The Half Decent Pharmaceutical Chemistry Blog

On March 31, Maria Rossi, my beloved grandmother, aged 82, was diagnosed with metastatic pancreatic cancer. Today, May 13, she passed away.

When I wrote this blog would have become much more personal towards its conclusion, I didn’t obviously think I would have ever written this post. At that time, everything was looking excellent: I had got the studentship I wanted, my thesis was going on nicely, my parents and grandma (who had always lived with us) were proud and happy, I was embarking on a new website and my head was literally full with ideas on future projects.
The news came as a massive blast. She had been suffering from abdominal pain and discomfort in the previous months and weight loss was going on since a couple of years (the picture above is from August 2005, when she was completely healthy): this phenomenon, however, had been associated by everyone with her (familial) problem with coping with dentures (and I still believe in it to be the main cause as she began to experience real issues eating certain types of food).

First, her doctor believed she was suffering from irritable bowel disease and gave her appropriate therapies to treat it. Given the lack of any improvement and because my mother insisted, she had a colonoscopy, which only proved no tumour was present there.
January arrived and she started to experience back pain too. Finally, ultrasonography and CT scan helped with a diagnosis: incurable, metastatic pancreatic cancer, the worst kind of pancreatic tumour.

Carcinoma of the pancreas is one of the most difficult to promptly diagnose, as it remains silent until it attacks other organs, liver in particular. Severe abdominal pain, jaundice and thrombophlebitis are widely thought to be the classic hallmarks of any pancreatic cancer. For what concerns the metastatic sub-type, the list of hallmarks features: pain, loss of weight, jaundice, nausea, anorexia, malaise, vomiting, back pain, diabetes mellitus, pancreatitis and ascites. Apart from jaundice, anorexia and diabetes mellitus (although glycemia was twice the normal towards her last days), she had all. Loss of weight, back pain and ascites are also the three signs of incurability.
According to the statistics, survival from the moment of the diagnosis is limited to 4 to 6 months.

I learned all these things a couple of days after the first exam, during an awful morning when I didn’t actually do anything in the lab, but spent my time searching for clear information about her disease (which had already been described to me as incurable). I was desperately looking for hope, any therapeutic protocol (even one under early investigation), or, at least, a date.
I instantly realised she couldn’t have undergone gemcitabine infusion or fluorouracil, as both cause unbearable side effects for an old woman.

Together with the doctors, we decided not to tell her about the incurable cancer and opted for a severe pancreatitis. Although I said she was suffering from many, dreadful symptoms, I recall she was, all things considered, relatively fine. She continued to cook for all and spent most of the days not in bed. However, even though she didn’t know the truth, things went downhill too quickly. She began to be given morphine to cope with her back pain and rapidly reached the point when she needed to stay in bed most of the time.

Nausea made impossible for her to swallow any kind of food or drink, last week, and we took her to a hospital last Friday, as renal failure was to appear on stage. For her last four days, we decided to take her to a private hospital, where she received all the support and comfort they could give her.
She died this morning, with my father and her daughter there. She had already lost awareness of the people around her on Sunday, although this might have happened even earlier but I prefer to think she was aware of me kissing and saying goodbye to her on Monday night, when I left her room.

By a large margin, she is the person I have loved the most in my life. I still can’t really realise that she won’t be there for my graduation or that I won’t talk to her about my life in London.
I thought I would have burst into tears the day she would have passed away. But I haven’t yet. Once we had arranged everything for the funeral, I went to the lab: I knew staying with other people and keeping my mind busy with some work was the right thing to do. In particular, I didn’t want to stay in the house we have lived together since I was born for too long as everything reminds me of her.

These events led me to the decision of dedicating my life and my research as a molecular biologist to cancer. Fortunately, my career will begin with a studentship offered by Leukaemia Research and a project aiming at increasing the amount of knowledge on pathways involved in the development of these tumours, in order to target them for therapy. I will obviously try to do something, in the future, about metastatic pancreatic cancer as it has killed a great part of heart.

I have also realised there are fantastic people among my relatives and friends: something I wasn’t that sure of recently.

So, I will now do what my grandma would certainly tell me to do: working and enjoying myself equally hard. I didn’t write a lot in these months as I wanted to look after her at my best. I never told her it, though, as she was also my first supporter as a blogger, although she never read this blog because she couldn’t speak English.

I am not a religious person and, thus, I find terribly hard to believe in life after death, but, nevertheless, I feel she will be supporting me: at least thinking about the happy moments with spent together makes me fell better and stronger, even if on the verge of tears, as I’ve just said. Even if I firmly believe that, if there is actually a life after death, dead people are completely detached from what happens to us, as this would be a source of trouble and distress to them.

Goodbye, granny: as you used to say, you’ll always be in my pocket to help me take the right decisions.

As many adolescences, at the beginning of high school I went through my rebellious period: interested in anarcho socialism and communism, curious about the “No-Global” movement, committed to many social issues such as immigration, pollution, equity, etc. If you are European, there’s a chance you, too, spent a bit of your adolescence dressing up like a squatter, participating in boring meetings and attending endless and pointless conferences.
That was a long time ago, when hormones can make a male teenager go crazy and oddly embark on silly projects for emulation or a physiological need to do something radical and, in his view, shocking.

Although adolescence, like acne, only lasts a few years and, fortunately, (most of) lads grow up from many points of view, this means I’ve turned myself into a globalisation-enthusiast, who likes fast-foods and famous sportswear brands, with no sense of social commitment (whatever that really means). This said, I just want to make clear I hoped Boris Johnson to become the new mayor of London.

However, my political views will be fully discussed in the Euchromatic Blog, as this post is solely about bioinformatics and, in particular, sequence alignment. To be honest, I can’t stand bioinformatics: I hate having to waste time in front of a computer doing BLAST searches on Gene Bank  or fishing for isoforms on Swiss Prot. Even worse, I’m not particularly excited by people who choose to carry out their research sitting at a computer instead of handling pipettes and getting dirty and smelly with media and reagents.

Unfortunately, last year, my current group leader insisted that I had to take a bioinformatics exam. The idea was to make practice with data bases but, sadly, the course annoyed me with depressing lectures on how Google works or the characteristics of an algorithm.
Nevertheless, the bit about sequence alignment was quite interesting. There are two levels of alignment: an algorithm can perform a pair-wise or multiple alignment. For example, BLAST utilises multiple alignments as it compares our target sequence to all the strings present in a data base.
More interesting is the distinction between global and local alignment: with the former you basically align the whole sequence(s), while the latter is based on dividing a string into segments and it is obviously the ideal option for a data base.

A global, pair wise alignment requires gaps in between the 2 sequences in order to obtain the best score. A global, multiple alignment is what you need when trying to predict secondary and tertiary structures.
A multiple, local alignment, on the other hand, aims at the so-called Maximal Segment Pair (that presents the highest matching score).
Therefore, the algorithm performs a multiple alignment, locally matching and retrieving only those with a score above a threshold.

Great stuff when you cannot sleep, eh?

I’ve created a new category for my future posts, “The Euchromatic Blog”, where to put everything related to the new blog. Today, for example, I feel the need to explain why, despite me being a co-owner of ChemBlogs, I have felt the need to register a web domain and use Wordpress.

First, Mitch, who actually had the idea of ChemBlogs as a platform for chemists willing to blog about their interests, has decided to close down the website as, well, I’m the only one who started a blog here (I wasn’t even that enthusiast about it) and has actually kept writing on it regularly. I could have gone solo and become the only owner of ChemBlogs, but I frankly can’t see the point of a (mainly) molecular biology blog, with the prefix chem- in the address line.
Moreover, nearly all the successful scientific blogs have their own, simple, address and domain.

The main reason for changing, though, has to do with spam. Yesterday I had been busy doing a Western blot, looking after cells and bacteria. Oh, and I also did one of things I like the most about molecular biology: a massive digestion with two different restriction enzymes, followed by a brief run on a highly resolving agarose gel.

Anyway, I could hardly check my inbox(es) every now and then, and certainly couldn’t check the comments to the blog I had to moderate. When I checked this today (after less than 24 hours) I counted 163 comments to moderate. As you can see, all of them have been cancelled because it was all spam. Today being Labour Day (or May Day: Wikipedia hasn’t helped me understand the difference), I have decided I have had enough of it: I’ve got better things to do than reading an endless list of spam messages.

This, though, doesn’t mean meaningful comments are not to be displayed after a while as usual: I just want to apologize for all the times your comments might be lost, simply deleted with the torrential flood of rubbish I receive.
You see, I believe both me and you don’t like this feature of my blog: you don’t immediately see your comment once it’s submitted and I have to dedicate a some time to go through the messages to fish for “real” ones. This badly influences the chances of people actually starting any interesting debate on what I write and, above all, is a bit of nuisance to me. However, of all the anti-spam plugins provided by LifeType, which powers this blog, this proved to be only reliable one, unless one doesn’t mind having tons of spam displayed among the comments to serious articles. Or needs to buy cialis or viagra or diazepam.

Wordpress has better systems of tackling the issue (I’ve tried them with another “draft” blog I’ve set up to work at the layout of the Euchromatic Blog) and many more toys to play with (perhaps even too many).

Summer is rapidly approaching and, apart from the warmer and sunnier days, if you’re a man, there’s a chance you have realised it by the sudden change in the diets of many women around you. Haven’t you noticed your girlfriend doesn’t you to take her out for dinner any more? Have you checked the ratio Diet Coke : Beer in her fridge? Haven’t most of your female colleagues at work started to sit around the table (where you have lunch all together) to eat, unlike you, carrots and yoghurt? Well, they are terrified, thinking about the day YOU will innocently propose to go the beach and they will have to squeeze themselves inside a microscopic bikini (perhaps, on that particular occasion they’d opt for a more forgiving swimming costume, pretending that it’s still too cold for them). Trust me, she hates you because you REALLY don’t care about your funny skin colour (after months in the lab, who could be bronzed?) and how fat you are.

Another common theme for a conversation among your female lab-mates (if you ever bothered listening) is the diet to try not to arrive…unprepared for the first Saturday at the beach. Magazines and tabloids are full of  last-minute, radical diets. Needless to say, most of these tips are for desperate, running out of time people and have no scientific rationale behind. So, they are useless and often dangerous.

Here at The Half Decent Pharmaceutical Chemistry Blog, we like to do things properly and, even when it comes to diet tips, we want to be taken seriously and put science behind what we say. So, for the diet I’m going to unveil for all the ladies reading this, I want you to believe me when I say I’ve tried it.

I’ve indeed what I call the Lowry diet on Wednesday and Thursday. In a few words, you skip the lunch break because you are in the middle of a protein extraction for a Western Blot: I know you’ll be thinking you should be in no hurry with the protease inhibitors and sodium orthovanadate and fluoride you have suspended your extract in. Don’t fool yourself, please: we all know you should be quick at boiling your samples and quantifying it (this, merely, because you’re curious to know how many microlitres you’ll have to load on your gel).
Whatever the reason, if you harvest at, say, midday (because it’s 24 hours after you performed a transfection), you’ll be right in the middle of your experiment, perhaps preparing your BSA standards for the calibration curve.
The Lowry assay, in fact, is a popular, maybe old-fashioned, certainly reliable and sensitive way of quantifying the concentration of proteins. It basically consists of two parts: in the former step you perform a Biuret reaction, which means you add an alkaline solution of Cu2+, that reacts with peptide bonds yielding Cu+ ions. Subsequently, Folin-Ciocalteau reagent is added: this mixture of phosphomolybdate and phosphotungstate undergoes a reduction to heteromolybdenum blue, which is coupled with the oxidation of aromatic amino acids in the presence of copper ions.

To sum up, the solution turns blue (picture to come soon, psi*psi) with an intensity that depends on the amount of tyrosine and trytophan and, therefore, the overall amount of proteins present in the cuvette. The precise concentration is determined by comparing the absorbance of the sample at 750 nm with those of standard solutions of BSA, used to draw a calibration curve.

This said, you generally need to wait 15 minutes after the addition of each of the two solutions, so you’ll be too busy to have lunch. It definitely works!

A sunny Sunday might not be the ideal moment to fill some official papers for my official enrolment at Imperial College: 22 degrees and a cloudless sky are an inviting opportunity for an afternoon spent lying in the sun with a beer and a nice book.
I must admit while typing these words I’m thoroughly considering this as an excellent solution, but before eventually heading for a park with a can of Guinness and, why not, the forms I received from Imperial, I want to write a remarkably (hopefully) personal and, at the same time, useful post about Oxbridge and their application procedure systems.

Let’s start from the end: to apply for the position I managed to get all I had to do was to send an email to my (at that time) potential supervisor, with a copy of my CV and the name of at least two academic referees. He invited me to London for a formal interview, at the end of which I was already offered the job. The MRC (Medical Research Council) provided me with two nights accommodation at a lovely and incredibly homely guest house in Hammersmith, plus all my travel expenses were paid back.
Then, I received forms from the MRC itself, followed by those from Imperial and, finally, Leukaemia Research, which is the organization actually financing my project.

To sum up, because this is what this post is mostly about, I haven’t paid a penny.

Back in December, I applied for a couple of projects at the MRC Laboratory of Molecular Biology, which, as my future employer (CSC) is part of Imperial College, is formally a part of the university of Cambridge. Therefore, you need to submit two, separated applications: one to the LMB and the other to the “host” university.
Whereas the former, perhaps due to the fact that this is another MRC-based organization, was for free and, once invited for the interviews, both accommodation and travel expenses got reimbursed, the on-line application to the university cost £25. What’s more, outdated as it may sound, I had to (quickly) send a paper copy of all the papers and forms I had already electronically sent to them through the post: this means I had to ask my refs not only to send their reference letters via email, but also to print them out, put 3 (!) copies in a sealed envelop (together with 3 copies of other documents I had to fill in and make them sign). Those envelops had to be ridiculously signed on the outside, just  across the seal, and the signature was to be protected with a bit of sticky tape. All these complicated procedures were meant to prevent anyone (me?) from forging the precious content and to keep it secret to me (ha ha ha!).
Nevertheless, although pathetically old-fashioned, Cambridge was rather quick in letting me know the (negative) outcome of my application and the LMB was very kind throughout my staying at Addenbrooke’s for the interview. All things considered, thus, I may still look at them for a PostDoc.
Sadly (?), I cannot say the same for the rival counterpart: Oxford. Sure, their £25 on-line application system is simpler, more user-friendly, incredibly quicker to complete and it doesn’t even require a paper submission afterwards.
However, unlike my interview at LMB, I have no photograph of Oxford to show you as my interview was over the phone. I know this is not that unusual, but it still sounds idiotic to me (and I really do mean that!).
Even more infuriating was their timing. On leaving Addenbrooke’s, I was told a final decision would have been taken shortly and, whatever the outcome, they would have contacted me no later than a certain day (two weeks time, to be precise): I was told the result one before the deadline.

It took Oxford (dep. of biochemistry) more than a month to come up with an answer: by that time (interview on February 29, no news until April 3), I had already gone through my interview at Imperial, accepted it and submitted all the official papers to the MRC. To make things worse, after my phone interview (which doesn’t really give you any opportunity to know anything about the group and place you might join) I was told they would have informed me “at least within a week”.

Therefore, I can already tell you The Euchromatic Blog will be hypercritical to Oxford University.

It’s important, anyhow, to make absolutely clear that the position I’ve got is, by a large margin, the best one from many points of view (the only drawback is that I’d have liked to live in small, peaceful and full-of-students Cambridge rather than in a huge city such as London). I applied to LMB mostly because of its fame and then turned to Oxford and Imperial for opposite reasons: the former to try both sides of Oxbridge, the latter because of the project itself. In fact, I haven’t talked about the other successful applications I turned down because fortunately already with a gorgeous project in my hands (which means I won't go to Switzerland...).