Science; not all beer and bacterial culture

Today was a long one! So time for a long post ^^ I was in labs 9am - 7pm treating / transfecting my cells. Unfortunately not all went to plan. My HeLa cells had aggregated at the centre of their wells as they were incorrectly mixed after seeding, a problem when you want uniform transfection and easy counting. To add to the days hiccups, thanks to an oversight / note making error on my part, I delayed the addition of my transfection reagent and added an incorrect volume to the wells! High school style error, needless to say I wasn't impressed with myself. Thankfully our main plasmid transfections went reasonably well and I was able to salvage what I could from the treatments. We'll find out tomorrow to what extent it's influenced our results, and if the experiments have to be repeated.

Wasn't on top form today

As a silver lining, I guess it's best to make these mistakes now while I'm learning rather than during a dissertation or whilst starting a new job! Certainly won't be making the same mistake twice.

The cell culture area

 Cell culture is new ground for me so I guess it's understandable there'd be some growing pains (no pun intended, heh) getting used to all the habits required to maintain an aseptic work area and keeping nasty organisms out of your cells. I'm sure it'll get drilled into me in time! Not tounching outside surfaces, keeping within the laminar flow zone, sterilizing with ethanol. And a thousand more things that you are required to do instinctively! Even writing up my own protocols and mixing my own reagents is new, but I'm sure I'll get used to it sooner rather than later. *fingers crossed*

A note on our cells

Meet Henrietta Lacks (or rather her cells), a tool no medical biologist can do without

In our experiments we're using HeLa cells. They are a biologists human cell line of choice for most applications and have been fundamental in the develompent of innumerable medical breakthroughs, which have saved the lives and improved the wellbeing of millions. They were the first immortalised human cell line, isolated by George Gey from the cervical cancer cells of Henrietta Lacks, without her knowlege during the 50's. She was a young black woman living in the harsh poverty-stricken reality of 1950s America, and never lived to witness the medical revolution she helped set in motion. Her story and that of the family she left behind is beautifully retold in the emotional book ''The Immortal Life of Henrietta Lacks'' by Rebecca Skloot (http://www.amazon.co.uk/The-Immortal-Life-Henrietta-Lacks/dp/1400052173), which I would thoroughly recommend to anyone. I felt compeled to know the story of the woman who gave us this amazing tool before I set about utilising them, to hopefully give her a measure of credit and respect that was never afforded to her during her all too brief lifetime.

Henrietta Lacks (1920-1931)

There has been some 20 tonnes of HeLa cells produced since Henriettas death, orders of magnitude more cells that ever existed in her body. Her cells have been exposed to innumerable chemicals and drugs to assess their effects on human cells, they have been exposed to the energy of nuclear weapons and launched into space. Without her cells our knowlege of cancer, HIV/AIDS, Polio and a vast catalogue of other medical conditions and scientific lines of inquiry would be massively impoverished.

Hopefully in the next few weeks they'll help me shine some light on autophagy!

That's that for tonight I think. Back to the lab first thing tomorrow. Hopefully everything will go swimmingly this time =) 

I think therefore I science

Whilst much I'll be learning revolves around methods of obtaining and processing scientific data, almost certainly the most important skills I will be learning are that of deep critical analysis of our evidence, forming hypothesis based on past evidence and trying to deduce relationships from what we find. As the late Carl Sagan mused, ''science is a way of thinking, much more than it is a body of knowlege''.

'I think'. The mark of great scientists past and present is the ability to follow the evidence wherever it leads, discard unsubstantiated notions, and to be open to enough to imagination, to allow for acceptance of new ideas.

So! In this entry I'm going to pick my brain to highlight some of my thoughts to you guys and maybe propose a few hypothesis.

Recent research (http://www.ncbi.nlm.nih.gov/pubmed/21726807) has implicated Nitric Oxide (NO) as an important physiological regulator of autophagy. It downregulates the activity of autophagocytic machinery through a few key routes:

1) Prevents phosphorylation of Bcl-2 by JNK-1 (two key proteins). This leaves Bcl-2 activated. Bcl-2 proceeds to bind key autophagy initiation factor Beclin-1 and deactivate it, preventing autophagy initiation.

2) Upregulates mTorC1 which blocks autophagy through a variety of means.

3)Prevents binding of Vps34 with Beclin-1, preventing formation of the inititaion complex.

It mediates these effects (especialy effect #1) mainly through its ability to 'S-nitrosylate' a specific amino acid called Cysteine located in the reactive site of many of these proteins.

Cysteine s-nitrosylation. the NO reacts with the thiol (HS) group on the cysteine replacing it with a nitrosyl group.

The resulting nitrosyl group is much less reactive than the thiol group it replaced, resulting in a reducation of activity on the effected cysteine. It is s-nitrosylation at cysteine number 116 on JNK-1, prevents it's ability to phosporylate, and hence deactivate, Bcl-2.

Hydrogen Sulphide, the chemical I am investigating, has a very similar mode of action to NO! It 's-sulfhydrates' cysteine residues, and in an interesting point, it massively increases the reactivity of the effected cysteine! (http://www.ncbi.nlm.nih.gov/pubmed/19903941)

Cysteine s-sulfhydration. The hydrogen sulphide reacts with the thiol producing a highly reactive sulpfhydryl group.

So this posses an obvious question. Does the increased reactivity of this important cysteine residue increase its ability to phosphorylate Bcl-2, and activate autophagy? And possibly work in other ways to activate the process, in an opposite fashion to nitric oxide?

An interesting hypothesis would be that Nitric Oxide and Hydrogen Sulphide work synergysticly to up and down regulate autphagy through interaction with cysteine residues.

We all know theres only one way to find out! . . .

And see what the evidence has to say! =)

Week one round-up!

Its the end of my first week and I've done as much practical work in four days than I get through in a whole semester at uni. Not that I'm complaining, getting free rein in a professional lab is excellent, and I've only managed to set fire to my hand once so far! I'd say things are going well. The first step of my project is complete. We've cloned and isolated enough DNA to start culturing our human cell line, which should be ready by monday! Exciting stuff.

Modifying bacteria with human DNA, cloning them, busting them open, taking out the DNA, and inserting it into the cervical cancer cells of a woman who has been dead 50 years . . . then making them glow in the dark. That's science folks!

 1) Inserting the CSE/CBS encoding plamids (circular DNA) into bacteria

2) Grow and select modified bacteria

3)Bulk-culture the modified bacteria to amplify plasmids

4) Burst open bacteria and isolate plasmids from genomic DNA, proteins and other inpurities.

5)Transfect human cells with plasmid DNA. Express proteins. Obsverve results via confocal fluorescence microscopy.

So that's what I have to do in a nutshell. We're up to step four, but step five is the big one, and will take the rest of my placement to complete. We'll also modify the human cells in other ways later in my placement. Some pictures from the lab!

Culturing the bacteria

Fluorescence microscopes

Confocal Microscope

Plasmid Isolation Procedure

So as you can see there's certainly plenty of science getting done! Thursday evening had myself, lab staff, scientists and academics attend a two hour science-thon on current discoveries by scientists at the institute, made all the sweeter by a spread of free beer, wine, cookies and a variety of other nibbles. My kind of evening!

Needless today I'm eager to return on monday and start on my work on the human cell line. But for now it's weekend o'clock!

How science works

First day on the job! Come at me science.

Seven hours, five coffees and a two inch high stack of scientific papers later, I've completed my first day at The Campus for Ageing and Vitality =D
It was no mean feat reading through, assimilating and annotating almost 14 papers, representing the best part of a decades worth of primary research into autophagys regulation, mechanisms and relationships to health and disease. I've certainly come out of it with a better understanding of why my research is necessary and how I'm going to go about conducting my work. But needless to say I spent most of the afternoon in bed resting up. I'm all scienced-out (-.-) Zzzz

A little bit of homework to be getting on with.

The campus is a brand new development evidently in the throws of establishing itself. A handfull of researchers and other academics have set up shop in the brand new kitted-out labs and are busily science-ing day and night, and more equipment and staff seem set to arrive over the summer in what will become a major research hub.  My first day consisted mainly of admin and getting acquainted with my surroundings, but I got to see some of the kit I'll be playing with over the coming weeks and tomorrow I should be throwing on the white coat and shadowing some researchers in the lab, hopefully whilst doing some wet-work of my own!

A confocal microscope; I'll be trained in its use tomorrow. The device is used to produce images like the cells seen in my previous posts.

It's all very exciting stuff. But for now, I best hit the hay (again) so I'm not falling asleep in my petri-dishes tomorrow!

And on that note:

If it's good enough for Neil Patrick Harris, it's good enough for me.

Introduction to autophagy, research details and pretty pictures of sciencey stuff..

So in an hour I'll be attending the first day of my research project. Before going any further I thought it best to do a short piece on what autophagy is and what exactly it is I'll be researching to give my future posts some context. So strap yourselves in and hold onto your hats! It's about to get sciencey up in here;

What's autophagy?

In a nutshell autophagy acts your cells internal demolitions and clean-up team. Cells are filled with long and short lived organelles and proteins, the molecular machines which drive our metabolic processes and constitute life. If these micro machines break down, are created defective, or simply pass their 'cell by date' (see what I did there?) they need to be degraded to prevent them causing trouble and allow their components to be reused.

Autopahgy in action!

 The above fluorescence micrograph shows the process of autophagy in appealing detail. What we're looking at is a cell mid-division that has been starved of amino acids. The cell quickly reacts by forming small bubbles or 'autophagosomes' around non-essential components. These vacuoles are then filled with chemicals which break down the proteins and release much needed amino acids back into the cell. The cells 'cytoskeleton' (the scaffhold by which the cell moves, retains shape and transports components) is stained pink, the nucleus green, and the autophagosomes red.

When autophagy goes wrong.

Breakdown in autophagy has been implicated in a massive range of diseases, most notably Alzheimers Disease and Parkinsons.

In alzheimers disease, autophagy fails to degrade by-products of protein production. These small 'waste' fragments of protein build up in the neurones and form aggregates or 'plaques' which then interfear with cellular mechanisms and cause cell death. This depletion of neurones gives rise to disease symptoms.

Autophagy may also play a key role in the longevity of healthy individuals and in the ageing process itself!

 My research project

The title of my project is:  'Effects of hydrogen sulphide on autophagy: a tissue culture study'
We'll be looking at how the chemical, Hydrogen Sulphide, effects the rate at which autophagy takes place. Prior research has shown that a similar molecular gas, nitric oxide downregulates autophagy. Specifically we'll be investigating how two enzymes (CBS and CSE) which produce hydrogen sulphide in cells, react with and regulate the process, if at all.

If hydrogen sulphide is shown to have effects on autophagy, it would increase our understanding of how the process works in healthy individuals and fails in those with related diseases, possibly leading to better treatments.

Hope that shed some light on what I'll be getting up to over the next eight weeks! Now for one final cup of coffee before making my way to the lab. . .