after the binding with theophyline, GFP production stopped, in other words, the biosensor was off when the target molecule was detected. And the picture is also showing how specific the biosensor is. The orange line is caffeine, an analogue of teophyline, as I told you before, while the green line is the biosensor only with no target and the red line is the biosensor plus an antisense RNA complementary to the binding site of the theophyline, so GFP was never produced here. As you can see, both, green and orange line shows that GFP was constantly produced, therefore, the biosensor wasn't detecting the target, because there was no target! Isn't this super interesting?  

Later Smolke explains a couple of other biosensors her students synthesized, and how they are applied. In the future, she concludes, biosensors can be engineered to detect unhealthy levels of molecules in blood samples, or to detect growth factors that drive cells to go carcinogenic. A very interesting promise. 

For now I’m going to stop here because this is already to long. But if you read the paper and would like to discuss more, not only about this experiment with the theophyline, but also the one Travis run with the caffeine, you can email me or write something in the comments. Also, if you find more recent applications of biosensors, or something cool related with this field of research, please, share it! :)

See you next week! 

The picture of the riboswitches was taken from Kim, J. N.; Breaker, R. R., Purine sensing by riboswitches. Biology of the Cell 2008, 100, (1), 1-11. Although I copied it from here. And obviously the picture of Travis' results was taken from Smolke paper. I mean, just saying. 

Wednesday, October 2, 2013


2 de Octubre ni se olvida, ni se perdona??
October 2nd, don't forget, don't forgive...

When I see the nationals news I just can say... For real???

Or is just my perception that Mexico is going under...??? Chaos everywhere... community police out of the law, due to the lack of gov in many towns; millions of kids with more than a month without classes; a bunch of "teachers" collapsing the Capitol (sorry capital); delinquency everywhere, and a very silent but unstoppable abatement of the freedom of speech... If you see/read Mexican news you have a perception, which is totally the opposite of see/read the news of Mexico from abroad... The economy, security, etc... honestly I fell if where living in 1984 (the novel).

Tax, education and energy reforms have polarized the already divided country... From my perspective I don't trust and I don't want the tax and energy reforms, is there a way in which a citizen like I can do something to avoid them??

45 years ago a group of youngsters gave their lives for what they believe, they express against the government, and the gov react... With the consequences that we know...

Now I have almost 30 years... I'm not a youngster anymore... Before I believed that doing the things right, give the best of my in my job, supporting the less fortunate was enough for make this a better place to live.... Now I think this is not enough ... I see the passivity, the lack of commitment, the prejudiced comments, the double moral of my society and I'm sick of it...

But I know that "feel sick of it" don't make any change... We have to be part of the solution, not of the problem.... Be prepared... Cause I'm tired of just being sick... I'll start to do something first with myself, with my weakness and then I have to do something more to this country that I love the most...

U're welcome to help me, any idea is welcome!!!


P.s. Doc maybe there's still those revolutionary youngsters in Mexico, of whom you read of them when you where in India.