in2scienceUK blog by Adeolu Busari

Despite the warning at the introduction day that the labs we will be entering will not resemble ‘a giant room filled with new state of the art tech and machinery littered with scientists in their lab coats constantly pipetting’. I still had small hopes that some aspects of the description would be true.

How I was wrong.

On the first day I was nearly convinced that Dr Hristova’s lab was a kitchen. There were 3 fridges, 2 weighing scales, an oven and a microwave (definitely a kitchen I thought), but then I snapped back into reality once the fridge was opened and I saw it packed full with all these different chemicals and the huge bottles of methanol and ethanol in the cupboard with the bright orange flammable symbol on it.

Despite the fact that the atmosphere and the appearance of the lab was different from what I anticipated, I’m glad it was. The lab had a friendly, warm atmosphere and was hardly ever packed. The masters’ students and Dr Hristova were friendly and would often engage me in conversation and tell me about their project.

Now onto the actual science: The project outline at the lab I was placed at was to study the effects of pharmacological inhibition of TGF-β1 on microglial cells after mild neonatal hypoxic ischemic insult. The study comes from unpublished data which has found the deletion of TGF-β1 in microglia to be protective. The aim is to see if pharmacological inhibition of TGFR-1 would prove neuroprotective.

On the first day I observed images of stained mice brains underneath a microscope, these images showed areas of the brain that had some physical damage to it. This damage was due to hypoxic-ischemic event, meaning the brain was partially oxygen deprived and that brain cells died. We measured the amount of physical damage noticed in them, physical damage meaning that the brain matter had been fragmented due to the dead neuronal cells.

The remaining of my two weeks in the lab I helped in prep for staining and staining the brains, preparation consisted of taking out frozen sections of neonatal brains and spreading them. Spreading is a process where the brains are rehydrated with bidistilled water (ddH2O), then spread using fine brushes to become flattened and fan dried at room temperature.

Later the sections were fixed in 4% formaldehyde and washed in 0.1M phosphate buffer for 5 minutes. Then defatted in acetone for 2 minutes each, washed twice in 0.1M PB, and then in 0.1% bovine serum albumin/0.1M PB (PB/BSA). After, they were placed on wet chambers and blocked in 5% goat serum in 0.1M PB for 30 minutes. The block was removed, and 90µL of primary antibody was applied to be kept on overnight.

On the second day of fixing, the excess primary antibody was washed off using PB/BSA, PB, PB and PB/BSA and a few more washes took place to insure there were no more reactions taking place.

Finally the sections were ready to be stained either via nissl or tunel. Tunel is a stain that binds to fragmented DNA, so is a specific marker for cell death. Whereas nissl stains cell bodies. If a cell was damaged it would not stain much with the nissl, it may not even stain at all. This is why two stains are used whilst analysing the effects of hypoxic-ischemic even on neonatal brains.

In order to stain via tunel the sections were spread and fixed as described above and then incubated in 3% H2O2 in methanol and then washed in 0.1M PB. Sections were incubated with 0.1% terminal deoxytransferase and 0.15% deoxyuridine triphosphate for 2 hours at 37°C. The reaction was stopped in tunel stop solution for 10 minutes. Then lastly the sections were washed thrice times in 0.1M PB and incubated for 1 hour in ABC solution.Nissl staining is not that different to tunel but is also complicated, so I will not be describing it due to the word count limit!

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Overall, I really enjoyed my lab placement and I feel very fortunate to have been allocated it. It has allowed me to witness the research side of science, which is far more interesting and practical than I thought it would be. Thank you Dr Hristova and In2science for giving me the opportunity.

Laing O’Rourke engineering placement: C520 Custom House Station

by Aneesa Ali

1My engineering placement was with Laing O’Rourke; an engineering enterprise based in the UK who provide engineering, construction and management services to countries all over the world. One of the projects they are currently working on is Crossrail; a new rail service in and around London which will reduce congestion on train and lessen journey times. Once open in 2018 it will run from Shenfield/Abbey Wood to Reading/Heathrow, going through 40 stations; 8 of which are newly built. This includes the Custom House Station, which is where I was situated for my placement.

On the first day, before I could do anything I had to sit through an extensive induction which emphasised the importance of health and safety on site. I was also given my orange PPE (Personal Protective Equipment) which everyone is required to wear when on site. This includes goggles, hard hats, gloves, boots and hi-visibility jackets and trousers.

2The rest of the week was split between being on site and talking to people in the office. Being on site allowed me to see for myself what was happening rather than having someone tell me. When on site I shadowed different engineers who showed me different aspects of the station. The electrical engineer showed me around the plant rooms and explained how the electricity would get around the station, whilst the civil engineers showed me around and explained the actual building of the substructure and superstructure. The photos below were taken whilst I was shadowing a package manager who’s in charge of the subcontractors on the project. The subcontractors, in this case DELTA, were installing the glass on the sides of the VDR (Victoria Dock Road) bridge using a mini-spider crane.

I was also able to see how slip testing (testing the resistance of the ground we walk on) is carried out and go on a site quality inspection with people from Crossrail and the quality manager on site. What I enjoyed most on site was surveying because it was more hands on; We used specialist equipment such as theodolites to measure the levels of the ground.

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In the office I worked with document control, commercial (quantity surveyors, procurement etc.), quality control and planning management. I was also able to talk to the environmental manager on site which I found to be very beneficial as the area of engineering which interests me most is environmental/energy engineering.

Something I feel like I’ve gained is the appreciation for all the work that goes into something we use every day, like stations. Whenever I’m at a train station now I can’t help but think of all of the engineering and construction that went into it.

Overall, it was an amazing experience and I’m really grateful to have had the opportunity to see what happens on a construction site. Most importantly I gained exactly what I wanted out of this experience, which was to see what engineering involves first hand.

Primers, DNA and enzymes – it’s a PCR! by Leighvi Pilcher

A PCR – Polymerase Chain Reaction – is a reaction that is used to amplify the genes from a sample of DNA. In my PCR, we were trying to identify the normal “wild type” samples, and the SOD samples. I ran 12 samples in total – 2 controls (1 wild type, 1 SOD) and 10 random samples.

The first step in a PCR (aside from acquiring the DNA – this is done by taking a sample, and digesting it in Proteinase K, which is an enzyme, and heating it at a high temperature) is making the reaction mixture. Within the reaction mixture, 4 primers are used. 2 of these primers are complementary to the wild type DNA, and the other 2 are complementary to the SOD DNA. dNTPs (free nucleotides) are also added. These bond to the newly amplified single DNA strands in order to make them double stranded again. A buffer is added to maintain the pH of the reaction mixture, with magnesium chloride added in order to remove the additional phosphate molecules from the dNTPs. Taq polymerase is added, which copies the target DNA. All of the reagents of the PCR mixture are added to sterile water, and enough is added to make 14 samples. After each addition, the tube is vortexted, which ensures that the mixture has been evenly mixed.

Once the reaction mixture is made, it is separated into 12 Eppendorf tubes. After this, the samples are added. Again, these are vortexted, which ensures an even distribution. The samples are labeled, and added to the PCR machine. In the PCR machine, the samples are heated. The heat “melts” the hydrogen bonds in the DNA. They are also cooled in the machine, which subsequently allows the hydrogen bonds to reform. After this, the samples are heated to 75 Celsius, which is the optimum temperature for taq polymerase, so the target DNA can start to be copied.

After running the PCR machine, a process called electrophoresis is carried out. Agarose powder is added to a buffer solution so that 10ml of a solution is formed. 50ml is used in the gel, and it is heated to approximately 100 Celsius before cooling to 60 Celsius. This is added to the electrophoresis chamber, which has slides put in it to help identify the holes that the PCR samples will be added to. Once the gel has set, the combs are removed, and the remaining buffer is placed on top. Bromophenol blue is added to each of the 12 samples, as this helps to identify them when placed under UV light in the transillumiantor. A control ladder is added to the first chamber in the gel, which is used to show the approximate placement of the DNA bands. The remaining chambers are loaded with the 12 samples. Once this is done, the gel is “run”. In this process, electricity is passed through the gel (50 volts) for 30 minutes. This stimulates the movement of the samples. The denser bands rest closest to the chamber, with the lighter bands resting further away from the chambers.

pcrNow that the hard part is out of the way, the samples are viewed in a transillumiantor. Here, a UV light is beamed from under the samples, which shows the movement of them. As you can see, the far left of the image is the ladder. Next to it is the wild type control, with the SOD control next it it. Samples 1, 2, 4, 6, 7 and 9 were all wildtypes, and the remaining samples were SOD samples.

When I was first given all of this information, I screamed internally, because it is such a lot to take in. It’s not necessarily a complicated procedure, however it is long and fiddly. So, I summarised in the following steps:

  • Reaction mixture
  • Addition of samples
  • PCR machine
  • Gel preparation
  • Electrophoresis
  • Transillumination

If only it was that simple!

Leighvi undertook her placement in UCL’s Sobell Department of Motor Neuroscience and Movement Disorders, at the Institute of Neurology, supervised by Dr Bernadett Kalmar in the group of Professor Linda Greensmith.

Mind control?!? by Kiran Pillai

Firstly I want to apologise. The majority of my week or even just a whole day was not based on mind control – it was only a small part. However what I did was as interesting as mind control. So at 8.20 Monday morning I arrive at the Royal London Hospital for Integrated Medicine to attend clinic with Dr. Sharma. What happens here is that patients are referred to the consultant neurologist – Dr. Sharma. He then begins to deduce through a number of different clues; mainly through the patients history and is able to draw on his vast knowledge to correctly diagnose the problem. Actually the most interesting case wasn’t a stunning work of intellectual ability. Rather it came from a patient who already had their diagnosis. They’ve been diagnosed with Cerebral Autosomal-Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy, which is bit of a mouthful (understatement much?). To save everyone’s time and mouth its called CADASIL. This disorder is caused by a mutation to a gene on the nineteenth chromosome. The effect of this mutation is far worse than its name. It dramatically increases the risk of having a stroke and also gives you plenty of migraines. The problem wasn’t exactly the risk of stroke – statins and a healthy diet were all helping to reduce the cholesterol levels. Rather it was the moral dilemmas that are so often skirted over. As it’s a genetic disorder the question was now to worry about the younger generation. The patient’s siblings all ready had the ball rolling in getting them tested. But what about the children? Would any one of us want to know we had this condition and could potentially die from it? The real world implications are huge. They’ll never get any insurance, a mortgage or even a bank loan. They’ll never be allowed to live in America and now a whole plethora of activities have been crossed out of their lives. How would children react if they knew they had the condition? Would it make them live their lives differently? It’s times like these when you truly appreciate the role of a doctor. The doctor has just dropped a massive life-changing bomb on the patient’s shoulders – but his work isn’t confined to just diagnosing. He’s already arranged therapy and counseling for the family and wishes to help them in anyway possible. To be a good doctor, you’re always going to have to balance the science with the social aspects of life and disease.

Moving on from that – Dr. Sharma isn’t just a consultant neurologist; he is also a senior researcher at the MRC Unit for Lifelong Health and Ageing at UCL and that’s where I spent the rest of my days after the clinic. His research is focused on the neurodegeneration of the motor system which he investigates using MRI scans, transcranial magnetic stimulation and the 1946 birth cohort. The first two are pretty science related but the last bit – the birth cohort is pretty amazing. It is a study of 5000 people who were all born in one week during March in 1946; they’ve been followed up throughout their 70 odd years alive. It is the biggest and longest running birth cohort study in the world and so much data; correlation and conclusions have come out of it. For example one such paper that was published using the results showed that the earlier a child would start to walk the higher the IQ. In fact for every month earlier the child learns to stand – on average they gain one half of an IQ point. This has further been proven with the complex link in the brain between the motor cortex and the cognitive ability and thus function of the brain (just want to point out – I could walk pretty much instantaneously after being born).

I bet by now you’re wondering where the mind control comes in. In fact I’ve already mentioned it. Transcranial magnetic stimulation. The act of using magnetic fields to stimulate an electric field in the motor cortex of the brain, which then creates impulses that causes the body’s nervous system to pass on electrical signal, which cause the muscles to contract and move. Currently the motions the body can be made to do are limited but the potential is there. It’s amazing to look at the science behind it, I remember learning in my Physics GCSE about Fleming’s left hand rule – which is exactly what the researchers use to stimulate the brain.

Another great thing about my placement was that my supervisor and me were both similar in one important fact. We are both horrendous in a wet lab. From a chemistry lab to the kitchen you can be guaranteed that I will make mistakes with my hands. So for me; working with computers in dry labs was a great experience. I learnt how to do some coding and use programmes that would actually allow me to do computer modelling of the brain. Using diffusion weighted magnetic resonance imaging I produced stunning images of the brain on my laptop. It’s really interesting how this specific type of MRI scanning works. We all know about the amount of the water our brain holds, but using diffusion MRI we can track motion of water in the brain and produce stunning images of the brain. These images were used to track changes in someone’s brain and then analyse the patterns that come out of it. For Dr Sharma it was how the brain changes with neurodegeneration experienced with motor disease.

The whole week was interesting, educational, inspiring and a number of adjectives that I can’t begin to list. Perhaps the best part was everyone I met whether it was Jane who gave me a cool electronic key to get into a Victorian house or Sanjay who helped me with my medical school entry. But ultimately I have to thank Dr Sharma who opened my eye to the potential for research in medicine; and how a background in both allows a person to approach any scientific problem with a unique perspective that has only motivated me more to become a doctor and now a doctor who is involved in furthering the collective scientific knowledge of the entire human race.