Again about signals and antennae

If you recall the post #2, you will remember that communication between different cells of the body occur via signals that are released and captured by antennae docked on the top of the cell dome (the cell membrane). Initially I said that these signals are then translated into effects as a result of further talk within the cells themselves. So the consecutive transmission of signals (through ligands, remember) coming from the outside to the internal side of the cells is termed “signal transduction” or, more friendly, “signaling cascade”, as the information goes in a sort of “top to bottom” approach, falling from the top of the cell dome towards the grounds. Like a secret whisper that is transmitted from neighbour to neighbour, our signal is passed along by intermediators that we call generally “second messengers”. Biochemically we define a second messenger as a molecule that increases upon a stimulus above a steady state quantity for a short time, to further return to its initial concentration when the signal has been transmitted (imagine the waves generated by the movements of the oceans: each wave might well represent a signal transduction from the horizon to the shoreline). Thus we now understand that this fluctuation is the core of the transmitting system…and guess what, in the ancient Latin language a fluctus is indeed a wave!

No need to mention that we (islet biologists) are very interested in understanding what waves are generated upon the binding of a ligand to a receptor as they are the intracellular intermediators of the results we observe in terms of insulin secretion. Examples of second messengers involved in the release of the insulin granules (please allow me to explain this terminology successively) are represented by small ions (like calcium) as well as newly synthesised molecules such as ATP (the cellular currency) and one of its derivatives, cyclic AMP. Other players are generated from the enzymatic conversion of fats that are components of the cell dome. Nevertheless, it should not surprise you if we actually g monitor their fluctuation and their generation within the islets of Langerhans, and in particular the beta cells.

I think this post has become heavy enough for this round…so, keep following me in my future publishing to discover how we perform this particular kind of magic! (and indeed there’s going to be some colourful pyrotechnic arts too!)

Juggling mode!

Hi there! Yes I know it, shame on me for such a long silence. But don’t you worry; here I am to catch up with you guys.

Since the last blog post, life in the lab has been pretty hectic. So much work to do and so much still to be done.

Can you imagine how fast does time flow when you are in a lab?

If you don’t pay attention to your timing and follow your daily agenda strictly, you might end up running for the last tube to go home!!!…or waiting for the night bus instead (for hours)!

So, what have I been doing so far? To answer, I have gone through my laboratory notebook and realised I have run quite a few independent experiments between dynamic insulin secretion, resistance of islets to external stress stimuli, receptor activation and generation of intracellular signals coupled with metabolic studies. Ah, don’t forget that we need islets to perform our ex-vivo experiments, thus everything begins with islets isolation from murine pancreases, of course!

A typical week of work can be resumed as follows: [please take something for granted at the moment, I will come back and explain successively]

DAY 1- islets isolation, which means dissecting the organs from a mouse, use some biochemical-based techniques to free the islets from the pancreatic tissues, collect the islets and make them as pure as possible (meaning that we try to reduce the presence of other tissues to the minimum)…then we leave the islets resting over night. Of course islets are not left stranded on a bench but in a special “bedroom” at 37 degrees Celsius, 100% humidity and with a mixture of air that contains 5% Carbon Dioxyde (this is the best condition resembling the body environment – warm, humid and thus happy!). Ah, islets are placed in a nutrient rich culture medium which provides them with all the ingredients to be happy and confy so that they can live and recover from the stress resulting from the isolation

DAY 2- I usually use most of the islets (rougly 60%) for my secretion studies. As you might recall from previous posts, I am a big fan of dynamic secretion, which is technically challenging and time consuming (but addictive!). It takes me about 1-2 hour to prepare all the solutions (the liquids) required for the experiment and another hour to set up the instrument. The secretion itself consists of something like a 2.5 hours run on average, plus another good half a hour to clean the apparatus. I usually generate 1400 samples per assessment and each sample needs to be tested for insulin (in duplicate). It takes roughly 5 hours to set up the insulin assay (between the writing of individual reaction tubes and the set up of the reactions themselves). The measurement of insulin needs 2 days of incubation (meaning that we set it up and let it rest for 48 hours in a fridge) then 4-5 more hours of work to transform it into a (cheerful/sad) number.

 DAY 3- I think you have noticed that DAY 2 is already invading DAY 3 and 4, but yes, I do have a DAY 3 anyway, and I usually set up other experiments such us the response of our islets to stress-related stimuli. In a typical set of experiments I aim to determine if our ligands and/or receptors (signals and antennae, remember) take part to the mechanisms regulating the life span of the islets, protecting them from damage or accelerating the effects of an induced damage. To evaluate this, a defined number of islets is exposed to a cocktail of molecules that are known to promote cell death and we measure the capability of our ligands and receptors to eventually reduce or foster these effects. It usually takes 48h to obtain our valuable numbers, but it is not something that requires continuous monitoring, so just 2-3 hours to begin the experiment, another 2 to terminate it and little more time in-between for the necessary passages proper of the method. So, at DAY 3 we are already juggling around!

 DAY 4- Juggling mode full ahead! There’s the insulin measurement from DAY 2 to be finished, babysitting the experiments from DAY 3 and prepare those of DAY 5, and of course, provide the islets we have in culture with the most confortable environment possible in the mean while (=refresh the culture medium as often as possible – and eventually talk to them to make them feel happier and less lonely!). So, if on DAY 5 we are running any microfluorimetry measurement, for example, we need to set up everything the day before

DAY 5- Assuming we are running a microfluorimetry experiment, between the set up and the run itself, it takes something like 6-7 hours to generate a good number of data to be useful for our scientific needs. Ah, and we do this in a room with dim lights. Do we ever fall asleep??? Shhhhh…don’t tell my boss!

Oh, did you forget we had an on-going viability experiment?! No worries, I did not! It’s done now!

Yes, it sounds complicated and it is, indeed! Because we work with animals, we try to obtain as much data as possible from the lowest amount of biological preparations. We  try not to waste any single islet to keep to the minimum the number of animals we use. Unfortunately, we can’t use any other model at the moment, but we (as well as other labs) are also working on generating valid alternatives to animals for the study of Diabetes (if you interested search for the 3R programs on the internet).

I did not include other things to this agenda, such as data analysis, experimental planning, students tutoring, department committees and reports. Oh, how could I forget the weekly lab meetings (I will dedicate a post to this I promise)!

Do you still wonder why the pubs around the campus are full of crazy people drinking as tomorrow never comes on a Friday evening?!?!
Ya, let’s try not to forget that we are British here (or we pretend to be, at least!).
Friday 5 pm it is drink o’clock!

;-)

{I wonder if Joggling would be the perfect title: a sport that combines juggling and jogging – I don’t think I do really hit my chair so much, indeed!} ;-P

The hottest week

If my previous post was more a weather update, this one won’t be…

I can definitely affirm that the week ending today has been one of the heaviest one so far…it was the “hottest” I have ever had, in terms of workload.
Reading through the pages I am sure it has filtered out that we look at islets biology from a 360 degrees approach and, yes, I am sure you had already been able to understand that we do work on animal models too. Moreover we are also lucky to be part of a research program that allows us to use human biological samples donated for research. Of course there are strict regulation on the usage of these samples and everything is done under a very tight ethical permission…so no worries, we are not any crazy psychos!

Well, in terms of experiments, this week has been very packed (or stressing if you prefer) because we have had the combination of already planned mouse experiments with an unexpected arrival of human samples: to cut a long story short the last non-work thing I can recall was on june 27th…and I realised only now it is July 4th! (ah, by the way, happy Independence Day to any American reader and happy belated Canada Day to those who are Canadians!). So if I have learned on my own skin that science is nothing easy, I have now learnt that the scientific agenda might become unpredictable as well and that it is mandatory to be able to juggle around a little bit to accommodate with all our eagerness of knowledge! (or if you prefer a more chemical terminology, we all need to be able to “buffer” the unpredictable!)

But please, don’t read this as a down-side feature: it really means I have had a great acceleration on my project(s)…more results to come in the very near future, sooner than expected!

PS: the post had remained in the “draft” folder for something like a month before I realised it wasn’t published…apparently I am still fighting with the blog platform!

Magic Sciences and Pyrotechnical Arts!

Among all the classes of my undergrad in Chemistry and Pharmaceutical Technology (…yes, I know, I used the fancy misleading translation of post 5), we were all sort of terrified attending a series of modules as the professor had the fame to be very tough and severe. Of course my opinion has changed as time went by (it sounds so childish to me now) and I do thank for having met such a kind of tough professors during my studies. Most likely some former students of mine would say the same thing about me and my courses of nutritional biochemistry…in any case, I sometimes recall images from those classes as he was one of the first guys who tried to make us understand that Research is not a kind of magic with spells and formulas – but it consists of a robust and reliable series of approaches that have to be reproducible in every part of the world according to the description we provide (and this is also known as the empiric experimentation and mathematical demonstration by Leonardo da Vinci, precursor of the most widely spread scientific approach generated by Galileo Galilei – Am I being parochial here?! Am I, am I ?!?)

So, how do I obtain those numbers that make me cheer (or be sorrow for most of the time)?!

Please, let me introduce you two (of the) main methods I have acquired so far, that have an important role in building up my pile of work…

From the initial pages of this blog you might recall that islets from pancreas release insulin in response to change in blood glucose. This phenomenon is called “secretion”.

To study secretion we use the islets outside of the pancreas and we put them in a different environment (thus we will say in vitro). To convince the islets to secrete insulin we accommodate them into a test tube and provide them with a solution of glucose during a defined amount of time – generally 30 minutes or one hour. Because this particular way does not involve any change of conditions during the time of performance, the technique is called static insulin secretion (static because it is a simple on/off situation).

If you think a little bit more about how our body works, well, it is everything but static!

So, you might realise that a static secretion is maybe the most convenient way to study insulin secretion, but sometimes it is not the most appropriate.

Here, at the Diabetes Research Group at King’s College London I have learnt how to design and perform experiments aimed at determining the response of islets to variable glucose concentration during time, a situation termed dynamic insulin secretion.

It represents the most fascinating part of my research; and although it is notably considered to be technically challenging and more demanding than static, believe me or not it creates addiction!

(if you don’t believe me please go and ask one of my crazy Spanish colleague that I keep fighting with to use the apparatus!!!)

But how does this (intriguing ?!?) method work?

It is still an in vitro technique, (thus outside of the body!): in an artificial way we are able to recreate the most suitable environment for the islets of Langerhans to “perform their magic” outside of a living organism, as they were into the organism itself.

The perifusion apparatus (as we friendly call it) consists of a series of independent chambers connected to various buffer reservoirs. Islets are loaded into the chambers where they continuously (remember it is dynamic!) receive a physiological buffer supplementation. This continuous supplementation enables the timed kinetics of insulin secretion to be determined in response to a potential stimulus.

If the static is an on/off situation, the dynamic secretion technique can determine whether an effect is acute (= short termed in action) or sustained for the duration of the treatment, and whether it is reversible or not.

So we profile islets’ capability to secrete insulin and we evaluate what stimuli, compounds, conditions can make the islets of Langerhans perform better or worse. It might sound banal but this is an important piece of work as it can help us understand the processes that take place in the complex mechanism of the pancreatic response to glucose.

If you remember the concept of antennae-signals expressed in one of the initial posts, this “profiling technique” coupled with the addition of a series of signaling molecules, allows us to understand the role of the antenna whose response we are trying to understand.

[And if I have raised a little bit of curiosity in you, well, shoot me an email and I will be more than happy to physically walk you through our facilities and experiments!]