The Y.O.R.F.

Of moss and me

2010-02-18T17:43:00.010Z

I recently had a look through some new papers published on moss (Physcomitrella patens) and found one published in Cell titled ‘Transcriptional control of gene expression by microRNAs’ [1]. This didn’t seem to fit the same pattern of a lot of moss papers I have read (ie in less high impact journals). This paper shows how useful moss is for studying gene expression using reverse genetics (so hopefully my PhD wont be a failure).

I think it is important to study important biological problems, such as RNA silencing and epigenetics, in a diverse range of organisms. I take this title from a short review published in Nature Medicine recently (as well as the Steinberg book) by Sir David Baulcombe (who holds a prestigious chair at Cambridge). He wrote about what we have learnt by studying things in plants (eg discovery of transposons) and their importance as a model, using his own research into RNA silencing and epigenetics as the main example [2]. I think this new moss paper goes some way to support this, but also to show the importance of this particular organism. My choice to work with Physcomitrella was because I wanted to stay in the plant world but wanted to work with something where I could knock out specific genes by homologous recombination and was simple to grow. Physcomitrella can be grown on simple agar media plates and joins the few species where genes can be knocked out.

Khraiwesh et al (2010) looked at what happened when you knocked out two Dicer-like (DCL) proteins in moss [1]. Dicer proteins in animals and plants cut long dsRNA to make small RNAs loaded onto Argonaute proteins (producing the effector complex). This effector complex is what causes the target mRNA to be cut or repress its translation. When PpDCL1a was knocked out you see a lack of microRNAs (miRNAs) being produced and the plant is restricted to its first developmental stage only. Not surprising. Strangely, when you knocked out PpDCL1b get production of miRNAs but not cleavage of the targets. It has been shown in animals, but not other plants, Dicer proteins can sometimes be used to help a small RNA in the effector complex to cut the target, as well as in miRNA or siRNA biogenesis. Very interestingly they found these targets had a lower level of expression in the mutant plants. If cleavage is not reducing the mRNA levels then what? The answer to this as many other odd questions in biology is epigenetics. Well DNA methylation at least. siRNAs have been shown to cause gene silencing but not much evidence for miRNAs doing this before. The authors went onto show the ratio of miRNA to mRNA target is critical. If you only have a little miRNA compared to mRNA target you get cleavage. If you have lots of miRNA to target mRNA it causes DNA methylation and silencing. But the methylation was only seen in the knockout so why should we care?

Finally they end the paper by showing ratio of miRNA to target mRNA is important in an endogenous gene involved with a hormone response. The plant hormone ABA mediates drought response and abiotic stress. In moss, when ABA treated you get a decrease in a transcription factor response gene. This is a target of a natural miRNA. This down-regulation is due to an increase in miRNA expression, changing the effect from cleaving the transcription factor’s mRNA to targeting its promoter for methylation and silencing expression. This study shows that miRNAs have the potential to cause DNA methylation, which had not yet been show, as well as suggesting a mechanism for it. Future studies can now go on to show the relevance of this in other systems from yeast to flies to mammals.

I end by simply saying MOSS ROCKS.

Khraiwesh, B., Arif, M., Seumel, G., Ossowski, S., Weigel, D., Reski, R., & Frank, W. (2010). Transcriptional Control of Gene Expression by MicroRNAs Cell, 140 (1), 111-122 DOI: 10.1016/j.cell.2009.12.023

Baulcombe, D. (2008). Of maize and men, or peas and people: case histories to justify plants and other model systems Nature Medicine, 14 (10), 1046-1049 DOI: 10.1038/nm1008-1046

Freedom of speech within science

2009-11-04T00:02:00.001Z

Not so much research but a rant. As you may have heard the chief advisor to the government about drugs policy Prof Nutt was told to resign recently after he gave a lecture stating his views on cannabis classification in the UK and how the government (former Home Secretary Jackie Smith) did not listen to their advice on the matter and increased its classification and punishment for taking it. I do not care about the drug’s classification to be honest. I don’t take it and never have but the current Home Secretary (Alan Johnson, tipped to be future Labour leader, among a couple of others) has had a disagreement with the scientist and dismissed him after he gave a lecture stating his opinion. The reason given was he had crossed the boundary between science and politics. What? A scientist who is an unpaid advisor states his opinion based on evidence then states the government didn’t listen to them when deciding policy is crossing the line?! It sounds like he was starting a debate, using his freedom of speech to discuss topics. The government has decided no, if you don’t agree with us and you are helping us then sod off. Whether you agree with Prof Nutt or not I don’t think it matters. What matters is whether advisors are allowed to criticise the government’s policy when they are not listened to. They believe not. Alan Johnson said: ‘he was not dismissed because of the work of the council but because of his failure to recognise that... his role is to advise rather than criticise’. My PM Gordon Brown said: ‘Prof Nutt had repeatedly undermined Labour's drug message.’ I think it is ridiculous to suggest a scientist cannot talk about such things in public and suggests to me the government is being hypersensitive over these matters. Perhaps because they cannot handle anymore bad press and people going against them. After all the chances of them winning the next election are slim to none.

So far Lord Drayson (a businessman) who determines science policy in the government’s Business department and coordinates with the research councils was strongly against this when he first heard it. Lord Winston FRS, a respected scientist in the public eye and Labour peer, is against the dismissal and Prof John Beddington FRS (who recently recovered an honorary degree from York University in the Summer 2009 graduation ceremony) has spoken the most sense on the matter (http://news.bbc.co.uk/1/hi/sci/tech/8340318.stm). He is the chief advisor to the government on all things sciencey. He points out most scientific committees do not have a problem with the government. This is reassuring but and the send of an email the Home Secretary (with no background what so ever in science) has dismissed an advisor and caused two to quit in sympathy because they don’t think they can work with a government that a) doesn’t listen to them and b) forces out the head of the committee for no good reason. I think this is unacceptable from the government hence I have written to the Home Secretary and feel I should email my local MP as well. Hopefully my next post will contain more science and less ranting. It’s being planned, just need to get some pretty photos.

Ig Nobels 2009

2009-10-12T13:13:00.003+01:00

For a few years now that I have been following the Ig Nobels as they are awarded. To continue the tradition, here is a post with the list of this year's winners:

VETERINARY MEDICINE PRIZE
Catherine Douglas and Peter Rowlinson of Newcastle University, Newcastle-Upon-Tyne, UK, for showing that cows who have names give more milk than cows that are nameless.
"Exploring Stock Managers' Perceptions of the Human-Animal Relationship on Dairy Farms and an Association with Milk Production," Catherine Bertenshaw [Douglas] and Peter Rowlinson, Anthrozoos, vol. 22, no. 1, March 2009, pp. 59-69.

PEACE PRIZE
Stephan Bolliger, Steffen Ross, Lars Oesterhelweg, Michael Thali and Beat Kneubuehl of the University of Bern, Switzerland, for determining — by experiment — whether it is better to be smashed over the head with a full bottle of beer or with an empty bottle.
"Are Full or Empty Beer Bottles Sturdier and Does Their Fracture-Threshold Suffice to Break the Human Skull?" Stephan A. Bolliger, Steffen Ross, Lars Oesterhelweg, Michael J. Thali and Beat P. Kneubuehl, Journal of Forensic and Legal Medicine, vol. 16, no. 3, April 2009, pp. 138-42.

ECONOMICS PRIZE
The directors, executives, and auditors of four Icelandic banks — Kaupthing Bank, Landsbanki, Glitnir Bank, and Central Bank of Iceland — for demonstrating that tiny banks can be rapidly transformed into huge banks, and vice versa — and for demonstrating that similar things can be done to an entire national economy.

CHEMISTRY PRIZE
Javier Morales, Miguel Apátiga, and Victor M. Castaño of Universidad Nacional Autónoma de México, for creating diamonds from liquid — specifically from tequila.
"Growth of Diamond Films from Tequila," Javier Morales, Miguel Apatiga and Victor M. Castano, 2008, arXiv:0806.1485.

MEDICINE PRIZE
Donald L. Unger, of Thousand Oaks, California, USA, for investigating a possible cause of arthritis of the fingers, by diligently cracking the knuckles of his left hand — but never cracking the knuckles of his right hand — every day for more than sixty (60) years.
"Does Knuckle Cracking Lead to Arthritis of the Fingers?", Donald L. Unger, Arthritis and Rheumatism, vol. 41, no. 5, 1998, pp. 949-50.

PHYSICS PRIZE
Katherine K. Whitcome of the University of Cincinnati, USA, Daniel E. Lieberman of Harvard University, USA, and Liza J. Shapiro of the University of Texas, USA, for analytically determining why pregnant women don't tip over.
"Fetal Load and the Evolution of Lumbar Lordosis in Bipedal Hominins," Katherine K. Whitcome, Liza J. Shapiro & Daniel E. Lieberman, Nature, vol. 450, 1075-1078 (December 13, 2007). DOI:10.1038/nature06342.

LITERATURE PRIZE
Ireland's police service (An Garda Siochana), for writing and presenting more than fifty traffic tickets to the most frequent driving offender in the country — Prawo Jazdy — whose name in Polish means "Driving License".

PUBLIC HEALTH PRIZE
Elena N. Bodnar, Raphael C. Lee, and Sandra Marijan of Chicago, Illinois, USA, for inventing a brassiere that, in an emergency, can be quickly converted into a pair of protective face masks, one for the brassiere wearer and one to be given to some needy bystander.
U.S. patent # 7255627, granted August 14, 2007 for a “Garment Device Convertible to One or More Facemasks.”

MATHEMATICS PRIZE
Gideon Gono, governor of Zimbabwe’s Reserve Bank, for giving people a simple, everyday way to cope with a wide range of numbers — from very small to very big — by having his bank print bank notes with denominations ranging from one cent ($.01) to one hundred trillion dollars ($100,000,000,000,000).
Zimbabwe's Casino Economy — Extraordinary Measures for Extraordinary Challenges, Gideon Gono, ZPH Publishers, Harare, 2008, ISBN 978-079-743-679-4.

BIOLOGY PRIZE:
Fumiaki Taguchi, Song Guofu, and Zhang Guanglei of Kitasato University Graduate School of Medical Sciences in Sagamihara, Japan, for demonstrating that kitchen refuse can be reduced more than 90% in mass by using bacteria extracted from the feces of giant pandas.
"Microbial Treatment of Kitchen Refuse With Enzyme-Producing Thermophilic Bacteria From Giant Panda Feces," Fumiaki Taguchia, Song Guofua, and Zhang Guanglei, Seibutsu-kogaku Kaishi, vol. 79, no 12, 2001, pp. 463-9. [and abstracted in Journal of Bioscience and Bioengineering, vol. 92, no. 6, 2001, p. 602.]

Looking for some science on swine flu

2009-09-06T18:26:00.007+01:00

As a medical association in Spain has recently communicated to the press, more than anything there seems to be an epidemic of fear going on. As I am currently living in Portugal, I do not know what the situation is in other countries, but I can tell you how the situation is here. Every news bulletin starts with more news about the flu, even though no one has died here yet. It then finishes with a previously recorded warning on how one should wash hands frequently and keep 1m away from other people while talking to them, quite an ordeal in a country where the common greeting is a couple of kisses in the cheek. In addition, we have ambulances going around with the paramedics dressed as in the photo, right out of films like ‘Outbreak’. Now, most people seem to assume that because I have just graduated with a Biology degree that I should know everything there is to know about the current flu. Besides the generic question ‘So, what do you think about this flu thing?’, I have had some more specific questions, from ‘I heard that it is impossible for this combination of genes to happen in nature, surely this must have been created by evil scientists with corporative interests?’ to ‘This virus is called H1N1, the 1918 pandemic virus was also H1N1, are we all going to die of Spanish flu?’. I try to explain people that at uni we are taught how to get informed about things, rather than becoming specialist on every single biological problems; and that frankly I am so fed up of hearing about this flu that I couldn’t be bothered to look it up. However, I think the hysteria might be affecting me, probably because I’ve just read the book Blindness (if you have read it/watched the film you will know why). So I decided to interrupt my holidays-away-from-science to read a bit about this topic, and thought I would tell you some of my findings.

My first port of call was the website of the World Health Organisation, usually a reliable source for information. I was pleasantly surprised by the lack of over-dramatic information. There is of course a whole section dedicated to this flu, right in the home page, but the answers given in the FAQs seem quite sensible. It tells us that 2009 A(H1N1) is spread as the normal flu, and that the current worries as based on the fact that being a virus which never circulated in humans before, there is no, or very little, immunity. In addition, as stated in their website, the virus is spreading fast in young people (10-45), from a majority of mild cases to some serious illnesses, the majority of which in patients with underlying conditions. The recommendations of the WHO are to take pain killers and drink loads of fluids if you have the flu. And only contact the medical services if you have serious symptoms like shortness of breath, or if the fever lasts for longer than 3 days. Quite unlike what recently happened in Portugal. When a man who had travelled to Britain was diagnosed with this flu, he saw his house invaded by doctors who quickly took him away to a hospital and confined the whole family to their house. Without telling them what to do or when they could leave.

Anyway, I then thought it would be a good idea to read some information from journals, a bit more scientific and hopefully less dramatic source of news. However, as you know, I am currently unable to access most papers, as I am in between universities and am yet to be provided with a username in my new institution. So what follows is based on a couple of papers I could access, and is by no means comprehensive.

As an introduction, some information from the Virology handouts we were given last October. Influenza viruses are RNA viruses that have a segmented genome (8 segments/genome). The glycoprotein spikes, haemagglutinin (HA) and neuraminidase (NA) are important in the entrance of the virus in the host cells. There are 15 known HA and 9 NA serotypes, and their combination provides the name that we see for the viruses such as H1N1. Birds and pigs are important reservoirs for genetic and antigenically diversity and reassortment in these viruses. In particular pigs, as their cells can be infected by both avian and human influenza viruses, making them nice ‘mixing pots’.

In July this year Garten et al., published a paper in Science in which they characterised the 2009 A(H1N1) virus both antigenically and genetically. They start the paper by giving a small introduction on the influenza pandemics of the last century, and how we got from those to the current 2009 A(H1N1). Then it goes on to characterise the actual viruses. The closest ancestral gene for each of the eight segments seems to have a swine origin, although having originally come from avians and sometimes humans in different occasions. This is quite interesting, as swine influenza viruses had not, until now, caused much disease or been incredibly good at human-human transmission. As the authors point out, this virus probably had been circulating for a while in pigs, unnoticed due to lack of monitoring.

Analysis of the virus genome showed that none of the molecular signatures of increased transmissibility or virulence of A(H1N1) viruses can be found in this strain, and that functionally important receptor binding sites on HA do not differ from classic swine influenza viruses. No markers were also found that indicate adaptation to the human host or features of previous pandemic virus. The main important difference seems to be a genetic marker for resistance to adamantine antivirals, but the virus still seems to be sensitive to the other type of antivirals, the neuraminidase inhibitors. So, this study concludes, we must be worried about this virus firstly because we don’t really know what makes it good at replicating in humans, and secondly because it has a genetic composition not seen before, so we don’t really know what to expect.

I then went on to read a couple of papers regarding studies done in mammal models such as ferrets (apparently classic models for influenza studies) and mice.

The first study was published in Science also in July, by Maines et al. This post is becoming quite long already, so I’ll just summarise their results (based on 3 independently isolated 2009 A(H1N1) viruses as compared with a seasonal influenza strain). They basically inoculated 106 p.f.u in ferrets and monitored different indicators such as body weight, viral titres, direct and indirect transmissibility, etc. The main conclusion from the study was that 2009 A(H1N1) caused higher morbidity (weight loss, etc, depending on which virus isolate), showed, unlike seasonal flu, high viral titres in the lower respiratory tract or the intestine, but that it was less efficient at indirect transmission (putting healthy ferrets in cages near to inoculated animals).

The second animal study I looked at was published online by Nature, in what they call a ‘near final version’. They also used virus isolated from infected humans, though not all of which were the same as in the Science paper. They studied the effect of these viruses in mice, macaques, ferrets and pigs, though not all of the virus isolates seem to have been used in all of the experiments. They also looked at more indicators, such as pro-inflammatory cytokines, lung pathology, etc. In ferrets the results were similar to those of the Science paper, except that this time indirect transmission was successful. In mice and macaques, the 2009 A(H1N1) virus seems to be worse than the seasonal virus (at least one of the isolates that they seem to mostly use throughout, CA04) although according with which criteria varies with the model. The inoculated pigs were asymptomatic, explaining why the virus had not been noticed before in this animal.

So, what are the conclusions I could get, overall, from these three studies? Overall it seems that the 2009 A(H1N1) virus is worse than the seasonal influenza viruses in mammalian models. However, how much worse is probably hard to say. I am not familiar at all with viral studies, so it is hard for me to critically analyse these studies, but it seems to me that in most cases the n numbers were quite small (n=3 in some cases in the Nature study), but this is perhaps not surprising, as they must have been quite quick studies, considering the current need to understand this new virus. And, as it is probably the case in most animal studies in general, I reckon the doses used were probably much higher than what humans would be exposed to in the real world. In addition, there is always the question on how well do these animal models really reflect what happens in humans.

In any case, it seems that so far these studies indicate that 2009 A(H1N1) is, in these models, worse than seasonal flu. Whether it is as bad as some of the pandemics that have been affecting us in the last 100 years it is unknown. The other worry is of course that the virus might evolve into something much more dangerous. Though all the virus isolates obtained so far tend to be quite similar, 5 minor genome variants have already been detected. Restricting the spread of the virus, even if it is not so bad right now, might be a good way of preventing it from acquiring any more deadly features. Whether this data justifies the measures being taken by most government, that is really, as PHD comics puts it so well, beyong the scope of my area of study. I will leave that to those who study the spread of pandemics and know of population health. I think we have had enough speculation for one disease.

Garten, R., Davis, C., Russell, C., Shu, B., Lindstrom, S., Balish, A., Sessions, W., Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J., Smith, C., Emery, S., Hillman, M., Rivailler, P., Smagala, J., de Graaf, M., Burke, D., Fouchier, R., Pappas, C., Alpuche-Aranda, C., Lopez-Gatell, H., Olivera, H., Lopez, I., Myers, C., Faix, D., Blair, P., Yu, C., Keene, K., Dotson, P., Boxrud, D., Sambol, A., Abid, S., St. George, K., Bannerman, T., Moore, A., Stringer, D., Blevins, P., Demmler-Harrison, G., Ginsberg, M., Kriner, P., Waterman, S., Smole, S., Guevara, H., Belongia, E., Clark, P., Beatrice, S., Donis, R., Katz, J., Finelli, L., Bridges, C., Shaw, M., Jernigan, D., Uyeki, T., Smith, D., Klimov, A., & Cox, N. (2009). Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans Science, 325 (5937), 197-201 DOI: 10.1126/science.1176225

Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, Gustin KM, Pearce MB, Viswanathan K, Shriver ZH, Raman R, Cox NJ, Sasisekharan R, Katz JM, & Tumpey TM (2009). Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science (New York, N.Y.), 325 (5939), 484-7 PMID: 19574347

Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y., Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watanabe, S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M., Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H., Takahashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Takahashi, K., Warshauer, D., Shult, P., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K., Nakano, K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M., Sugaya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K., & Kawaoka, Y. (2009). In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses Nature DOI: 10.1038/nature08260

Investigating the Smallest Bacterial Genome

2009-08-16T05:38:00.011+01:00

During the first two terms of my final year at university I took on a dry project based on bacterial genetics. In retrospect this was quite a cheap move since it avoids so many of the problems involved in normal lab work, but in my defence it was the project that interested me most and I really wanted some computing experience before making PhD applications. My work was based on bacterial endosymbionts of insects, which have been of recent interest due to their extremely small genomes. Bacteria in these symbiotic relationships are given a protected, nutrient-rich environment and in return they allow insects to survive on unbalanced diets by synthesising scarce biomolecules.

Primary endosymbiotic bacteria live their entire lives inside insects and are vertically transmitted from generation to generation, a process that leads to coevolution between the bacteria and the insect. One of the results of this coevolution was major changes to the original bacterial genome, which contained many genes that are essential for free-living bacteria but are unnecessary for life within an insect. Consequently, common features of endosymbiotic genomes compared to those of free-living bacteria are severe gene loss, genome compaction and skewing of GC content.

Electron micrograph showing bacteriocytes taken from P. venusta

1 – Bacteriocyte; 2 – C. ruddii; 3 – Unidentified electron-dense mass

My project focused on Carsonella ruddii, the only bacterial endosymbiont of the psyllid, Pachypsylla venusta. It was hailed as the smallest bacterial genome chracterised when it was sequenced in 2006 and still holds that record. Its genome contains only 182 ORFs, less than 3% intergenic DNA and has a GC content of 16.5%. The bacteria appears to be provided with many nutrients by its host and its metabolism has been reduced to a few pathways: ATP synthesis, a section of the pentose phosphate pathway and biosynthesis of certain amino acids.

The early stages of my project involved a reannotation of the C. ruddii genome followed by a sequence-based functional analysis of its metabolic enzymes. Using the enzymes deemed functional in this analysis I built an updated model of the C. ruddii metabolism which could be divided into six pathways involved in amino acid biosynthesis, five of which were incomplete. The only fully intact pathway led to the production of isoleucine and valine. These are both essential amino acids for insects and are severely under-represented in the adult psyllid diet.

Four of the incomplete amino acid pathways were missing only one reaction and the conservation of the rest of each of the pathways suggested that they might still be functional in C. ruddii. The ‘missing’ reactions might occur spontaneously under some conditions or could be catalysed by unidentified enzymes. For three of these four missing reactions I found an example in the literature of a different bacterial endosymbiont which had lost that reaction but had retained the rest of the pathway. This seemed to suggest that the enzymes catalysing these reactions might be expendable and subject to loss during genome reduction in endosymbionts. Based on this and some other evidence from similar situations in endosymbionts I predicted that these pathways are probably functional in C. ruddii and that its main role symbiotic role is to provide the psyllid with essential amino acids.

The fourth of these incomplete pathways was the most interesting because I was unable to locate another endosymbiont which was missing the same reaction. The reaction was catalysed by the product of a gene, AS, which was present on the C. ruddii genome but which I had labelled as a pseudogene during functional analysis. Although it’s difficult to conclusively say that an enzyme is inactive solely by sequence analysis, multiple alignments showed that this copy of AS was extensively degraded and was missing both of its key catalytic residues as well as its substrate binding residues. However, later in the project when I was scanning an EST set taken from the insect host of C. ruddii I located another copy of AS which also had bacterial origin but which was not present on the C. ruddii genome. Sequence analysis showed that this version of AS seemed to be active and could potentially fill the gap in the pathway.

Where did this copy of AS originate from? It aligned well with the version of AS from P. aeruginosa and appeared to have a bacterial origin but was not found on the C. ruddii genome or the psyllid mitochondrial genome, both of which have been sequenced. Several lines of evidence ruled out the presence of a second bacterial endosymbiont in this symbiosis and since no plasmids had been reported during DNA sequencing of C. ruddii the source of this sequence appeared to be the nuclear genome of P. venusta itself. The presence of this bacterial sequence in the eukaryotic genome suggests that LGT may have taken place between a bacterial genome and the insect nuclear genome. This would be one explanation for the fact that C. ruddii has only 182 ORFs, which is significantly lower than the predicted minimal bacterial genome. However, it is also possible that C. ruddii uses mitochondrial proteins to survive and so LGT is not the only explanation for the low ORF count.

This was my favourite line of investigation during my project but the symbiosis between C. ruddii and P. venusta had many more interesting features that I read about over the year. One of the questions I got in my viva was whether C. ruddii should be labelled as a bacterium or an organelle. I think this question is only really important when considering a minimal bacterial genome and if C. ruddii does turn out to be importing essential proteins from elsewhere then I think that the label organelle is definitely more appropriate. However, the definition of an organelle doesn't seem to be well-established and so whether or not C. ruddii really does have the smallest bacterial genome is a matter of opinion.

Nakabachi A, Yamashita A, Toh H, Ishikawa H, Dunbar HE, Moran NA, & Hattori M (2006). The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science (New York, N.Y.), 314 (5797) PMID: 17038615

Gil, R., Silva, F., Pereto, J., & Moya, A. (2004). Determination of the Core of a Minimal Bacterial Gene Set Microbiology and Molecular Biology Reviews, 68 (3), 518-537 DOI: 10.1128/MMBR.68.3.518-537.2004

Glass, J. (2006). Essential genes of a minimal bacterium Proceedings of the National Academy of Sciences, 103 (2), 425-430 DOI: 10.1073/pnas.0510013103

Thao, M., Moran, N., Abbot, P., Brennan, E., Burckhardt, D., & Baumann, P. (2000). Cospeciation of Psyllids and Their Primary Prokaryotic Endosymbionts Applied and Environmental Microbiology, 66 (7), 2898-2905 DOI: 10.1128/AEM.66.7.2898-2905.2000

Non-B DNA structures and disease

2009-07-15T13:31:00.008+01:00

People seem to have started contributing actively to the blog again so I thought it was my turn to try again. And this is an achievement, as facing any science, in particular anything written by me about it, would have been impossible a month ago.

I decided to keep things simple for this one and just give a short introduction on the theme of non-B DNA structures and how they influence disease, the topic of my open essay.

When we think about DNA, we think about its B-DNA structure, the one that Watson and Crick described in that famous Nature paper more than 50 years ago, i.e. a right-handed double helix, 20Å diameter, in which the base pairs are almost perpendicular to the axis of the helix, 10.5bps per turn. Although most DNA will be in this structure, it can also form other structures, the so called non-B DNA structures. Some of these are shown in the figure. These alternative structures have a few common characteristics: firstly, whether or not a structure is formed, and which specific structure, seems to be sequence dependent, as it often involves formation of new pairings of bases. Secondly, most of these structures are in higher energetic state than normal B-DNA, for example, because they require the separation and reformation of hydrogen bonds. Overall, therefore, DNA with a favourable sequence tends to remain in its B-DNA form, requiring events such as DNA replication, transcription or protein binding to be transiently converted to these unusual structures. Sequences with propensity to form unusual structures are quite common in the human genome, and it seems that in some cases they are necessary for the normal functioning of the cell. However, they have also been implicated in disease, and this is what I’ll be focusing till the end of this post.

There is a considerable list of diseases thought to involve, as part of their pathology, the formation of non-B DNA structures, but the mechanisms by which this is though to happen can vary. For example, certain sequences/structures have been thought to cause genomic instability, e.g. by causing chromosomal rearrangements due to the propensity of these structures to promote double strand breaks. Another interesting form of genomic instability associated with these unusual structures is repeat expansion, implicated in motor diseases such as Huntington’s disease. Non-B DNA structures, namely Z-DNA, have also been associated with viral infections.

Now, there isn’t really enough space here to talk about everything, so I’m going to give one examples of a situation in which a non-B DNA structure is though to be involved in disease. Friedreich ataxia (FRDA) is a disease caused by the expansion of GAA•TTC tracts in intron 1 of a gene encoding the protein frataxin, essential for mitochondrial function. In this disease, repeat expansion is associated with loss of protein expression. One of the current models by which frataxin production is thought to be reduced in expanded GAA•TTC repeats is based on their ability to form triplexes (an alternative model suggests that epigenetic changes may also be important). Duplex opening within the repeated region, due to the passage of RNA polymerase, is thought to allow one of the separated single strands to form Hoogsteen hydrogen bonds with the purine strand of a B-DNA duplex within the same repeated sequence. This leads to the formation of a 3-stranded helix. Its formation on the non-template GAA strand in frataxin probably interferes with RNA polymerase progression. The free template strand is then thought to base pair with the newly synthesised RNA transcript, forming stable RNA/DNA dimers and preventing further transcription.

Now, this is only a model, for a specific type of structure within the context of a specific disease. And although different sequences have been shown to form these unusual structures, and these structures to be somehow associated with specific diseases, to neatly demonstrate how exactly one influences the other seems to be quite hard. This is particularly difficult because different groups often use different model organisms or protocols, leading to sometimes contradictory results. Overall, although I liked writing about this topic, since I had never considered how the structure of DNA could have an impact on disease, I got the feeling that a lot of work still needs to be done before convincing evidence is given for how exactly these structures impact on our wellbeing, and what sort of therapeutics can be developed from this knowledge.

Red, GAA repeat strand; blue, GTT complementary strand; orange, RNA transcript; yellow, RNA polymerase. 1, Intramolecular triplex; 2, Stalled RNA polymerase; 3, DNA/RNA hybrid

Hebert, M. (2008). Targeting the gene in Friedreich ataxia Biochimie, 90 (8), 1131-1139 DOI: 10.1016/j.biochi.2007.12.005

Bacolla, A. (2004). Non-B DNA Conformations, Genomic Rearrangements, and Human Disease Journal of Biological Chemistry, 279 (46), 47411-47414 DOI: 10.1074/jbc.R400028200

WELLS, R. (2007). Non-B DNA conformations, mutagenesis and disease Trends in Biochemical Sciences, 32 (6), 271-278 DOI: 10.1016/j.tibs.2007.04.003

Wang, G., & Vasquez, K. (2009). Models for chromosomal replication-independent non-B DNA structure-induced genetic instability Molecular Carcinogenesis, 48 (4), 286-298 DOI: 10.1002/mc.20508

Learning how to tell time with cyanobacteria

2009-06-29T16:11:00.000+01:00

Most living things can innately tell the time. If you take a person and put them into continuous darkness for months by themselves they will follow a sleep-wake cycle of about 24-25 hours (as well as going a little mad). Circadian rhythms allow life to predict the raising and setting of the sun to allow us to change our gene expression metabolism and behaviour with the changing of the day. Predicting changes in temperatures and light levels are very important. Circadian clocks have three important features. First of all they are free-running at about 24 hours (in constant conditions like light or darkness they will keep going at ~24 hours). Secondly they are temperature compasatory meaning that changes in temperature do not greatly affect the clock. Finally they must be able to be reset by environmental inputs. The changes of the seasons mean days get longer or shorter so to accurately predict the raising of the sun your clock must be reset. Plus for those who fly great distances we must reset according to the new environment and sadly suffer from jet lag while we are slowly adjusting (Johnson et al., 2008).

These clocks have evolved independently multiple times. In animals, plants, some fungi and recently discovered in cyanobacteria (Johnson et al., 2008). It was once thought because prokaryotes divide so raplidly that a 24 hour rhythm would be useless to them but that is not the case (Johnson et al., 1996). A great deal has been learnt about rhythms in bacteria but there are still many unanswered questions. The fact circadian clocks have evolved multiple times shows it offers a great advantage to those that posses one but was no present in the last common ancestor of life. Changing behaviour to only occur at a specific time of day is a great advantage to organisms, plants to photosynthesize and animals to look for food at the right times (Bell-Pedersen et al., 2005).

Cyanobacteria also rely on the sun for energy so predicting when the sun will rise allows them to start synthesising photosystem proteins before the sun raises while not producing them all night or waiting for the sun to raise. It has been shown when you make a mixed culture of bacteria with a mutant clock (no rhythmic changes in gene expression) and wild-type then wild-type will soon outcompete the mutants. Those that have a different length period (eg 16 hours rather than 24 hours) will always lose to wild-type in 24 hour day conditions but wild-type will lose in conditions that match the internal clock of these mutants (Yan et al., 1998). Interestingly in cyanobacteria the whole genome shows rhythmic expression! (Liu et al., 1995) This is in contrast to most organisms that control 15%-35% of their genome in a circadian dependant manner (McDonald and Rosbach, 2001; Correa et al., 2003; Michael and McClung, 2003). This is controlled by the supercoiled state of the whole genome! (Smith and Williams, 2006) I eagerly await the paper that explains how this achieved.

What is rather fascinating about this system is the oscillator. In most organisms gene expression is negatively regulated by its product in a way it cycles its levels over 24 hours and controls output (Bell-Pedersen et al., 2005). In cyanobacteria, this is not the case. Rhythmic expression of clock proteins occur but they do not repress there own synthesis. Also an in vitro oscillator can be setup. In the test tube the clock protein KiaC will cycle through different phosphorylation states if with KaiA, KaiB and ATP. In the organism this forms the post-translational oscillator (Nakajima et al., 2005). This is temperature compensatory and this is built into the ATPase activity of KaiC (Terauchi et al., 2007). It has been shown even when locked in one phosphorylation state you get a weak rhythm so a transcriptional-translational feedback loop as in eukaryotes is needed for a robust system (Kitayama et al., 2008) posing the question of whether this occurs in eukaryotes.

And to finish off I will add one final oddity about the circadian rhythms of cyanobacteria. Instead of using a photoreceptor to sense light inputs like other organisms they read the state of the metabolism. When light is high photosynthesis is occurring and the redox state of the cell changes and is seen as an input to reset the clock (Ivleva et al., 2005).

References

*Of importance
**Truly significant

Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L. and Zoran, M.J. (2005) Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nature Reviews Genetics, 6, 544-556.

Correa, A., Lewis, A.Z., Greene, A.V., March, I.J., Gomer, R.H. and Bell-Pedersen, D. (2003) Multiple oscillators regulate circadian gene expression in Neurospora. Proceedings of the National Academy of Sciences of the United States of America, 100, 13597-13602.

Ivleva, N.B., Bramlett, M.R., Lindahl, P.A. and Golden, S.S. (2005) LdpA: a component of the circadian clock senses redox state of the cell. Embo Journal, 24, 1202-1210.

Johnson, C.H., Golden, S.S., Ishiura, M. and Kondo, T. (1996) Circadian clocks in prokaryotes. Molecular Microbiology, 21, 5-11.

Johnson, C.H., Mori, T. and Xu, Y. (2008) A Cyanobacterial Circadian Clockwork. Current Biology, 18, R816-R825.

**Kitayama, Y., Nishiwaki, T., Terauchi, K. and Kondo, T. (2008) Dual KaiC-based oscillations constitute the circadian system of cyanobacteria. Genes & Development, 22, 1513-1521.

Liu, Y., Tsinoremas, N.F., Johnson, C.H., Lebedeva, N.V., Golden, S.S., Ishiura, M. and Kondo, T. (1995) Circadian Orchestration of Gene-Expression in Cyanobacteria. Genes & Development, 9, 1469-1478.

McDonald, M.J. and Rosbach, M. (2001) Microarray analysis and organization of circadian gene expression Drosophila. Cell, 107, 567-578.

Michael, T.P. and McClung, C.R. (2003) Enhancer trapping reveals widespread circadian clock transcriptional control in Arabidopsis. Plant Physiology, 132, 629-639.

*Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyarna, T. and Kondo, T. (2005) Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science, 308, 414-415.

Smith, R.M. and Williams, S.B. (2006) Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences of the United States of America, 103, 8564-8569.

Terauchi, K., Kitayama, Y., Nishiwaki, T., Miwa, K., Murayama, Y., Oyama, T. and Kondo, T. (2007) ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 104, 16377-16381.

Yan, O.Y., Andersson, C.R., Kondo, T., Golden, S.S. and Johnson, C.H. (1998) Resonating circadian clocks enhance fitness in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 95, 8660-8664.

The Life and Death of Elysia Chlorotica

2009-06-25T03:36:00.022+01:00

Since the end of university I’ve had some trouble letting go of science as a constant occupation. Fortunately I was given the chance to post here about an old journal club presentation rather than go cold turkey, so here is a short piece on one of the most interesting organisms I’ve read about.

The picture on the left is of a sea slug of the genus Elysia which has evolved to blend in with its surroundings by looking almost exactly like a small leaf. The green colour that makes this camouflage so convincing is produced when the slug feeds on algae and steals their chloroplasts, subsequently storing them throughout its body. The use of chloroplast pigments to produce a leaf colour is common among sea slugs but the subject of this post is exceptional among the rest of its family. While in the majority of sea slugs the chloroplasts stop working within days or weeks of incorporation, those taken up by Elysia chlorotica can remain stable and active within the slug for at least ten months. As a result the slug is a rare example of a photosynthetic animal and it can survive without food for a long time, as long as it has light and carbon dioxide.

E. chlorotica has coevolved with a specific species of algae named Vaucheria litorea, which is a source of food and chloroplasts for the slug. The metamorphosis of E. chlorotica larvae into their slug form takes place only if the larvae are attached to filaments of V. litorea and in the absence of this algal species the larvae will die. Immediately after metamorphosis the slugs feed on the algae to which they are attached and take up their chloroplasts, incorporating them into their branched digestive tract.

As mentioned previously, most species of sea slug lose their chloroplast functions soon after obtaining them from algae. This is expected to be because the majority of genes required for chloroplast survival and function are encoded on the algal nucleus. Since animals do not have genes involved in chloroplast maintenance the question of how E. chlorotica manages to sustain its stolen plastids was investigated by several labs. In a recent study, the chloroplast genome of V. litorea was sequenced to determine whether it had greater genetic autonomy than previously sequenced chloroplasts, however, the chloroplast genome was found to be largely normal and was missing many genes essential for photosynthesis.

One of these missing genes, psbO, encodes a component of photosystem II which is vulnerable to damage during photosynthesis and needs to be regularly resynthesised for chloroplast function. Rumpho et al. were able to amplify the whole of this gene from sea slug DNA using primers based on seqeuence databases. The amplified sequence was shown to correspond exactly to the version of psbO from V. litorea, indicating that lateral gene transfer had taken place between the algae and the slug. The same sequence could be amplified from sea slug eggs which had not yet encountered algae, confirming that the sequence had entered the E. chlorotica germline. The authors behind this study speculate that all of the other genes needed for chloroplast survival and function have also been transferred to the slug germline, explaining the unique ability of E. chlorotica to maintain its chloroplasts for its entire life. So it seems that the sea slugs have stolen both DNA and organelles from algae, which is quite an achievement.

Another interesting property of E. chlorotica is that its generations are separated from each other. Whether slugs are collected in the lab or monitored in their natural environment, the entire adult population undergoes a synchronous death every year after they have laid their eggs. Pierce et al. observed that viral particles could be seen in the cytoplasm and nuclei of slugs just before their mass death and suggested that they may be responsible for the mortalities. The morphology of the viruses and the presence of reverse transcriptase activity suggest that they are retroviruses and, since the particles were found even under controlled laboratory conditions, it is likely that the virus is encoded on the genome of the sea slug. The group who made this observation suggested that the viruses may respond to annual environmental changes and kill the sea slugs in the spring, though this hypothesis has not been tested so far.

Unfortunately, the last paper I found on research into the viruses within the sea slugs was in 1999 and so research into this interesting aspect of the organism may not be ongoing. However I'm hoping to find some more about it in the future.

Rumpho, M. (2000). Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis PLANT PHYSIOLOGY, 123 (1), 29-38 DOI: 10.1104/pp.123.1.29

Pierce, S., Maugel, T., Rumpho, M., Hanten, J., & Mondy, W. (1999). Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance Biological Bulletin, 197 (1) DOI: 10.2307/1542990

Pierce, S., Massey, S., Hanten, J., & Curtis, N. (2003). Horizontal Transfer of Functional Nuclear Genes between Multicellular Organisms Biological Bulletin, 204 (3) DOI: 10.2307/1543594

Rumpho, M., Worful, J., Lee, J., Kannan, K., Tyler, M., Bhattacharya, D., Moustafa, A., & Manhart, J. (2008). From the Cover: Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Proceedings of the National Academy of Sciences, 105 (46), 17867-17871 DOI: 10.1073/pnas.0804968105

Flu flu go away, come again another day (when I don't have exams)

2009-05-01T14:50:00.003+01:00

Right I should be revising for those finals but with my arm hurting I am using it as an excuse not to write practice essays today and typing doesn't put as much strain on it (I think).

So Swine flu/Mexican flu/H1N1 flu has brought Mexico to a halt and scared the rest of the world into a panic. Big mouth Vice-president Biden of the USA has done it again saying he wouldn't trust travelling etc. fair point but you are trying to calm the masses as a leader. From what is humble virologist wannabe can see is this new hybrid strain made from genes of avian, swine and human flu is doing is spreading very quickly among people. For some reason it has killed lots in Mexico (allegedly) but only causing mild cases elsewhere. Today however the researcher at Mill Hill have suggested this strain is not very deadly. This paints a rather paradoxical picture. We see many deaths in Mexico from a fast spreading flu. Yes the world should be worried and has done the right thing by preparing for the worse. But we have only seen mild cases in most people outside Mexico and its genome suggests it is only mild. It doesnt have the make-up that causes cytokine storm that kills young people (like Spanish flu did). That is one thing not to worry about (right now). Plus it only infects cells near the top of the respiratory track so doesn't cause infections deep in the lungs, making it easy to spread but not as deadly as some.

So two things need to be thought about 1) why has it killed so many people in Mexico? and 2) what shall we call it? I am not going to tackle the first but I have some thought. The second I think should be Mexican flu. Swine flu is hurting the pig farming industry (some countries have banned pork imports!) while H1N1 is just rubbish. Loads of flu strains are called H1N1. Some seasonal flu is H1N1 and Spanish flu of 1919 was H1N1!!! I personally think the H and N naming system is out dated and inaccurate. It is not the H and N proteins that make a virus what is is per se but the many other changes with them.

Closing thought though, Flu has a high mutation rate with its RNA genome (no where near HIV but still more than what we would like) so changes may occur soon that could be game changers.

http://news.bbc.co.uk/1/hi/health/8028371.stm

When a bit more fat is not that bad

2009-04-07T21:06:00.012+01:00

Nothing is as simple as it looks in Biology. We can’t even rely on gene or protein sequences to tell us what is going to happen. At the chromosomal level, the new field of epigenetics is showing how the nucleotide sequence does not define everything. At protein level we have to consider post-translational modifications. My final year project, now at the final write up stages, concentrated on an aspect of Trypanosoma brucei molecular biology which I very pompously described as a potential drug target and the cure for African sleeping disease. This bit was probably ‘paper talk’ though. In any case, the project focused on protein palmitoylation.

Palmitoylation is part of a group of several lipid modifications that can occur in proteins. Examples include N-myristoylation, prenylation and GPI-anchors. N-myristoylation is the reason why I thought it was not pushing it to much to say that we can kill parasites by targeting these modifications. N-myristoylation corresponds to the co-translational addition of myristate to an N-terminus glycine, and is catalyzed by N-myristoyltransferases (NMT). It so happens that NMTs were shown to be essential for parasite viability, and several antifungal NMT inhibitors apparently work on reducing NMT activity in T.brucei as well(1).

But back to palmitoylation, the post-translational addition of palmitic acid to a cysteine residue (reviewed in 2 and 3). It can occur in any place in the protein, and so far no consensus palmitoylation motif has been identified. Often it occurs in an N-terminus cysteine, just next to a myristoylation site. These proteins are said to be dually acylated. Palmitoylation can also happen in close proximity to prenylation sites. Palmitoylation can perform many functions in the cell, the most obvious being protein tethering to membranes and cellular localization. It has also been shown to interfere with protein-protein interactions and even with protein degradation. For example, the yeast SNARE Tgl1, when is not palmitoylated, can interact with the ubiquitin ligase Tul1(4).

Those paying attention (or still reading) will think that it is a bit strange that palmitoylation can regulate protein degradation like that. In fact, most of these lipid modifications, N-myristoylation for example, last for the life time of the protein. Well, palmitoylation is characterized by a unique feature: it is reversible. This means that it can determine processes that other lipidations cannot. Dynamic trafficking of proteins of proteins, for example. This is the case of Ras protein, which through an deacylation/reacylation cycle is able to exchange between the plasma membrane and the Golgi apparatus (5). Reversibility can also be important for signalling. Regulator of G-protein signalling 2 (RGS2) was shown to increase or decrease its GTPase-activating ability depending on which of its cysteine residues are palmitoylated(6).

The process of palmitoylation in itself is catalyzed by the enzymes palmitoyl-acyltransferases (PATs). These were pretty hard to identify, apparently. Not only they were difficult to purify, but the reaction itself can occur spontaneously with biological significance(7), which made many sceptic on whether these enzymes existed at all. However, they were identified, first in yeast, now in mammals and in my dear T.brucei.

And what was supposed to be a short post if already long enough. I finish by adding that my project was about finding proteins that were palmitoylated and that would change their localization in the absence of this modification, in an attempt to identify substrates for RNAi PAT studies in T.brucei. I must say that the project was not particularly successful, but I developed the important skill of writing long discussions based on few results, which I bet will come handy in the future.

I now challenge Mel to continue in this post-translational modification topic and tell us about his project on glycosylation.

References:

1. Price, H. (2002). Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites Journal of Biological Chemistry, 278 (9), 7206-7214 DOI: 10.1074/jbc.M211391200

2. Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007;274:5202-10.

3. Greaves J, Chamberlain LH. Palmitoylation-dependent protein sorting. J Cell Biol 2007;176:249-54.

4. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J 2005;24:2524-32.

5. Rocks O, Peyker A, Kahms M, et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 2005;307:1746-52.

6. Ni J, Qu L, Yang H, et al. Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2. Int J Biochem Cell Biol 2006;38:2209-18.

7. Kummel D, Heinemann U, Veit M. Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 2006;103:12701-6.

Faith of a different sort

2009-03-31T13:03:00.001+01:00

So having finals upon us should be no excuse to stop the advancement of scientific and philosophical thought on here. So when I wandered into town today to do a favour for my dad (30 min walk for me, 3 hour drive for him) I saw a stall with a sign talking about truth. I still have no idea what a truth is so I thought I would have a look at what truths they were talking about. It was a stall full of Jehovah’s witnesses and I was very nice and polite before you say anything! I always am to there face. Being nasty and telling them, ‘you are probably was wrong as you can be’ doesn’t help win them over to your cause! Richard Dawkins QED. But over our chat I mentioned I supported evolution and was an atheist. But they suggested trusting some of the gaps in evolution had an explanation needed faith. This is an interesting one. It doesn’t but it requires something. I know very little physics and geology. If someone was to question the age of the earth in front of me I would have to turn around I say ‘I don’t know how they worked it out and don’t understand the sciences behind it’ so is it faith in these people I have in other scientists? Probably. But is it bad or wrong for me. I think it is a different sort of faith. First of all it is not blind absolute trust. I know I can easily be wrong about many things (it doesn’t happen often enough though) and I know other can be.

We all need to have this kind of faith to get through life. We cannot know everything so we need to trust others or ‘the system’. Imagine a friend offers you a life in there car but you have never seen them drive before in your life. You don’t know if they are good or bad or will get you killed or not. But you get in anyway. Perhaps because you are so lazy you don’t care if you could die as long as it gets you there faster like me. Or because you have faith that having passed a driving test and having belief in there abilities means you can trust them. It is like knowing peer reviewing produces mostly good knowledge and the author is confident in the results even if you don’t understand the experiments or the maths used to analyse it. This evidence based faith is not a bad thing but needs use to take the results with a large pinch of results. It is not perfect. Sometimes someone who you think would be a good driver turns out not to be while taking a ‘fact’ at face value because you trust the scientist you get it from could mean you are wrong but sometimes we need to take these ‘leaps of faith’. Just know that is what you are doing when you do.

Agree or not?

Drosophila: Virgins, balancers and jump-outs

2009-02-16T04:14:00.009Z

A wise man once said that the sooner you find your model organism, the better. Ignoring the fact that the wise man I am quoting is actually me, I think I generally agree with him. This year I am working on an exciting project which extensively uses the model organism Drosophila melanogaster, geneticists’ favourite little invertebrate. In the 15 short weeks I have been working with flies, I have learned a great deal from these wonderful creatures and experienced emotions from adoration to amusement to frustrated rage in my dealings with them.

Briefly, this is your average everyday “fruit fly” (even though a more appropriate name would be “vinegar fly”) with the complete package of a head, a thorax with 2 wings and 6 legs, an abdomen, and everything else that makes flies flies. I will spare you the tediously detailed anatomy and physiology lecture and will instead jump straight to the cool stuff that interests us geneticists and you readers of genetics blogs.

One of the most interesting things about Drosophila is in fact the people that use them in research. These people are commonly known as “fly pushers”, since they do in fact spend a lot of their time pushing flies around under a microscope using an old half-mangled paintbrush. One thing that you will inevitably hear about once you start hanging out with fly pushers is how interested they are in virgin females. Flies, that is. You see, since experiments in fly genetics involve crossing male flies from one line to female flies of another in order to obtain progeny of the required genotype, one must ensure that the females have not already been fertilised by males before picked out and crossed to a different line. Collecting virgins involves looking for flies that have a dark spot in their abdomen called the meconium, which is visible for 2-3 hours from eclosion (i.e. emergence of the adult from the pupa). Females will not be receptive to males for up to 8 hours from eclosion so any fly that has a meconium can be safely considered a virgin. The most efficient way to collect virgins is to empty a vial of eclosing flies in the morning, selecting females with visible meconium, and returning in the evening. Any females in that vial will be virgins even if the meconium isn’t visible, since less than 8 hours will have passed since your last collection. Much like the world we live in these days, virgins can be rather hard to find, so if you ever hear a scientist worrying about the fact that they need as many virgins as possible, you now have an idea of what they are talking about.

Now let’s delve into some actual genetics. One of the multitude of reasons Drosophila are an excellent test tube for genetic experimentation is something called a “balancer chromosome”. A line of flies carrying a recessive lethal mutation can be maintained by tracking the mutation using dominant markers, or by “balancing” it over a different lethal mutation on its homologous chromosome. This way each chromosome “rescues” the mutation the other is carrying. However, if recombination takes place between these chromosomes, it is likely that you will then have one chromosome carrying both mutations over a wild type chromosome, which will quickly take over the stock and the mutations will be lost. You could always outcross males every generation (there is no recombination in male Drosophila) but that would be very laborious. In order to get around this problem, fly geneticists initially discovered and subsequently intentionally generated balancer chromosomes. These are full chromosomes which contain multiple inversions (essentially all the genes are still there and intact, but their order is jumbled up) in order to suppress recombination with its homologous chromosome and make any gametes with recombined chromosomes non-viable due to aneuploidy. They also carry some dominant visible markers such as Cy (curly wings), Tb (tubby), Sb (stubble) and others, and some recessive lethal markers, meaning that they can never go homozygous.

So, to illustrate this, if you have a recessive lethal mutation on chromosome 2 (let’s now call that chromosome “Yorf”), in order to establish a line that will self-perpetuate and not need constant attention, all you need to do is cross it to a line that carries a balancer second chromosome, for example the chromosome CyO. You will now have a line whose flies will always have the same genotype, i.e. Yorf/CyO. As the Yorf/CyO flies interbreed, any Yorf/Yorf progeny will not survive because the mutation will be lethal, and any CyO/CyO flies will also not survive because of the recessive lethal marker that CyO carries. There will also never be any recombination between the Yorf and CyO chromosomes because of the multiple inversions in CyO that prevent homologous pairing. Wonderful, isn’t it?

Another great thing about Drosophila is insertional mutagenesis. P-elements are transposons, short stretches of DNA which, in the presence of transposase and absence of inhibitor, are excised from the genome and reinserted randomly into another site. Some P-elements are rendered inactive by having the transposase gene deleted. This temporarily immobilises the transposon, until that fly is crossed to a fly carrying an active transposase gene. In the progeny, the P-element will jump out, and if it inserts into the coding sequence of a gene, it can disrupt its function. A mutagenesis screen can then be carried out and the insertion locus can be mapped using standard complementation or cytological mapping. There is however an application of P-element mutagenesis that has accelerated Drosophila genetics in recent years.

The targeted gene knock-out system developed in mice is an excellent way to study the role of single genes, but the long generation time and ethical concerns make the mouse a rather cumbersome model organism. An analogous targeted gene disruption tool has been developed in Drosophila, taking advantage of a property of P-element transposition. When a P-element is transposed, its excision from the genome can sometimes be imprecise, taking some of its flanking DNA along with it, thus causing a deletion in that gene. Since an extensive library of mapped P-element inserts already exists (in over half the fly genome), a line carrying a P-element insert in a particular gene can be obtained and the P-element “jumped out” by crossing to a transposase line. In the cases where the jump-out has been imprecise, the result will effectively be a targeted gene knock-out.

This is, of course, only scratching the surface of the range of genetic tools that Drosophila offers. The best way to understand fly genetics is to try some crosses on paper yourself. Some quite amazing things can be achieved within a few weeks’ time simply by performing a series of crosses and studying the progeny. The waiting time is still a little too long for impatient people like myself, but careful time planning can ensure that you always have some flies to play with and are never stuck staring at a pupa, waiting for the damn fly to emerge.

Meaning out of nonsense

2009-01-22T13:30:00.001Z

Imagine you are a eukaryotic cell. You have many genes and make many mRNAs. One problems is mutated genes, pseudogenes and mistakes by RNAPII all make mRNAs which contain Premature Stop Codons (PTC). These then make truncated proteins which could be toxic to the cell. Eukaryotes have evolved a mechanism to recognise these and degrade them. I guess prokaryotes do not have an analogous system (that I know of) because the half-life of these mRNAs are minutes long not hours long like most eukaryotic mRNAs. Eg the average mRNA half-life in Arabidopsis is between 4-6 hours and is very variable between mRNAs. Personally I am not convinced this is an important enough reason to have a quality control check mechanism. The system I am referring to is called Nonsense-Mediated mRNA Decay (NMD). Mediators of it have been found in the early branching Giardia and is conserved between fungi/animals to plants. A PTC is recognised as wrong rather than a correct stop codon at the end of a ORF during the first round of translation by the ribosome. When the ribosome reaches a stop codon it has two choices, terminate correctly or stall and cell effectors of NMD to take it away for degradation by whatever nucleases that cell uses. It was first found in yeast and the mutants were called upf1-3 (for up-frame shift 1-3) and these yeast grow fairly normally but have increased levels of transcripts with stop codons. Raises the question why did it evolve if not very important when you KO it. In C. elegance SMG1-7 were identified having genital defects. Strangely, these were involved NMD with SMG2-4 corresponding to UPF1-3. Homologues of most of these proteins are found in many other eukaryotes. UPF1 is generally phosphorylated when ribosome stalling takes place and condemns it to degradation. Interestingly, it is a DEAD box RNA helicase (SF2 I believe).

The two questions I find interesting is 1) how do organisms tell the difference between a PTC and normal stop codon and 2) how this can be used to regulated gene expression in cell signalling. You may ask ‘James, you charismatic stallion, how can something degrading useless mRNAs be useful or regulated for controlling gene expression’ and I would reply ‘wait and read the rest of this post you $#£!&%’. The first question is rather interesting as different organisms do different things. Yeast and invertebrates such as C. elegance and the fly pushers Drosphilia use something called the 3’ faux model. Something in the 3’ end allows normal termination when the stop codon is here but NMD starts when the stop codon (PTC) is distant from the 3’ end. It has been found the distance from the poly-A tail and therefore the Poly-A Binding Protein (PABP) determines this. When a long 3’ UTR is present the mRNA is degraded. When PABP is tethered close to the stop codon termination occurs normally. This also works in plants and mammals. However, another mechanism also works in both of them. When an introns is spliced out an Exon-Junction Complex (EJC) is left behind and if a EJC is found near a stop codon by the ribosome NMD starts. Why this operates in both plants and mammals but not yeast and invertebrates is curious. Did both mechanisms operate in the last common ancestor of plants and animals and has been lost a few times in the fungi and animal kingdoms or is it convergent evolution? I hope to better understand that.

Another and perhaps more important question is what is NMD role in plants and animals. Does it provide another function other than keeping toxic protein levels low? Short answers, yes. It affect development in C. elegance and embryo lethality in mice. Clearly it plays a role in animal development. In yeast the cells senesce sooner in upf1-3 mutants because telomeres shorten. In mammals, amino acid deletion causes problems for the ribosome and NMD is down regulated and genes for amino acids biosynthesis are up-regulated so more amino acids will be made. In Arabidopsis, when upf-1 is completely KO we see embryo lethality but Knock-down alleles see some developmental phenotypes (such as in the flowers) and altered stress responses.

I will finished up now but feel free to ask questions. I have just finished writing a 4-page grant proposal on this. Just to piss Mel off, I will say this. I didn’t do any reading for this. This was all from memory when I got home. No PDF files were opened – not one!

Arch nemesis

2009-01-01T02:24:00.009Z

Evidence is the cornerstone of science. It is what allows scientists to reject or accept hypotheses, and a robust piece of evidence can force the staunchest defender of a theory to completely reject it. People tend to like evidence when it supports their "beliefs", and dislike it when it refutes them, but scientists have no choice, they need to be apathetic and disinterested towards any results, so as to avoid introducing bias. However, scientists are also (rumoured to be) people, and the tendency to like or dislike a piece of evidence can be quite hard to resist.

How could a scientist maintain an interest in their research while avoiding interest in the data generated from it? It seems almost self-contradictory. What goals do scientists usually have when carrying out research? For a scientist working at a pharmaceutical company (assuming they wanted to keep their job), this goal would be to get a drug on the market. For a graduate student, the goal could be to find something surprising or revolutionary in order to jump-start their career. For a hardened senior professor, it could be to prove their own hypotheses right and boost their reputation (and maybe get in line for a Nobel prize?). All these goals are fundamentally selfish, and you would be hard pressed to find pure altruism in even the "most moral" scientist (whatever that means...). Perhaps that's because scientists tend to be ambitious, competitive animals, and that's a selfish motivator in itself, but I would say it's because people's goals tend to be selfish in nature.

These selfish goals scientists have certainly pose the most danger in making them introduce bias into their data. However, the drive that these goals give to scientists is what has led to so many historical discoveries. I'm sure some discoveries had altruistic motives but let's just say Watson and Crick weren't really thinking about saving humanity from terrible doom. Not to paint a sad picture of scientists, but sometimes the only thing that scientists get up for in the morning is the tiny chance that today's experiment will get them even slightly closer to achieving their goal.

I think that scientists manage to minimize the danger of introducing bias by finding the one person on Earth they most disagree with and establishing an intimate, life-long professional relationship with them, manifested mostly through heckling at conferences and angry e-mail exchanges. This works much more efficiently in academia than in industry, because industry scientists are usually either unwilling or not allowed to talk to outsiders about anything they do. Academics are also secretive to a certain extent in order to avoid being scooped, but the spirit of collaboration and criticism is much stronger. This kind of mutually abusive relationship is what every scientist needs, and I personally can't wait to meet my arch nemesis!

P.S. Happy 2009!

Kinetoplastid DNA

2008-10-17T13:17:00.011+01:00

It seems to be that time of the year in which we post about something to do with our posts, as these are the topics we are supposed to be reading about now. Therefore, a post on parasites. That’s where the connection with my project finishes, but anyway.

As you might know, tryponosomatids are organisms from the family Troponosomatidae, which include some pretty nasty unicellular parasites such as Trypanosoma brucei, causer for African sleeping disease, and Leishmania, causer of leishmaniasis. As other higher eukaryotes, these parasites are characterised by two genomes: a nuclear genome and a mitochondrion genome, 10-30% of all the cell DNA, situated in the kinetoplast (the name given to the mitochondrion in these organisms).

Fig.1 A- Schematic minicircle organization. B- in vivo network organization, seen sideways.

(Adapted from reference 1)

So far nothing extraordinary. Things get interesting, when one looks at the structure of the kinetoplast DNA (kDNA) in these species. The kDNA is constituted by thousands of small DNA circles, the minicircles, and a few dozen of large DNA circles, the maxicircles. These are interlocked, as in a chain mail of medieval armour. Each circle is interlocked with 3 other neighbouring circles. The minicircles and maxicircles are stretched into a disk-shaped structure, situated near to the flagellar basal body (Fig.1). The maxicircles are those more similar to normal mitochondrion genome. They include rRNAs and genes encoding proteins associated with the respiratory processes that takes place in this organelle. However, the maxicircles mRNA requires extensive editing, namely the introduction or deletion of uridylate. The minicircles encode the guide RNAs used as templates for this editing. Since there are many types of editing required, as well as maxicircles with different sequences, there is the need for the thousand minicircles with different sequences. Seems a bit of a wasteful system, but what do I know!

More interesting is to think of the replication process. The minicircles must stop being interlocked, replicated, interlocked again, and in the end originate two new networks of thousands of minicircles that separate into the two new daughter cells. Let us look at the different steps, following an individual minicircle (Fig.2):

Fig.2 kDNA replication model. (From reference 2)

1- A minicircle must first be released from the network into the so-called kinetoflagellar zone (KTZ), where several proteins involved in the process exist. Here the unidirectional replication of the circle occurs, although the details of this process are not well known.

2- The two daughter minicircles move to opposite antipodal sites of the circle, where there are two protein assemblies. Here a variety of reactions occur: the RNA primers are removed, the gaps between Okazaki fragments are filled by DNA pol, and nick as sealed by DNA ligase. An important characteristic, however, is that the minicircles maintain at least one gap in their structure. This is thought to be a self-check feature of this system, as a way to guarantee that no minicircle is replicated twice.

3- Minicircles are attached by topoisomerase II to the network periphery adjacent to the antipodal sites.

The way the minicircles are attached to the disk varies with the species of trypanosomatid. In T.brucei, for example, the minicircles accumulate at the network poles. In T.cruzi, and C.fasciculata, the minicircles seem to be uniformly attached around the periphery of the disk, creating a ring of new minicircles, of increased thickness. How this uniform attachment is done is not certain, but it is though that either the antipodal protein complexes or the disk itself must rotate! (Fig.3)

Fig.3 Replication in C.fasciculata. New minicircles have been labelled with fluorescent nucleotides. The arrangement of the new minicircles around the periphery of the disk is obvious (from reference 2).

4- Regardless of the method, new minicircles are attached to the disk, and the valence must increase to 4 to 6 attachments per minicircle, in order to ensure twice as many minicircles in the same space (the mitochondrion membrane has not doubled at this point). When the space increases, topoisomerase II ensures that valence returns to 3.

5- Finally, the nicks are repaired and the network splits into 2, although how exactly this division occurs is not well known. Most importantly, it is not known for sure how to guarantee that the two daughter networks have the exactly same minicircles. Considering their importance in maxicircle mRNA editing, the loss of one minicircle could have dramatic consequences.

There is much to be explained in this process, namely identify exactly how the minicircles are moved to the different antipodal sites, or the details of individual minicircle replication, but such understanding will require the identification of the proteins involved. Considering the complexity of the process, many must be involved, but only a few have been identified so far.

This is probably not life-changing research, but it is surely interesting. It is important to remind ourselves once in a while that though we learn the textbook pathways and processes, Nature seems to like using hundreds of alternatives. Just to make scientists lives harder, I assume!

Liu, B., Liu, Y., Motyka, S., Agbo, E., & Englund, P. (2005). Fellowship of the rings: the replication of kinetoplast DNA Trends in Parasitology, 21 (8), 363-369 DOI: 10.1016/j.pt.2005.06.008

(2) Smith D. and Parsons M. (1996). Molecular Biology of Parasitic Protozoa, Kinetoplast DNA: Structure and Replication, Oxford University Press, USA

Apical dominance, MAX and my project

2008-10-12T21:54:00.005+01:00

One of the most important things the plant hormone auxin does is play an important role in is apical dominance. This is where the Shoot Apical Meristem (SAM), the growing part of the above ground half of the plant, produces a signal that inhibits growth from other auxiliary meristems. The SAM and auxiliary meristems contain the plant’s ‘stem cells’ and generate new leaves, stems and flowers. They both have the same ability to generate new organs. When the plant grows and leaves emerge from the meristem, an auxiliary meristem is left behind with the leaf, which may or may not grow out, just above where the leaf meets the stem. For years it was known auxin from the SAM inhibited their outgrowth. Auxin is produced in the SAM and moved down the plant and when it is stopped, the auxiliary meristem can grow out. The best person to tell you this is a gardener. When pruning, they remove the SAM on a plant, like a rose bush, to allow dormant growing parts of the plant grow out and make it bushier. It is also a pain for, let’s say for example, tobacco farmers. They ‘top’ tobacco plants so it does not grow up and make the leaves fatter. However, this stops auxin travelling down the stem and auxiliary meristems grow out. The addition of unpleasant chemicals is used to solve this problem.

Auxin does not travel up into the auxiliary meristem, so how does auxin inhibit bud outgrowth? A second signal must be involved. This ‘second messenger’ is not an easy thing to understand. It turns out to be a complex interplay of plant hormones that I will try to explain now control bud outgrowth. I will start by explaining the players involved. The hormone cytokinin (Ck) often plays the opposite role to auxin in plant grow. When it is applied to an auxiliary meristem it will grow out. Ck is generated both in the roots and locally in the stem but it is unclear which is more relevant to shoot branching (I think it varies from species to species). The auxin signalling pathway does regulate Ck synthesis but this is not the whole story. Auxin signalling pathway mutants do show some increase in shoot branching but it is lower than those affected by mutations in MAX genes (see below) and the effects of these are additive to auxin signalling.

Mutant screens reviled the MAX genes. max mutants have increased shoot branching. To cut a long story short, the MAX pathway does not produce the inhibitory signal but helps auxin regulate shoot-branching. MAX3 and 4 produce enzymes that alter a caratinoid. MAX1 is a P450 that acts downstream of MAX3 and 4 and alters the chemical further, producing the MAX-dependent hormone (more on the identity below). MAX2 does not produce the graft-transmissible signal but helps perceives the MAX-dependent hormone. In fact, it is an F-box protein like TIR1 from my post on auxin signalling. However, I think it is unlikely to bind the MAX-dependent hormone directly as TIR1 does auxin. I suppose this cannot be looked at until the hormone has been characterised better. MAX2 is also involved with leaf senescence and some features of light perception but its roles in these are not very well understood either.

The Leyser lab has been working on finding the identity of the MAX-dependent hormone but two papers published in the same September issue of Nature (just after my subscription ended!) suggest the identity in pea and rice. Ottoline has just written a short review on them (below). They found a chemical, called strigolactones, was involved. Previously shown to be involved with germination, formation of mycorrhiza with fungi and growth of parasitic plants, now it appears they are a key regulator of shoot branching. The exact identity of the biological active strigolactone is still yet to be found.

The MAX pathway’s mode of action is through limiting auxin transport. This work was published with a PhD student at York as the lead author in the Leyser lab (he was a very well known blue coat). Auxin is transported from the SAM to the roots. To move in and out of cells, auxin needs proteins to transport it. One important class are the PIN proteins. They can become localised to a particular part in a plant cell, such as the basal side, to ensure auxin only moves in one direction. This is very important in creating a vascular system in the plant. First of all, auxin is made by the auxiliary meristems but it is their ability to transport auxin out that allows them to grow out. It is best to imagine the auxin transport network as roads. Auxin (the cars) leaves the SAM and moves down the stem via PIN proteins (lanes on a motor way). In a normal plant, not all lanes are open. So auxin from the SAM fills most of the PIN proteins and only a little auxin from the buds can enter, letting some grow out. max mutants have an increase transport capacity because more PIN proteins are present. This is like opening extra lanes on a motor way so auxin from all the buds can enter and move freely, letting them grow out! Integrating the actions of all hormones leads to something like this: auxin moves down the plant and its movement down is limited by the MAX-dependent hormone from the roots. This limits the amount of auxin auxiliary merisetems can transport out, so large amounts of auxin accumulate in these buds. This (somehow) inhibits Ck production to limit growth of the bud. The integration of multiple hormones has been described as the brain of plants.

The reason max mutants and auxin signalling double mutants have an additive effect is because auxin works both through its classical signalling pathway but also through auxin transport. This is where, for me, things get confusing. How does auxin in the bud know not to be exported and form vascular tissue to connect to the main flow in the stem? This has been a long standing question, not for shoot branching but for the formation of a vascular network, yet there is no answer (or good one at least).

My final year project is in the Leyser lab, for what I hope are obvious reasons now. They have done lots of great work. A modifier of the max1 phenotype was found in a mutants screen and mapped to a location on chromosome 1. My job is to widdle the candidate genes down from around 20 to one! I don’t want to give too much away about the project on here but the mutation alters the max1 phenotype and has its own slight developmental phenotype. Hopefully understanding it will help in the long term goal of understanding plant development. I doubt it will have a direct role synthesis or degradation of the MAX-dependent hormone but I believe it will affect downstream events. I hope this has been enlighting.

Here is a very good review and explains these things better than I can do by someone in the Leyser lab:
http://www.ncbi.nlm.nih.gov/pubmed/17728300?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Here is an ahead of print short review on the identity of the MAX-dependent hormone by Ottoline:
http://www.ncbi.nlm.nih.gov/pubmed/18804430?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

IgNobels 2008

2008-10-03T15:06:00.007+01:00

'For achievements that first make people LAUGH and then make them THINK'

As I seem to be spending all my holidays memorizing hard words in english (Melly will know what I mean) I actually haven't had the time to read anything 'scientific' like you guys. I had, however, time to have a look to what Nature regards as, and I agree, 'the highlight of the scientific calendar'. Yes, my friends, the Ig Nobels are out again. As you will remember, I wrote a post last year when I realized that these amazing prizes existed, and have been eagerly waiting for this year's winners. So, below, enjoy the list of the IgNobel winners 2008!

NUTRITION

Massimiliano Zampini of the University of Trento, Italy and Charles Spence of OxfordUniversity,UK, for electronically modifying the sound of a potato chip to make the person chewing the chip believe it to be crisper and fresher than it really is.

PEACE
The Swiss Federal Ethics Committee on Non-Human Biotechnology (ECNH) and the citizens of Switzerland for adopting the legal principle that plants have dignity.

ARCHEOLOGY Astolfo G. Mello Araujo and José Carlos Marcelino of Universidade de São Paulo, Brazil, for measuring how the course of history, or at least the contents of an archaeological dig site, can be scrambled by the actions of a live armadillo.

BIOLOGY Marie-Christine Cadiergues, Christel Joubert,, and Michel Franc of Ecole Nationale Veterinaire de Toulouse, France for discovering that the fleas that live on a dog can jump higher than the fleas that live on a cat.

MEDICINE
Dan Ariely of Duke University, USA, for demonstrating that high-priced fake medicine is more effective than low-priced fake medicine. (a study on placebo effects)

COGNITIVE SCIENCE Toshiyuki Nakagaki of Hokkaido University, Japan, Hiroyasu Yamada of Nagoya, Japan, Ryo Kobayashi of Hiroshima University, Atsushi Tero of Presto JST, Akio Ishiguro of Tohoku University, and Ágotá Tóth of the University of Szeged, Hungary, for discovering that slime molds can solve puzzles.

ECONOMICS
Geoffrey Miller, Joshua Tybur and Brent Jordan of the University of New Mexico,

USA

, for discovering that a professional lap dancer's ovulatory cycle affects her tip earnings.

PHYSICS Dorian Raymer of the Ocean Observatories Initiative at Scripps Institution of Oceanography, USA, and Douglas Smith of the University of California, San Diego, USA, for proving mathematically that heaps of string or hair or almost anything else will inevitably tangle themselves up in knots.

CHEMISTRY Sharee A. Umpierre of the University of Puerto Rico, Joseph A. Hill of The Fertility Centers of New England (USA), Deborah J. Anderson of Boston University School of Medicine and Harvard Medical School (USA), for discovering that Coca-Cola is an effective spermicide, and to Chuang-Ye Hong of Taipei Medical University (Taiwan), C.C. Shieh, P. Wu, and B.N. Chiang (all of Taiwan) for discovering that it is not.

LITERATURE
David Sims of Cass Business School. London, UK, for his lovingly written study "You Bastard: A Narrative Exploration of the Experience of Indignation within Organizations."

Hehe, as usual, delicious to read... For more information on the Ig Nobels or this year's ceremony (I actually don't know yet what's this year's topic. Last year it was chicken and involved dressing proper Nobel laureates in egg outfits), see the link below

http://improbable.com/ig/winners/#ig2008

One of last year's winners, Dan Meyer demonstrates his skills, after winning the 2007 IgNobel for medicine in collaboration with Brian Witcombe, for their study on sword swallowing and its side-effects

The wonders of auxin

2008-10-03T14:43:00.003+01:00

I am a complete sell out. I have worked for a plant biotech company and getting funding from Gatsby (a charity with a goal, alone with others, is to get scientists interesting in plants). These people have been very nice by helping me pay rent, buy DVDs and science magazines as well as sending me to Mexico for a conference. Therefore, I thought I should give something back by educating my friends in a bit of plant biology. Don’t worry faint hearted, I will keep it nice and molecular as that is the way I like it.

After returning from my Mexico trip I realised a few things, English seems to be spoken by everyone, don’t eat food from Mexico City airport and auxin is a fascinating plant hormone and virtually everyone in the plant community agrees. Most talks there were on auxin biosynthesis, receptor function and structure, its control of shoot branching and transport around the plant. However, I do not recall all of it. Or hardy any of it as I was jet lagged with little prior knowledge of plant hormones (plus it has been over a year since the conference).

Auxin was the most interesting of all the plant signalling molecules. It has been studied for over 100 years and only today we are starting to understand how it controls plant development and cell biology. Auxin is a small molecule made from an amino acid and its most biologically active for is called IAA. Auxin controls cell elongation and division, and can promote these or stop them depending on the tissue type auxin enters. Auxin causes lateral root growth and patterns the vascular system of plants. Auxin starts shaping the plant in the embryo. What is interesting is how it regulates many of these things.

It controls gene expression by activating Auxin Response Factors (ARFs). This is done by destabilizing proteins called Aux/IAAs. Genes activated by the addition of auxin contain Auxin Response Elements (ARE) which an ARF binds to. When no auxin is present, an ARF is bound to an ARE but it is diamerized with an Aux/IAA, which represses transcription of this gene. When auxin enters the cell, it gets ride of this Aux/IAA and the ARF diamerizes with another ARF. This causes transcription of the auxin regulated gene. How does auxin cause breakdown of Aux/IAA? (I thought I would sound more intelligent if I asked a lot of questions) Auxin doesn’t have a nice receptor at the cell membrane that activates a second message or a phosphorylation pathway, no that would be to simple. In 2005 Ottoline Leyser and Stefan Kepinski (then post-doc, now lecturer at Leeds) published in Nature that TIR1 was an auxin receptor. tir1 mutants had been known to be deficient in auxin signalling for a long time but not thought to be a mutation in the receptor. TIR1 is an F-box protein, which are not famous for being receptors (until now). Normally they simply act as an E3 ubiqutin (Ub) ligase, meaning they take Ub from one protein and add it to another creating a poly-ubiquitin tag that sends a protein for degradation by the 26S proteasome. Ubiqutination and targeting to the proteasome is found animals and fungi and plays an important role in signalling, including regulation of the cell cycle! TIR1 adds Ub to the Aux/IAA when auxin enters the cell. It was assumed that auxin was perceived by some other protein in the cell and caused some modification of the Aux/IAA or TIR1 to cause this to happen, probably by phosphorylation. Now we know that auxin binds toTIR1 and acts as a molecular glue, bridging the gap between TIR1 and Aux/IAA so it can be broken down. I think this is a brilliant method. I am glad to see plants are being original and creating cool new signalling pathways instead of being just like boring old animals and fungi that love there MAPK so much they should just go along and marry it. Please see my simplified diagram of the auxin signalling pathway, it is not perfect, but who is......except maybe Colombo. But then how does auxin have very different effects on different cell types. This appears to be because different tissues expresses different ARFs and Aux/IAAs and these turn on different genes but little is known right now.

One final thing, is TIR1 the only receptor? A good question indeed. Like I said earlier, I like asking questions to make me sound smart. Other F-box proteins appear to do the same job. The same only story of redundancy. However, this style of signalling cannot explain all the effects auxin has on plants. Some auxin responses occur very quickly after auxin addition. These happen so quickly, it is unlikely proteolysis followed by transcription and translation of effectors can account for them. Things like guard cell outward K+ current up, increased cytoplasm calcium, cell wall acidification starts (to help increase cell wall expansion) and elongation growth. How can we explain auxin’s influence over these physiological changes? There is debate and investigation over what causes these things. Some are caused by changes in membrane potential and perhaps Auxin Binding Protein 1 (ABP1) has some control over some changes but I am not convinced and other features are unrelated to this. After auxin addition MAP kinase activity increase but this is unrelated to both TIR1 and ABP1. There are still a lot of unanswered questions.

Here is a short, concise review by Ottoline’s old post-doc (now at Leeds), which I found yesterday and does a far better job of introducing people to this that I have!
The anatomy of auxin perception
http://www.ncbi.nlm.nih.gov/pubmed/17876776?ordinalpos=57&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Another one talking a more detailed look at auxin receptors and the debate around them. I have not read it fully, sadly.
Receptors for auxin: will it all end in TIRs?
http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=Receptors+for+auxin%3A+will+it+all+end+in+TIRs%3F

I have not referenced here for two reasons. First, I can’t be bothered and secondly, a lot of this is from conferences or meetings I have been to. I hope you enjoyed this little rant from the plant person.

Rats at a rave

2008-10-01T16:55:00.009+01:00

The illegal recreational drug ecstasy, also known as X, or MDMA (an abbreviation of its chemical formula), is popular in the rave scene and is used to induce euphoria and enhance the experience of dancing and loud music. The drug is considered relatively safe by the average raver due to its short term effects, despite having received publicity for various dangers identified by scientific research.

Ecstasy pills

The most widely known (undesired) effect of the drug is overexertion and sweating, leading to the ecstasy user drinking large amounts of water and being in danger of hyponatremia (the depletion of solutes from the blood plasma), which can eventually lead to cerebral edema. The drug has also been known for causing “holes in the brain”, Parkinsonian tremors, and permanent brain damage from single use, however these claims have been discredited* and retracted from the literature (1). Some research has shown that the drug may not be as toxic as once thought when used in moderation, but that does not address whether environmental effects could change the action of the drug and potentially make it more dangerous than a “traditional” toxicity study might suggest.

Research conducted in Italy and published in 2006 (2) has shown that not only does the drug enhance the rave experience for the user, but this works both ways, with the loud music physically enhancing the effect of the drug. Rats were given various doses of MDMA and treated to a surely exciting 4 hours of white noise at 95 dB (the loudest level permissible in Italian night clubs) while electrocortical activity in their brains was monitored by electroencephalography. Controls were conducted with rats treated with saline instead of MDMA, as well as both treatments without sound. ECoG monitoring was repeated over 5 days without administration of the drug or sound in order to study long term effects. The ECoG “spectrum power” was taken to be indicative of higher neural function in rats.

The data showed that a low dose of MDMA combined with sound caused decreased ECoG spectrum power significantly different from control, but no significant effect when MDMA was administered without sound. A high dose of MDMA combined with sound produced a dramatic effect that lasted for 5 days, while all other treatments had no long term effects. This powerful data shows a synergistic relationship between exposure to loud noise and the effect of MDMA on higher neural function, and suggests that higher doses only have long term effects when combined with loud noise. The authors do not suggest a possible mechanism for this effect, but they do warn that the drug may be more dangerous than commonly thought since most ecstasy users combine the drug with loud music.

* As a side note, some legislation around ecstasy was driven by the above mentioned discredited “findings”, which is disconcerting. An example is the RAVE act (3), introduced in 2002 by current Democratic VP candidate Joe Biden as part of the War On Drugs, which allows the law to shut down clubs and raves if ecstasy use is suspected to occur on their premises. The drug is still a dangerous one, but one would hope that laws would be revised after research they were based upon is discredited, which they were not.

References
1. Ronald Bailey: “The Agony of Ecstasy Research”. ReasonOnline.
2. Michelangelo Iannone, Stefania Bulotta, Donatella Paolino, Maria Zito, Santo Gratteri, FrancescoS Costanzo, Domenicantonio Rotiroti (2006). Electrocortical effects of MDMA are potentiated by acoustic stimulation in rats BMC Neuroscience, 7 (1) DOI: 10.1186/1471-2202-7-13
3. http://en.wikipedia.org/wiki/RAVE_Act

Environmental refugees?

2008-10-01T01:14:00.004+01:00

As we seem to be starting a new, shorter, form of post to guarantee that someone keeps writing in this blog, I decided to follow James' example. Here I just liked to mention a short article I read at Harvard's university website. The president of Kiribati, a south Pacific island nation has given recently a lecture at Harvard where he has presented his plan preparing his country for the eventuality (or should we call it certainty) of extinction. Apparently, with the sea-levels planned to raise by 1 meter in the next century due to climate change, the islands are starting to run out of space, and to eventually leave the islands for good seems to be the only option. However, the president seems to want to avoid creating what he describes as 'environmental refugees', a new word that we will perhaps become more and more familiar with in the future, as climate change starts having 'real' consequences that we cannot pretend to ignore anymore.

Read the short article here: http://www.news.harvard.edu/gazette/2008/09.25/13-kiribati.html

Facts about genes

2008-09-28T12:40:00.002+01:00

As I am the saddest of the three musket-geneticists by far I have decided to post something. I bought a book called ‘a short guide to the Human Genome’. The booked is aimed at people with a background in genetics. Good for lecturers to liven up lectures with facts. The book is a series of questions on various parts of molecular biology and ‘omics’ with short, one page long, answers. These are interesting questions but difficult to find the answers to them without a lot and more importantly, knowing where to look. So here I will look at a few of the more interesting and simple questions.

How many genes are there?
22,740 predicted and known genes. All genes minus predicted transcripts gives us only 18,357.
What is the typical size of a gene?
The median sixe is 16,995 nucleotides.
Which are the largest genes?
CNTNAP2 is 2.3 Mb (remember the genome of E. coli is only 4.63 Mb) and it generates a mRNA of 9.9 Kb. DMD which makes dystrophin is the second largest gene at 2.22 Mb making an mRNA of 14.1 Kb.
Largest proteins?
TTN gene which makes titin, which is 33,423 amino acid residues long. Mucin 16 is the second largest at 14,507 amino acid residues.
How much of the genome is made up of transposable elements?
45%: SINEs 13%, LINEs 21%, LTRs 8% and DDNA transposons 3%.
How many pseudogenes are there?
There are ~5000 pseudogenes with a median size of 1200 nucleotides.
The books seems good but I would suggest you borrow it off me rather than buy a copy.

Cross presentation: presenting unexpected antigens to the cell-mediated immune response.

2008-08-10T21:29:00.004+01:00

Please cast your minds back to basic immunology. I am sure you remember there is the innate immune response (boring) and the acquired/adaptive immune response (very interesting). Perhaps the most interesting part of it is the gene re-arrangement to generate antibodies and T cell receptors (TCRs). But this re-arrangement is random; how can they tell self from non-self? The very basic explanation is seems to be that antigen (Ag) presenting cells (APCs) shows lymphocytes these Ag in the local draining lymph nodes when the body knows it is ill (ie the innate immune system is acting up). (Sorry for this long introduction to the topic but I need to remind myself of this while I type on the train myself.)

A problem comes when we remember a bit of basic immunology. Peptide Ag presented on MHC class II molecules to CD4+ T cells (T helper cells) are taken up by APCs and are exogenous Ag. However, peptides shown on MHC class I to CD8+ T cells are generated from endogenous proteins. So how can a virus that infects only the liver like hepatitis or the respiratory system (like rhinovirus or flu) show their peptide Ag to naive CD8+ T cells (cytotoxic T cells or CTLs for short) which live in the lymph organs. Unless all viruses also affect the APCs as well then it seems impossible! Let’s forget how ridicules that idea is first and remember CTLs also attack tumour cells expressing mutant proteins. Cross presentation appears to be the answer. Somehow, APCs such as dendritic cells (DC) take up proteins from other cells and process these in a manner that means they are treated like endogenous proteins and therefore processed to be peptide Ag loaded onto MHC class I. This is called cross presentation, when CTLs are activated this way they are cross primed. The fine details of this appear to be missing but a lot of evidence is mounting that shows this must be the case. Other than this I can see no other way apart from the naive CTLs circulating the body and activating their; but no evidence has been found for this that I can see (I am pretty sure this also flies in the face of most accepted ideas in immunology). There are four ways for DC to capture extracellular proteins; (i) endocytosis (ii) pinocytosis (cell drinking) (iii) phagocytosis and (iv) macropinocytosis. For example, phagocytosis can be the uptake of a bacterium or cellular debris such as an apoptotic body. When cells are signalled to undergo apoptosis the dying cell starts to bleb and releases intact fragments of the cell called apoptotic bodies that express ‘eat-me’ signals. It is not a huge leap of the imagination to think this could be a major way tumour proteins are presented to naive CTLs.

So how do these exogenous proteins within sub-cellular compartments such as the endosome or phagosome get loaded onto the MHC class I molecules? It is a good question and various routes have been suggested and evidence for some of these have been found. In DC exogenous proteins have been shown to be exported into the cytosol before they are degraded in the lysosome. This means they can be substrate for the proteasome and broken down to short peptides to be exported across the ER membrane by TAP. When here they can bind to the peptide binding cleft of MHC class I molecules and move to the cell surface like normal endogenous proteins in the classical presentation pathway. Another pathway acts in the endosome. This TAP/proteasome independent pathway uses cathepsin S to generate some peptide Ag. It is unclear whether other peptides here also act but currently appears unlikely. What I find amazing is how this protease produces peptides of the correct size of 8 or 9 amino acids residues long to load onto MHC class I molecules. This pathway even produces the same peptide Ag as the much more complex proteasome based pathway. When both pathways are knocked out most of the cross-presentation in vivo is lost.
Interestingly, tolerance to an Ag can be generated using these mechanisms when no stimulator of the immune system is present, and this has been called cross tolerance. It is now clear cross presentation is not some strange phenomenon like originally thought when discovered but is a key part of the immune system. When you knockout these pathways for cross-presentation none occurs. The next step is to manipulate this with vaccines to stimulate the cell-mediated immune response. Often only antibodies are produced because the antigens are not cross-presented.

A short and sweet review:
Brode and Macary (2004) Cross-presentation: dendritic cells and macrophages bite off more than they can chew!

A full and great review:
Rock and Shen (2005) Cross-presentation: underlying mechanisms and role in immune surveillance.

Uncommon ways of looking at common problems- gene therapy for mitochondrial DNA diseases

2008-07-24T17:13:00.010+01:00

I’m always very interested in less conventional approaches to conventional problems and a recent review found while looking for a paper for a journal club provided a good example.

As you know, gene therapy aims at correcting a genetic deficiency by the introduction and/or substitution of the mutated gene by a functional copy into relevant cells. The classic examples for gene therapy are normally diseases such as Cystic Fibrosis- monogenic, recessive, autosomal diseases, as these are, for obvious reasons, those that theoretically offer higher chances of success. There are, however, other types of genetic diseases that offer more space for original, unconventional gene therapy approaches.

Mitochondrial DNA diseases are a good example. The human mitochondrial genome is small (16kb) and is housed in the mitochondrial matrix. As most of its genes encode proteins involved in oxidative phosphorylation necessary for cellular respiration and ATP production, is not too surprising that mutations in such genes can cause a variety of diseases, many of which with symptoms associated with progressive defects in muscle and nerve systems.

There are two obvious strategies to tackle this problem using gene therapy. One is to introduce the functional copy of the mutated gene into the nucleus. The protein, with an appropriate mitochondrial targeting sequence, can then be transported to the mitochondria. This is the so-called allotropic expression of the gene, and has been shown to produce promising results in culture. Other option is to target the plasmid containing the functional gene into the mitochondria itself, as these organelles contain all the ribosomal RNA and tRNAs necessary for intra-mitochondrial protein synthesis (assuming that these are not the mutated genes). These strategies have been tried out with some success both using a wild type sequence of the mutated gene, or a gene from another species, known to perform its function in the pathway more efficiently (xenotopic expression).

However, as you might have already noticed, a mitochondrial DNA disease is characterised by the fact that not all the mitochondrial genome copies in a cell are necessarily mutated (the presence of different genome types within the same cell is known as heteroplasmy). This has two main implications: firstly, mtDNA diseases are threshold diseases: only when around 60-95% of copies in a cell are mutated will symptoms appear. This threshold will obviously vary depending on the disease. Secondly, rather than trying to introduce a functional copy into the mutated mitochondria, a much more interesting approach can be to just manipulate the relative amounts of the mutated and the wild type populations of mitochondria.

One approach is to promote specific types of exercise training to promote mitochondrial biogenesis. However, as the geneticist that I am, the alternative strategy- to manipulate the mutated mt genome- is much more interesting. A brilliant idea, in my point of view, is to take advantage of the fact that some mutation types introduce restriction sites. That way, rather than trying to express a functional copy of the mutated gene, it may be more successful to express a specific restriction enzyme that will degrade the mutated genome altogether. This is limited, obviously, to those diseases in which a unique new site is introduced, or in those where the number of restriction sites is higher enough in the mutated population to have a significant effect.

Another clever idea is to methylate the mutated mt genome so that it is not expressed, allowing the wild type population to proliferate and overcome the mutated one with time. The use of mitochondrial targeted PNAs (peptide nucleic acids) were a potential method, but were shown not to cross the inner membrane of the mitochondria. However, the use of zinc-finger peptides with DNA sequence specific binding capacities have been used to successfully methylate differently the mutated mitochondrial genome. If these domains are linked to a nuclease, it may be possible to degrade the mutated genome as well.

These are all new strategies, only attempted in cell culture and their usefulness in in vivo models and ultimately in patients is yet to be determined. Nevertheless, I think these novel approaches just show that even standard problems can be approached in innovative ways.

References:

Kyriakouli, D., Boesch, P., Taylor, R., & Lightowlers, R. (2008). Progress and prospects: gene therapy for mitochondrial DNA disease Gene Therapy, 15 (14), 1017-1023 DOI: 10.1038/gt.2008.91

Alexeyev, M., Venediktova, N., Pastukh, V., Shokolenko, I., Bonilla, G., & Wilson, G. (2008). Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes Gene Therapy, 15 (7), 516-523 DOI: 10.1038/gt.2008.11

(the most recent paper I could find in this field, using the restriction enzyme approach in culture)

Yet another twist in the world of gene expression - transcription factories

2008-07-16T22:28:00.009+01:00

When I went to a conference on post translational modification and chromatin, one talk really grabbed me, ironically it was the one that didn’t relate to the topic of the conference. I was going to facebook people about it and then remembered this existed.

First of all I will ask you, did you know that transcription only happened at a few sites within the nucleus? In mouse cells from the animal there are between 100-300 of these but in cultured cells such as HeLa cells there are many more. Transcription factories also known as RNAPII foci are where most, if not all mRNA is produced. This amazed me and raises the obvious questions of why and how. The why may be obvious. It is a good idea to keep the nucleus as I see it ‘tidy’ but in more technical terms it is a way of keeps gene expression organised and regulating it (see below). The how this work I don’t think has been addressed! All I can say is watch this space.

Another interesting feature of mammalian gene expression that has come out of these studies is that most ‘active genes’ are not on all the time like classically thought. Most genes appear to show a pulse fashion of expression. Travelling to transcription factories and being expressed then returning to a dormant stage until they travel back to transcription foci for another round of expression and so on. However, some genes show the classical continuous expression pattern and are seen at these sites all the time. One example is Beta-globin. Genes like these are relatively rare compared to those showing the pulse type expression but both are continuously on in there cell type. If elements of the beta-globin promoter is deleted then not only is overall expreesion decreased but it shows the pulse type expression.

One of the most interesting things is that some genes will co-localize to the same transcription factory more often than you expect by pure chance. What is more interesting is these genes can be separated by several megabases! or even be on different chromosome. Using the 3C and 4C assay you can join different pieces of DNA together from different parts of the genome that have transcribed within the same transcription foci. These have shown along with RNA FISH the physical movement of genes to the same place to be transcribed. One of the two Beta-globin alleles within a cell was found to enter the same factory with some other genes at a rate of ~25% with a background rate expected to be less than 2%. These gene’s promoters were searched and a simple motif was found: CCACC. This is actually a binding site for a regulator of Haem biosynthesis and haemoglobhin production called Eklf. When Eklf is knocked out the association between these genes stops! This transcription factor must recruit to these genes to the transcription factories it is located at. I can see two mutually exclusive models here (tell me what you think or extra ones to consider). Either 1) something, perhaps Eklf recruits genes to these sites and physically moves them there or 2) genes with this motif move to all transcription foci and only stay to be transcribed at ones that have Eklf present. I would guess the second is more likely but I see no evidence for either model at this time. Another question I would like answered is whether other or maybe all transcription factors work in this way. I think it is unlikely but perhaps that is the case!

One more interesting thing related to this is the story in B cells of mice. It has been shown that the proto-oncogene Myc is expressed in the same foci as immunoglobulin heavy chain (IgH) a very high proportion of the time. The interesting part is that these two genes have been show in many blood cancers to be translocated to be on the same chromosome so perhaps there is a link between transcription factories and cancer. Well that is what the people doing the research on it want to think. After all related to cancer equal more funding as I told a friend of mine earlier. A new lab has been set up by the post-doc who led most of the described research to explore the cancer link.

And I now realise one very important thing, I should have been writing my result section now I have a deadline as well as lab work, S#£!!%$! Hell!

Cameron S. Osborne, Lyubomira Chakalova, Jennifer A. Mitchell, Alice Horton, Andrew L. Wood, Daniel J. Bolland, Anne E. Corcoran, Peter Fraser (2007). Myc Dynamically and Preferentially Relocates to a Transcription Factory Occupied by Igh PLoS Biology, 5 (8) DOI: 10.1371/journal.pbio.0050192

Cameron S Osborne, Lyubomira Chakalova, Karen E Brown, David Carter, Alice Horton, Emmanuel Debrand, Beatriz Goyenechea, Jennifer A Mitchell, Susana Lopes, Wolf Reik, Peter Fraser (2004). Active genes dynamically colocalize to shared sites of ongoing transcription Nature Genetics, 36 (10), 1065-1071 DOI: 10.1038/ng1423

Scientists have (too much) sense of humor- The IgNobels

2008-01-07T12:39:00.002Z

I know it is my time to post something, but because I spent my Christmas doing nothing, I’ll have to write a non-scientific post… and yet related with Science. Basically, about something I found out a few months ago and which shows how scientists can have a sense of humour- the IgNobel prizes. Unlike the Nobel prizes, the IgNobel prizes are not awarded to life changing research, but rather to research “that first makes you laugh… and then makes you think”. The ceremony normally happens in Autumn, more or less at the same time as the official Nobels. The mood is, however, much more relaxed and funny, perhaps a bit too much! Every year the ceremony has a topic, and this year’s topic was… chicken! Therefore, the evening was full of references to chickens including a mini opera with two chickens on stage and several talks on chicken, including one by a Google engineer with the title, and I quote, “Chicken, chicken, chicken, chicken, chicken, chicken”. Besides these very educative talks, there were also a few classics namely the 24/7 lectures, in which a scientist is invited to first give a technical description of his/her field of research in 24 seconds, and then a clear summary that anyone can understand in 7 words. Tricky, but very funny to watch!

Among much laughter and jokes, the highlight of the evening are the actual awards. But that doesn’t mean the end of the fun! The IgNobel prizes don’t have to fit into the Nobel categories, nor do they have to be the same every year. Basically, the prizes are designed to fit whatever unusual research is done every year. Some of the highlights of this year’s prizes. The IgNobel prize for medicine, for example, was given this year to DrWitcombe, for his study “ Sword swallowing and its side effects” (!). The IgNobel for Linguistics was awarded to Dr.Toro and his team from the University of Barcelona, for finding out that mice cannot distinguish between a person speaking Japanese backwards and a person speaking Dutch backwards… (!!). This year’s IgNobel prize for Peace is one of my favourites, awarded to the labs of the USA Air Force, for developing the so called “Gay bomb” which apparently makes enemy soldiers irresistible to each other (!!!). The IgNobel for Aviation, on the other hand, was given to the discovery that Viagra helps hamsters to recover from jet lag (!!!!). And the awarding of the prize is always accompanied by a little joke. The IgNobel for Chemistry this year was awarded to Dr. Yamamoto for the extraction of vanillin, the flavouring present in vanilla, from cow dung. When the prize was awarded, the Nobel (yes Nobel) laureates present on stage were invited to eat a glass of ice cream with the previously mentioned vanilla…

The IgNobel prizes, which have been awarded ever since 1991 by the Annals of Improbable Research in Harvard, are a source of laughter, especially when we think that they have been given to real researchers, for their authentic research. Some prizes are also awarded to inventions, such as an alarm clock that runs away (so that the person is forced to get out of bed to stop it) or to a scientist who patented the wheel… in 2001! Some prizes are also ironic. A man in Lithuania was awarded the IgNobel of peace due to the creation of a theme park called “The world of Estaline” while the Department of Education of the state of Kansas was awarded the IgNobel for the education of Science after having forbidden children from learning Darwin’s theories in school…

It would be wrong to think, however, that only unknown scientists participate in the ceremony. In fact, Nobel laureates are able to appreciate and participate in the fun. And not only by physically giving the IgNobel to the (un)lucky winners. A good example is the already classic contest “ Win a date with a Nobel laureate”, in which anyone from the audience can win a romantic date with a Nobel laureate. This year’s victim was Robert Laughlin, the winner of the Nobel Prize for Physics in 1998. Another curious example involves the already classic tradition of throwing paper planes on stage during the ceremony. Professor Roy Glaubert, “The Keeper of the Broom” was for years responsible for sweeping the planes from the stage. Well, in 2005 Professor Glauber couldn’t perform his normal task… as he was in Stocholm receiving the Nobel prize for Physics!

I advice all of you to have a look at their website when you have a bit of time. An afternoon when I didn’t have much to do I just laughed my head off reading the list of prizes since 1991. And you can actually watch the 2007 ceremony there. Have a look at http://www.improbable.com/

Operons breaking the rules

2007-10-28T21:31:00.002Z

For every rule in biology there is an exception. This causes problems in teaching. Do you give the rule and leave out the exception, effectively lying to the people you are teaching or do you give them the rule and exception and making rule mean much less? This is a real problem. Well I have just discovered eukaryotes have broken a golden. They have operons. I was always taught eukaryotes don’t have operons, only prokaryotes do.

For anyone needing to be reminded what an operon is it is several genes all on the same mRNA molecule. One name for this is polycistronic mRNA (cistron is an old name for what we know as a protein coding gene). Several genes have a single promoter and a terminator of transcription controlling them all. On this polycistronic mRNA are several Shine-Dalgarno sequences (bacterial ribosome binding sites) so a ribosome binds in front of each gene and translates it until it reaches a stop codon and falls off. The classic example is the lac operon. It makes 3 proteins used by E. coli to break down the sugar lactose. The reason why you would want a single promoter to control the expression of several proteins is because if they have related function like in the lac operon you want them all to be turned on at the same time and at the same level of expression therefore making the same number of proteins.

Classically eukaryote have monocistronic mRNA (one gene per mRNA) but their is more to this story. Strange transcripts have been found in animals from the simple nematodes to complex mammals such as us. One type of mRNA that has been called operons by some people in C. elegans uses something called trans-splicing. A gene is expressed with one promoter but contains 2 protein coding regions. But this does not mature into a polycistronic mRNA but during RNA processing the introns are removed and the two coding regions are separated. But how does the downstream RNA resist degradation by RNases? It has a SL-exon from another transcript trans-spliced onto it. This way one ‘gene’ makes 2 mRNA (see diagram). This is not a conventional operon like those found in bacteria. This is not a minor thing in the nematodes genome – 15% of all genes in C. elegans are found in operons of this type. We still have monocistronic mRNA here so eukaryotes are behaving fairly well so far.

But in Drosophila and higher organisms dicistronic mRNAs have been found. No trans-splicing involved what so ever. This is definitely breaking one of the golden rules in biology. Here a promoter causes the transcription of 2 genes onto the same transcript. Each one of these gets translated. It is unclear at the moment if both genes have there own ribosome intonation site are if the ribosome that translates the first gene also translates the second one by not falling off at the stop codon of the first gene. One example (that is conserved between man and mouse) is the dicistronic mRNA coding the growth and differentiation factor 1 (GDF-1) and a membrane protein of unknown function (UOG-1). Whether they are co-transcribed because they have related function is obviously unknown.

Living creates are strange. We think we understand a bit about them, create rules that we think life follows so we can understand life better. But life doesn’t always follow the rules we think it does. So the best we can do is make rules or models up and test them as this is how science works. I just hate it when something I thought I new turned out to be wrong.

T. Blumenthal (2004). Operons in eukaryotes Briefings in Functional Genomics and Proteomics, 3 (3), 199-211 DOI: 10.1093/bfgp/3.3.199

The Fanconi anaemia DNA repair pathway

2007-10-08T01:23:00.001+01:00

Fanconi anaemia (FA) is an autosomal recessive chromosomal instability, characterised by congenital abnormalities, defective haemopoiesis, a high risk of leukaemia and development of solid tumours. FA patients have a 10% incidence of leukaemia and are 50 times more likely to develop solid tumours (particularly hepatic, oesophageal, oropharyngeal and vulval) by early adulthood. The disease consists of 13 complementation groups, each connected to mutations in 13 corresponding FANC genes (A, B, C, D1, D2, E, F, G, I, J, L, M, and N) which make up the FA pathway. Two thirds of FA cases are caused by inherited mutations in FANCA.

Interstrand crosslink damage

FA cells are characterised by hypersensitivity to damage by DNA Interstrand CrossLinking (ICL) agents such as mitomycin C and diepoxybutane. Cells fail to repair ICL damage, resulting in polysomies, radial chromosomal structures and unrepaired breaks (Figure 1). ICL agents tether two DNA strands together, creating a physical barrier to replication and transcription, which causes arrest of DNA replication. In normal cells, several mechanisms come in place to minimize damage and maintain the replication event. This necessitates removal of the physical barrier, repair of the gap and re-establishment of the replication fork. This complex repair mechanism involves the Fanconi anaemia proteins, translesion synthesis (TLS) polymerases, and the homologous recombination (HR) machinery. The FA pathway has also been implicated in spontaneous DNA damage repair, despite early speculation that the pathway was specific to ICL damage. In addition, there is some uncertainty as to whether the FA pathway also functions in intra-strand crosslink repair.

The FA pathway

The FA proteins are part of a complex DNA repair pathway, which is not yet fully understood. They are commonly arranged in three distinct groups according to their function. The first group is the FA core complex, which consists of FANC A, B, C, E, F, G, L and M. This complex stabilises stalled replication forks, processes the ICL site and activates a second group, which consists of FANCD2 and FANCI. Finally, group 3 consists of the remaining FANC proteins, BRCA2 (which is the same gene as FANCD1), FANCJ and FANCN, which have accessory or uncertain function in the pathway (Figure 2). The most recent model of this pathway is described in Wang (2007), Kennedy & D’Andrea (2005) and Niedernhofer (2007), and a detailed description of the model follows.

An ICL physically blocks replication by preventing separation of the strands by the advancing DNA helicase, which causes the enzyme to arrest. The replication fork arrest activates the ATR kinase, which subsequently phosphorylates FANCD2. A double strand break (DSB) upstream of the ICL is carried out by XPF-ERCC1, MUS81-EME1 or MUS81-EME2. Simultaneously, the monoubiquitin ligase FANCL, part of the FA core complex, monoubiquitinates FANCD2 and FANCI. This stage is regulated by the deubiquitinating enzyme USP1, and the monoubiquitin acts as a chromatin localisation signal. UPS1 is itself regulated by cell cycle controls, and self-cleaves in response to DNA damage. The integrity of the whole of the core complex is essential for this stage, as proven by the lack of chromatin localisation in FA core complex mutants. The downstream function of FANCI-Ub is uncertain, but FANCD2-Ub is arguably the most important component of this pathway, acting as a “fire captain” to recruit the proteins needed for the rest of the pathway (Figure 3).

As group 2 is activated, the FA core complex is also carrying out DNA processing in preparation for the next step in the pathway. FANCM acts as a DNA translocase. It anchors the rest of the FA core complex onto DNA, which stabilises the broken replication fork and facilitates removal of the ICL. A nucleotide excision repair (NER) endonuclease “unhooks” the ICL (which remains attached to the one strand), and translesion synthesis (TLS) polymerases recruited by the FA core complex fill in the gap. The TLS polymerases’ ability to pass through the damaged site has the trade-off of being error-prone, introducing random mutations in the repair site. The ICL adduct is then completely removed by a NER exonuclease, and the result is a replication fork broken by a DSB, with the ICL removed and the gap filled in (Figure 4). The next step is to repair the DSB, which is carried out by homologous recombination (HR) proteins.

HR-mediated replication fork re-establishment

FANCD2-Ub forms a complex with BRCA2 and chromatin. All the FANC proteins, along with BRCA1, NBS1, PCNA, RAD51 and other related proteins are recruited by the FANCD2-Ub/BRCA2-chromatin complex into nuclear foci at the damage site. BRCA1 forms a complex with BRCA2 and is essential for the formation of nuclear foci, even though its exact function is unknown. FANCJ, also identified as the helicase BRIP1 (BRCA1-interacting protein), depends on BRCA1 to translocate to the DNA damage site and unwind the replication fork in order to promote HR. The MRN complex, which consists of MRE11, RAD50 and NBS1 and has 3’-5’ exonuclease activity, processes either side of the DSB to prepare it for HR.

DSB repair can occur in two ways. Firstly by single-strand annealing, an error-prone non-homologous end joining (NHEJ) pathway, where the two strands are trimmed until homologous repetitive sequences are reached, and the strands are then annealed at that region of homology. This is mutagenic since the non-homologous intermediate region is deleted, but this is preferable to the arrest of replication. Secondly by strand invasion HR, where RAD51 is loaded onto chromatin by BRCA2 to form a nucleoprotein filament, within which Sister Chromatid Exchange (SCE) occurs and a Holliday junction forms between the strand being repaired and a homologous strand (Figure 5). Holliday junctions are intersections of four strands of DNA, which appear most commonly during meiosis when “crossing-over” occurs, and are resolved by endonucleases such as MUS81-EME1. FA cells show high levels of SCE in FANCC mutants, while other FANC mutant cells have normal SCE levels, suggesting a regulatory role for FANCC over SCE formation and a generally significant role in HR. HR thus re-establishes the replication fork and at the end of the S-phase, USP1 deubiquitinates FANCD2 to turn the pathway off.

In summary, ICL damage causes replication arrest, one of the strands is separated by a DSB, the FA core complex stabilises the broken fork and TLS repairs the ICL. At the same time, the structural integrity of the FA core complex is essential for the monoubiquitination of the “fire captain” FANCD2, which forms a complex with BRCA2 and chromatin and recruits HR and accessory proteins into nuclear foci. Finally, the MRN complex processes the ends of the DSB for HR to repair it and allow replication to restart.

There has been increased interest in the FA pathway in the past decade or so due to the discovery of its connection with cancer and complex relationship with the HR pathway. The pathway model is still in its early stages but already there are important questions raised about cell cycle regulation and its connection with DNA repair.

References

Kennedy, R.D. and D'Andrea, A.D. (2005) The Fanconi Anemia/BRCA pathway: new faces in the crowd. Genes Dev. 19: 2925-2940.

Niedernhofer,L.J., Lalai,A.S., and Hoeijmakers,J.H.J. (2005) Fanconi Anemia (Cross)linked to DNA Repair. Cell 123: 1191-1198

Patel,K.J. and Joenje,H. (2007) Fanconi anemia and DNA replication repair. DNA Repair 6: 885-890

Tischkowitz,M. and Dokal,I. (2004) Fanconi anaemia and leukaemia - clinical and molecular aspects. British Journal of Haematology 126: 176-191

Wang,W. (2007) Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins. Nat Rev Genet 8: 735-748

Bioluminescence

2007-09-28T17:42:00.002+01:00

In many biological studies it is essential to be able to translate into an observable and measurable signal the specific process under analysis. This is normally possible through the use of assay systems based on radioactivity, photon absorption or photon emission. Radioactivity and photon emission are less and less used nowadays, the first due to the restrictions and cares associated with the use of such hazardous materials, the second due to its low sensitivity.

Photon emission can occur by two different processes- either fluorescence or chemiluminescence. Both processes involve the emission of photons associated with transitions between energy states, but the way the excited state is generated varies. In fluorescence, the absorption of light (i.e. of photons) produces the excited state, while in chemiluminescence this is due to exothermic chemical reactions (see diagram- fluorescence is on the right, luminescence on the left).

Both of these processes have their advantages and disadvantages. Fluorescence (of which the most famous example is probably GFP) normally shows a much stronger signal, as the source of the excited signal are photons which can be introduced in the sample at very high rate. However, this jamming of photons tends to create a high background signal which influences the sensitivity of the assay. Chemiluminescence does create lower intensity signals, but as no photons need to be provided there is hardly any background signal. Whichever of the two systems is used will depend on the machinery required in each assay. If the efficiency of light collection is limited, then fluorescence is the best option, this being the reason why until recently fluorescence was the main assay system used. The development of more sensitive machinery, however, has made the background noise the most important problem to solve, therefore justifying the increased interest in chemiluminescence systems.

Bioluminescence is a form of chemiluminescence that naturally occurs in certain living organisms. The enzymes that catalyse this reaction are called luciferases and their substrates luciferins. Note, however, that these are very general terms as bioluminescence seems to have evolved several times independently. This is why the several existing luciferases have such different molecular structures. In fact, luciferases have been cloned from several different types of organisms, namely jellyfish (Aequorea), sea copepod (Gaussia princeps), corals (Tenilla), click beetle (Pyrophorus plagiophthalamus) and several bacterial species. The most successfully luciferase, however, is the widely used firefly luciferase, from the firefly Photinus pyralis. It is commonly used in its humanized variant (codons optimized for mammalian expression), requiring ATP and magnesium in the oxidation of its substrate luciferin, and yielding a yellow-green light with a maximum luminescence of 560 nm.

Firefly luciferase (FLuc) is normally used as a reporter gene, which may mean very different systems of reporting. The most obvious is, of course, the insertion of the luciferase gene in the same plasmid and under the same regulatory promoter as the gene of interest. In my current department (Gene Therapy department, NHLI), for example, firefly luciferase is the main reporter gene used in CFTR transfections. However, FLuc can report more than the expression levels of transfected genes. As it depends on ATP for its activity, it is ideal in assays of ATP concentration. It can also, for example, be an indicator of how well G-protein coupled receptors (GPCR) work. This is possible by using a cAMP response element (CRE) upstream of the luciferase gene. GPCR activation causes an increase in intracellular cAMP concentration. This increase leads to Protein Kinase A activation, which in turn phosphorylates CRE binding protein. The CRE binding protein will bind to the CRE upstream of the luciferase gene, leading to increased transcription. Overall, an increase in CPCR activity leads to increased luminescence signal. This type of report system, for example, is useful in the study of GPCR agonists. The complexity of the system, however, makes it prone to interference, which can lead to false positive results. To prevent this, a dual-reporter system can be used involving a second luciferase, Renilla Luciferase (RLuc), which is an internal control to detect aberrant data. A dual luciferase system can also be used to detect two different processes or expressed genes simultaneously (as FLuc and RLuc have different spectral maximuns, namely 560 and 480 nm).

As a reporter of transfected gene expression, FLuc shows a few limitations, namely the fact that it is an intracellular protein. This means that measuring of transfected gene expression requires, in in vitro cell culture the lysis of the transfected cells, and in in vivo studies the killing of the animal in order to access the transfected tissue. In patients under clinical trial, luciferase assays imply invasive obtainment of tissues (I am still trying to find out how this is done exactly). This is why the development of new luciferase, namely a secreted luciferase is important. Until now, around 4 types of secreted luciferases have been found, but the only one commercially available for cell supernatant assays is that produced by the copepod Gaussia princeps (see figure). Gaussia luciferase (GLuc) is naturally secreted from cells and used by this animal as a defence mechanism. As Gaussia princeps lives at a depth of between 350 and 1,000 m, the sudden production of light is a good distractive mechanism against dark-adapted predators. As a secreted luciferase, Gaussia is reported to give very good signals when medium supernatants are assayed. The important question right now (at least for me) seems to be if it can be assayed in something more… well, you will have to wait for my year-away talk next year for more information!

References

Fan F., Wood K. (2007). Bioluminescence Assays for High-Throughput screening, Assay and Drug Development Technologies, 5. pp 127-136

Markova S., Golz S., Frank L., Kalthof B., Vysotski E. (2004). Cloning and Expression of cDNA for a luciferase from the marine copepod Metridia longa, The Journal of Biological Chemistry, 5. pp 3212-3217

Roda A., Pasini P., Mirasoli M., Michelini E., Guardigli M. (2004). Biotechnological applications of bioluminescence and chemiluminescence, Trends in Biotechnology, 22. pp 295-303

Serganova I., Moroz E., Moroz M., Pillarsetty N., Blasberg R. (2006). Non-invasive molecular imaging and reporter genes, Central European Jornal of Biology, 1. pp 88-123

Tannous B., Kim D., Fernandez J., Weissleder R., Breakfield X. (2004). Codon-optimized Gaussia Luciferase cDNA for Mammalian Gene expression in culture and in vivo, Molecular Therapy, 11. pp 435-443

Wiles S., Ferguson K., Stefanidou M., Young D., Robertson B. (2005). Alternative Luciferase for monitoring bacterial cells under adverse conditions, Applied and Environmental Microbiology, 71. pp 3427-3432

Reaction or catalyst? Which started life?

2007-09-23T16:36:00.001+01:00

If only the question was as simple as ‘which came first the chicken or the egg’ (answer: the egg). The question here is which came first - DNA or proteins. as we all know DNA stores the code to make proteins but proteins are needed to read this code and make more DNA. Obviously you can’t have one without the other. So the RNA world hypothesis was born. RNA can do both store information and catalyse reactions, perhaps even catalyse its own replication. I believe this is a beautiful theory. However beauty is only skin deep. There are real problems with this explanation of the origins of life apart from the obvious difficulties in testing it, which is true for any explanation of the origin of life. RNA is famous for being chemically unstable. It can break itself down quite easily (especially in alkaline conditions). Also where did the RNA come from? I don’t mean did it come from outer space (because radiation levels in space are so high RNA or cells could not have survived). I mean how the molecules were created on earth. Like it or not RNA is a complex molecule and no experiment trying to recreate the primordial soup has ever found RNA nucleotides. Or any really complex molecules, only the simplest of amino acids have been made.

This has lead to another explanation for the origins of life. Perhaps thinking of life as a bunch of replicators has blinded use to it. Living organisms can also be thought of as chemical factories. Genes and their products are simply there to control these reactions. Some believe it was chemical reactions that came before the proteins or RNA that catalyse the reaction. This is called the metabolism first theory. People who follow this theory have described what is needed for a chemical system to be the beginnings of life. 1) A boundary or form of membrane is needed to keep life and non-life away from each other. The 2^nd law of thermodynamics states the universe my decrease in order but life increases in order so inside the boundary entropy decreases but this generates heat that causes an increase in entropy outside it. 2) An energy source must have existed. Perhaps some sort of redox reaction to power the chemical reactions in the metabolism first model. Radiation may have been used. 3) The energy source must be linked to the other chemical reaction. For me this is difficult to see how this could happen without proteins there to help things along but perhaps if I knew more chemistry it would be clearer. We use ATP as our energy currency but how could redox reactions or radiation be linked to these ancient chemical reactions? 4) The chemical reactions must be able to change and evolve. If a cycle of reactions was created where A became B and B became C and C became D and D became A again we have something to expand upon. If we had a carbon input such as E we could take compounds off the cycle and expand it (see diagram). These reactions could be powered by a redox reaction of X to Y. Eventually complex molecules could be created 5) One final requirement for these reactions to have been the origins of life is need and that is to be able to replicate. It is hard to imagine how this is possible before a lipid membrane existed for it to divide into two. If possible this would have allowed for Darwinian evolution through the competition for recourses.

The RNA-first approach has some support for it. Minerals have been found that contain boron in ‘containers’ or ‘bowls’ in Death Valley which could help create the ribose sugar in RNA. If such pores with boron existed billions of years ago RNA could have been created. Laboratory experiments have shown some randomly generated RNA molecules can catalyse the addition of an ATP molecule to itself. This is tested by using an ATP molecule with a sulphur atom not an oxygen atom and using a column that pulls out the sulphur containing RNA molecules only. RNA can carryout many reactions such as making and breaking DNA and RNA links and amide bonds and even make links with sugars. So their is diversity in RNA’s ability to catalyse reactions but its limit appears to be speed. It is possible one reason proteins took over from RNA as life’s catalyst because proteins are faster as well as because protein is more stable. Metabolism first has the great weakness of not having much lab experiments to support it but only computer simulations.

I believe these two theories could work together. Perhaps it is only because I find the rna world aesthetically pleasing i want to save it but these reactions could eventually become so complex they make rna. Over time these built up and started to take control and then made proteins to do much of there job when dna then took over as the info store and rna was simply the messenger and helps out in only a few reactions today. This nicely explains why the formation of the peptide bond in the ribosome is still done by rna. We could go even further into theory and suggest there was a polymer before RNA that could act as catalyse and self replicator. PNA has been suggested. Instead of having the sugar-phosphate backbone like RNA and DNA it has peptides attached to bases forming a backbone. Sadly such a molecule does not exist in our cells today or leave fossils in the ground for us to examine so we cannot test if PNA was really the first molecules that lead to life. If PNA did exist it all became RNA and then DNA.

At the moment we have many ideas about what may have been involved with the start of life on earth but it is difficult to prove anything. What we can do is explore the potentials of these molecules or chemical systems. After all theories about the origins of life cannot be tested directly but the do make predictions and by testing these predictions we can hopefully learn a lot.

References

Albert et al. (2002) Molecular biology of the cell. 4th edition.

Shapiro (2007) A simpler origin for life. Scientific American 296: 24-31.

The Lives Of Stars

2007-08-23T09:39:00.002+01:00

I am currently reading Carl Sagan’s classic popular science book Cosmos, and felt inspired to write about a chapter I particularly enjoyed. Chapter IX, ‘The Lives of Stars’, takes us on a journey through space and time, looking at the Sun as well as some of its distant cousin stars, all of which behave in strange and wonderful ways.

Probably the most surprising thing about stars is that it all boils down to simple chemistry. Four hydrogen nuclei will combine, under very high gravitational pressure and temperature, to form a helium nucleus and emit light as a gamma ray photon. This is the almost disappointingly simple answer to a question that has tormented humankind since we first realized that there was actually a huge hot bright disc up there, a question that has led millions to invent all sorts of farfetched hypotheses and religions to explain it away. It really gets interesting when you travel backwards and forwards in time to see where it all comes from and where it will all end up. Let’s take it from the top, then.

The big bang was an explosion and rapid expansion of the fabric of spacetime, which consisted of some matter in the form of protons, neutrons and electrons, as well as a huge amount of nothing. The rapid cooling that followed due to this expansion caused these elementary particles to form hydrogen and helium gas clouds. The explosion itself was uneven, so clouds began to form clusters of various sizes, collapsing into themselves under the force of gravity. These massive clouds of gas are the birthplaces of millions of stars, eventually forming the galaxies we see and live in today, such as the Andromeda galaxy pictured on the right. Stars consist of that same gas having collapsed into itself at various points in space.

Stars are essentially massive engines that burn hydrogen. When temperatures in the core of a star are high enough (over 10 million degrees), the collapse stops as the outer layer is held back by the combustion taking place in the core. The photons emitted by the reaction take a million years to reach the outer layer. The sun has been a simultaneously exploding and collapsing hydrogen bomb for about 5 billion years, and it will continue to behave that way for about as long. Eventually, all engines run out of fuel, and so do all stars, but that does not always mean their death.

As the hydrogen runs out, the reaction will begin to cool and the star will expand outwards, engulfing the inner solar system. However, it will soon begin collapsing again under its own gravitational force, this time until temperatures get high enough to burn helium. Sagan compares this beautifully to a Phoenix rising out of its ashes, except this is not just an ancient myth but a real event that is constantly happening throughout the universe. The remaining hydrogen left over in the expanded region of the star will burn while helium burns at the core at higher temperatures. This is a red giant, with a hot carbon and oxygen-producing helium reactor in its core and a planet-engulfing hydrogen-burning outer region.

When the helium runs out, it does mean the end for most stars. A new expansion will take place, and the star will shoot out concentric shells of gas that will form the planetary nebula (pictured below). At this stage, the Sun would engulf Pluto. A few more massive stars can recollapse and burn carbon and oxygen for a while, but this is not very common. After the sun expands for the last time, the solar system will become a blue and red-fluorescent dead world. Billions of years later, the exposed core will become a white dwarf, and eventually a cold, dead black dwarf.

A planetary nebula

There are so many different aspects to this story that rival any storyteller’s wildest imagination. The poetic elegance of the lives of stars masks their terrible and devastating effect on the observing civilizations of their orbiting planets, but the universe is of course entirely indifferent and apathetic. I strongly recommend Cosmos to anyone who wants to catch a glimpse of the amazing things astronomy has discovered, especially since the invention of the radio telescope which can take us right to the edge of the universe.

Someone else in the universe already posted this but in a galaxy far far away

2007-08-02T20:55:00.001+01:00

I would like to start off by saying I know nothing about physics apart from the excess of Sci-Fi I watch when I was younger and the random conversations I have with my physics friend Bob. Nevertheless I do like it despite it never making any sense. As a biologist there is a reason behind just about everything (ie evolution has shaped it all). It unsettles me when I don’t know why something is the way it is (so most of immunology then). Before reading the rest of this you need to assume a couple of things about the universe for what I am going to say to make sense, they are apparently correct but what do I know. Firstly space is infinite or extremely large (according to Prof Max Tegmark evidence for a small universe or donut shaped one is weak and he believes the universe is infinite) and secondly that matter is evenly spread out throughout space (not just clumped around us).

For the first part we will say our universe is the part of space we can see (the outer edges are determined by the age of the universe and how fast light it, as it gets older we can see more because more light has had time to travel to us). This is also called our hubble. Level I multiverse or parallel universes are simply hubbles out there that are like ours (and many more not like ours). If space is infinite all arrangements of matter that are possible exist! I like to think star wars and LOTRs obey the laws of physics so they could exist. Everything in level I has the same laws of physics as our universe because it is only an extension of ours. Out there, there are more hubbles just like ours but very far away. One estimate says one just like ours in every way could be 10 to 10¹¹⁸ metres away! Closer will probably be some hubbles that are very similar to ours but slightly different. Their will be an infinite number of all hubbles because space is infinite (from my understanding). I think these estimates are far to low and don’t take into account some things but it gives you an idea that even a low estimate is amazingly far away.

That was the easy stuff. Level II multiverse (or parallel universes) is less accepted than level I but still apparently explains a lot of things in physics. It helps biologists in their fight against pro intelligent design arguments. It is where many multiverses (same as in level I) exist but each multiverse is separate and has different physical constants or number of dimensions. This explains why our universe just so happens to be able to support life – it isn’t custom built to support life but is just one possibility of how the universe works. We have 3 spatial dimensions and one time dimension but if we had more time dimensions events would be completely unpredictable and if we had more spatial dimensions atoms would be unstable. If the mass of an proton was slightly larger it would decay to fast for molecules to be made so obviously nothing for evolution to act on and life to form. Each multiverse is still infinite within a sea of inflating infinite empty space (don’t ask me how you can have infinite space inside infinite space, because I just don’t understand). We can never travel between these multiverses even at the speed of light because they are moving away from each other faster! That sounds like science-fiction to me.

Level III is the one that interested me the most as a child (yes I have always been this way, Egon Spengler was my favourite Ghostbuster…need I say more). Every choice means you have to choose a path to follow. But in level III all outcomes exist but the other choices exist in another universe and not in space as we know it but ‘elsewhere’. However the choices I am talking about take place on the quantum scale! This goes back to Schrödinger’s cat (if you don’t know what it is, it would be best to look it up before continuing). From what I understand all it means is all states or positions exist until someone observes what state it is in. So something is both on and off or dead and alive until someone checks. This is called a superposition. An alternative theory is that both do exist but new universes are created to accommodate the other possibilities. It is like rolling a die that is only governed by the rules of the quantum world and not the overall rules of the universe. Because it is only on the quantum scale the outcome is completely random (from what I understand). According to level III multiverse it will not land on a 1 or a 2 or a 3… but will land on all six values at once. How; each one exists in a different universe, easy! And we thought biology was screwed up, at least it usually makes some sense. The outcome of level III is the same as for level I and II, more universes most slightly different from ours. The difference is how it is made.

Level IV is the one I feel least confidence about explaining. It is there to explain why our universe works under a set of specific mathematics and not controlled by other models. Universes that work using different mathematical models may exist outside our spacetime and work in completely different ways. They work using different laws of physics even more different to ours than by multiverses in level II. Level I, II and III were created by the same big bang. Level IV exists outside spacetime and will have had there own starting events but more level I and II (and III) could have been created by other big bangs as well. In level I and III you will have a Doppelgänger but in II and IV space will be so radically different you will not. Well I think you might if a parallel universe created in level II or IV is very similar and works off the same rules as ours.

All of these theories do make some predictions so they no longer lay in the realms of metaphysics but of real science. So in the next few years we may see some of these confirmed or rejected as fact. Whether they exist will have little impact on our lives. Will it comfort you to know someone out there in the infiniteness of space is in exactly the same situation as you or better of, or in a worse situation! I don’t think it will keep me awake at night. Nothing we can do will have an affect on them. I only discuss this because I find it interesting. But I find most things about our world its place in the universe interesting. We didn’t evolve to understand things like this but we did evolve to ask question about our environment.

For more information see Scientific American special report on Parallel Universe by Max Tegmark or his website:

http://space.mit.edu/home/tegmark/multiverse.html

this has more references.

Please ask me questions, no promises I can start to answer but I have thought about the topic for a while and would like to hear your thoughts and see of you have the same questions as me.

Colds and the cold

2007-07-03T22:37:00.001+01:00

My mum always told me if I didn’t wear socks and shoes and had cold feet I would get a cold! Some evidence suggests that this old wives tale may be true. Adults have about 3-5 cold infections every year (unless you are a student and it is considerably more than this) and the symptoms are so well know and easily recognised that people self diagnose and there are no special tests doctors perform to say you have a cold, they go off these same old symptoms as the public. Saying what is a cold or flu is not easy but flu is much worse than most common colds, but a bad cold could be easily confused with a mild case of flu. Someone’s reaction to an upper respiratory infection depends more on the person (such as there stress level) than the virus that infects them. There are more than 200 serotypes of viruses (viruses with different antigens) that cause the common cold. Once infected by one of them you have antibodies to protect you from it causing another cold but there are plenty more waiting in the wings to hit you the next time exams are approaching. The rhinovirus is the most common cause of the common cold. The symptoms we experience when we get a cold are not the virus damaging us but our body reacting to the virus. Histamine triggers nerves in the noise to fire and tell the brain to make you sneeze and a sore throat is from a small peptide (bradykinin) signalling to nerves to tell you something is wrong. The colour of you lovely mucus changes from clear to yellow to green as more leukocytes (such as neutrophils) are recruited to fight the infection (Eccles, 2005). The majority of infected people are believed to not have any symptoms or only very mild ones. These are called cub-clinical infections and they can spread to others who will develop a full blown cold (Eccles, 2002).

The question is does the cooling of the body’s surfaces increase the chances of you getting a cold. The name cold suggests a link to me. The usual answer to why we get more colds in winter and cold weather is because we all crowd around in close spaces indoors and breath the same air. However I disagree with this. I do not change my habits during the winter and summer, I live in the same house with the same people who stay in the same no matter what the weather and go to school/uni and sit in the same classes with the same amount of people no matter what the weather. So how can you explain why I get more colds in the winter? I guess my mum is right. Because my feet aren’t warm enough…well there is now evidence to support my mum’s theory. Eccles and Johnson (2005) did find that when people put there feet into cold water and were exposed to a virus they were more likely to develop cold symptoms than those who did not have cold feet. But what are the mechanisms that mean cooling of the body’s extremities to let the virus get the upper hand? Vasoconstriction happens when you get cold, therefore less blood flows to the upper airways. This restricts the supply of heat and nutrients to leukocytes that eliminate viruses in a non-specific manor and reduces phagocytosis. Virus replication may also be increased, rhinoviruses replicate better at 33^oC than 37^oC. This could all cause a sub-clinical infection to become a full blow cold! Runny noise and all. Vasoconstriction helping cause a cold may also explain why some people get more colds than others. It has been shown that people who get more colds a year have a greater vasoconstriction response than those who only get a couple of colds per year (Eccles, 2002).

There are a lot of questions about colds and how they cause disease and how we catch them. But these diseases do not cause a lot of deaths, only reduce work output and the symptoms can be treated directly. I am just interested in what happens when I am ill.

Guest article: Economics...

2007-06-25T23:36:00.000+01:00

As we struggle through the last exams of term, I thought that leaving the 'biology bubble' for a while could be a good idea... so I have invited Miguel Faria e Castro, a promising economics student at the faculty of Economics, Universidade Nova de Lisboa (New University of Lisbon) to bring a bit of fresh air to the blog...

When the invitation to write an article for this blog first came, I felt that anything written by an Economics student would feel a bit out of place. It happens, however (and most fortunately, in my own personal opinion) that Economic Science is not restricted to, as some people think, heavy statistical paperwork and impossible mathematical models with two thousand variables. A wide, varied field, it incorporates a lot of knowledge from other scientific subjects, namely psychology, sociology, physics – even biology!

It is not my intention to bore you with sophisticated technical terms or theories which are able to explain whether you opt for a cup of tea or a chocolate bar under the right conditions. That is why I have decided, in this brief article, to give you a first glance at what really is economic science.

Curiously, the term “Economics” comes from a very picturesque Greek expression: Oikosnomos, or, in plain English, “how to take care of your house”. Coined somewhere in the ideological turmoil of the 18^th century, it came as a brief, yet clear, reference to the founding pillar of the entire science, the concept of scarcity: How can I satisfy my endless needs with the limited resources that are available to me? This is commonly known as the fundamental problem of economics, this is the problem that more or less 6 billion people face every day, at every moment.

Everything gets more complicated when we notice that not only people have different interests and, therefore, different needs, but also different qualities and quantities of resources available to themselves. This was what Adam Smith, a late 18^th century Scottish philosopher who is commonly recognised as the first modern economist, attempted to analyse when working in his great (quite literally, three volumes of 1,000 pages each) work: The Wealth of Nations. Basically, the entire work was a defence of two fundamental economic theories, and I am almost sure you have already heard of at least one of them somewhere: The Invisible Hand, and The Division of Labour. While the former states that each individual, while attempting to prosecute his own self-interest will ultimately contribute to the society’s greater good (or, “after all, being greedy is not that bad”), the latter is based on the principle that certain countries appear to have a natural advantage on the performance of certain tasks over others, and that the entire world would gain if each country focused on what it’s good at. Another founding father of economics, the British David Ricardo would further develop this theory, by proving, with a simple mathematical example that it would be better for both countries if Portugal only produced wine and England textiles, rather than having them producing both resources. Look in wikipedia for “Comparative Advantage” and you’ll learn a neat trick with which to impress your friends (this last part sounds quite nerd, but we were actually told it by a professor).

As with every science, new theories on how to better satisfy people’s needs appeared. The so-called Classics, which I have just mentioned, tended to follow a very liberal orientation. Throughout the 19^th and 20^th centuries, economics, initially seen as a weird mixture of politics, law, philosophy and mathematics, increasingly became a matter of interest for politicians – the rise of Marxism as a political ideology only happened because Karl Marx had launched the theoretical basis for his own economic principles (usually called Marxian economics, as to avoid any embarrassing confusions). Until the 20’s, economic science focused almost exclusively on the behaviour of those who produced every day’s goods and those who bought it – Consumer and Producer Theory, the basis for what is today known as Microeconomics (it studied the individual behaviour of economic agents). In 1929, however, with the Great Depression, the Western “Civilised” World was hit, for the first time in History, with widespread inflation and unemployment. Two occurrences which, as the economists of that time conceded could barely be explained by the tools that they were using.

This is where another great mind enters. An Englishman named John Maynard Keynes – a brilliant thinker (the philosopher Bertrand Russell once said that Keynes was the most intelligent man he had ever met) and fierce investor who would publish his thoughts on what had happened during the Great Depression in the United States of America and the rest of the world. His work led to the creation of an entirely new field – macroeconomics, or the study of the aggregate behaviour of all economic agents (and now the State plays a special role…) when faced with certain circumstances and conditions. Keynes was the first to identify the fact that a phenomenon such as inflation (which is, by the way, the continuous and generalised rise in the price level or, in English, the occurrence thanks to which things are much more expensive today than they were when your parents were eighteen) could never be explained by studying what an individual consumer does or a single firm does not do. Only by studying the economy as a whole, can we grasp the real magnitude of such an event. A recurring joke about economists tells you that one of the advantages of being one is that, when you are in the unemployment line, you will at least understand why you are there. Thank Mr Keynes for enlightening you on that. But enough of history.

A common misconception is that economics only deals with money. Money is, as surprising as it may seem, a very small part of this grand show. Do you eat money? Do you drink money? Do you drive money? The answer is no – money is merely the means for you to get whatever you need. This is where we get at another important concept – the Classical Dichotomy, which basically states that real, not nominal, variable are important. What is the difference? Imagine you are a happy German with a monthly income of €5,000. Then, there’s that Polish guy who makes only €5 a day. It happens, however, that an apple in Germany costs €1,000 , while the Poles can eat them for €0,5 each. Which means that, nominally, you earn €5,000. But your real wage is 5 apples, while the Pole earns 20 apples a month. His real income is four times yours, even though you nominally earn a thousand times more. Interesting, eh?

Guest article: Decision-making

2007-06-24T00:33:00.002+01:00

Our guest writer this week (well, maybe this will become weekly) is Christina Ieronymides, student and aspiring thylacine hunter. Hailing from Cyprus, she has just completed her BSc in Zoology at Imperial College, and will be doing her MSc at the Institute of Zoology (London Zoo) starting this autumn. Without further ado...

As a guest writer on this blog I feel the need to point out that my grasp of genetics does not extend much further than the basics, and molecular biology, in fact, is one of the subjects I tend to steer clear of. This does not mean I dislike the field; it merely indicates that I may find myself in need of a molecular geneticist friend at some point in my career.

In this article I’d like to introduce a couple of my pet subjects. The nature-nurture debate is well known to most people involved in the biological sciences, and despite all the controversy, “a bit of both” is the answer. This gives rise to the phenotypic gambit: that there is a genetic element in animal behaviour, and as such what you see is behaviour that is adaptive and under selection. This is the basis of behavioural ecology, a field to which I was first introduced by Richard Dawkins in his classic work The Selfish Gene.

In his book, Dawkins uses game theory to look at the evolutionary outcome of social interactions between individuals adopting different behaviours, and more specifically to explain how cooperation can evolve amongst social conflicts. He explains how cooperation results in the best overall outcome for the individuals involved, when a win-win outcome is possible. Cooperation is, however, a fragile state, dependent on small population size, repeated interactions over time and communication. These conditions are vulnerable to outside influences, such as immigration, population growth and change in the social circumstances.

Externalities imposed on such a system of reciprocal altruism inevitably lead to the break down of cooperation. This is the root of the Tragedy of the Commons. Open-access resources are doomed to overexploitation because people only think in the short term, and this is what we see happening in the world’s fisheries today.

Achieving sustainable resource use is only possible in the light of the incentives that individual resource users face. In the case of the bushmeat trade, conservation, development and animal welfare collide. Overexploitation of tropical forest fauna for food has both biological consequences and consequences for people. This bio-economic system operates at a number of scales (from the decisions made by an individual hunter to consumer demands and the wider economy) and is dynamic and heterogeneous (species abundances and population dynamics within the forest), and is therefore particularly difficult to tackle.

Resource management is in general complex and policies put in place to ensure sustainability are usually unsuccessful, as conservation clashes with the human element. In many cases, it is necessary to convince the public that management is worth while. Scientists are generally quite poor at communicating their knowledge to the people they are serving. This lack of communication between science and the public carries much of the blame for the distrust towards science that we see growing among lay people. Laurie Marker’s work on conservation of the cheetah in Namibia is an excellent example of understanding the social, economic and biological issues and cooperating with the locals in such a way so as to introduce acceptable and effective solutions. Marker’s work highlights the fact that accessible information, education and the fostering of trust are paramount for the successful implementation of any resource management scheme or policy.

Why dream?

2007-06-22T14:58:00.001+01:00

We all know what dreaming is, experiencing sensations such as images and sounds during sleep that we usually cannot control. Lucid dreaming is when the dreamer is aware they are dreaming and can sometimes alter their actions and the dream world. Anxiety is the most common felt emotion during dreams. Dreaming is associated with the REM (Rapid Eye Movement) stage of sleep. We go through ~4 cycles of REM sleep. All the muscles in our body are paralysed apart from the muscles in your eyes hence the name.

But why do we dream at all, nearly all mammals and birds appear to do it. Suggesting it must be evolutionary beneficial but how? Some people have thought dreams were messages from god or predictions of the future. Early psychologists like Freud were interested in dreams and their meanings. He believed dreams were wish fulfilment. Your unconscious mind doing things your conscious mind does not allow it when you are awake, but your ‘superego’ sensors your dreams. Part of this is symbolism, items and events represent what you really want. Freud has said not everything is a symbol, ‘sometimes a cigar is just a cigar’. He thought if you understand what they meant and resolve the deep arguments within yourself then all is well. I have never thought this made evolutionary sense…how many psychoanalysists were there in cavemen times to help them by charging +£60 to listen to them speak? Not many I would assume but I have no data on the matter.

Neuroscientists have also tried to explain why we dream. The great neurobiologist Crick (yes I do mean the on and only Crick) put forward the theory of reverse learning as a reason for why we dream. This theory says we dream to forget! The theory says we take in too much info in during the day so at night we erase these memories. Otherwise they would become damaging to you. This theory has little experimental support. It doesn’t explain why newborns sleep and spend so much time in REM (assuming they are dreaming). Also why do dreams have a story if they are simple a deleting mechanism? Why not simple flashes of sound and images, or has our brain come to make sense out of this, what function could that have. Lloyd et al. (unpublished) criticises it by showing more dreams are experienced when revising and yet much of the information stays. He suggests dreams are either a way of rehearsing or organising the information. But he fails to explain why dreams usually have little to do with what has been learnt. But it is clear information is not being forgotten.

Dreams have also been seen as a way of solving problems. Are you in fact thinking during dreams and coming up with ideas? This seems hard to grasp – you are actually thinking but have no control. Kekulé figured out the structure of benzene through dreaming of snakes biting its tail making a ring. Lloyd et al. also provides evidence of this. The subject woke up from a dream with the answer to chemistry homework that they were unable to solve beforehand. However there is no evidence people who do not pay attention to dreams have more difficulties in life than those who do.

It is hard to figure out what is going on with dreams, we cant figure out what memories are on the molecular level let alone how dreams arise and what function they have.

Just remember you do not know you are dreaming in non-lucid dreams then how can you prove you are not dreaming right now! (warning the devil is in me)

Hypothermia and paradoxical undressing

2007-06-15T17:50:00.002+01:00

The temperature of our body is normally around 37ºC… however, it is only necessary a 2º decrease for hypothermia to initiate… As body temperature decreases even more the consequences become more and more negative, so that between 25 and 28ºC the heart just stops and dead is the logical outcome...

Considering that hypothermia is caused by a decrease in body temperature, how to explain that so many people are found dead due to this condition and yet with no clothes on? As this scenario was very often observed in poor people in cities, the logical explanation was raping and theft. Yet this doesn’t explain why victims of hypothermia refuse warming clothes when rescued… Overall, this phenomenon is commonly referred to as ‘paradoxical undressing’…

How to explain this apparently contradictory behaviour? A quite logical theory tries to give an explanation. When hypothermia initiates, the organism tries to prevent vital organs from cooling too much, and as a consequence the ‘available’ heat is concentrated to central areas of the body. This is possible by reducing peripheral circulation, namely by contraction of blood vessels. Vasoconstriction, possible by contraction of those muscles situated around blood vessels, requires energy, namely glucose provided by circulation itself.

Vasodilation, on the other hand, doesn’t require energy. Therefore, after a period of stress as that of hypothermia, with reduced income of energy and accumulated tiredness, the muscles lining the vessels tend to relax and allow once again the flow of blood into the restricted areas of the blody. The sudden flow of blood kept warm by its concentration elsewhere is thought to be responsible by the sudden sensation of warmth which leads hypothermia victims to undress. Obviously, the undressing turns out to help the hipothermial process, and victims eventually die. In fact, there is no register so far of any hypothermia victim able to survive without help after reaching the ‘paradoxical undressing’ state.

To finish off it is interesting to note that in 20% of the lethal cases of hypothermia another perhaps not so strange situation is registered, the so called ‘hide-and-die’ syndrome, which leads to the finding of hypothermia victims hiding in the most unlogical places, namely under beds of behind closets. This is probably the remains of an old instinct that seems to be present in a variety of animals and can basically be translated into the following piece of wisdom- ‘when things get really bad, find somewhere to hide’. I wonder if this works with the exams too… :-)

Based on ‘Paradoxical undressing; 21st April 2007, pag 50, New Scientist

Yawning makes you cool

2007-06-15T04:01:00.002+01:00

Yawning is a semi-voluntary action performed by pretty much all vertebrates. It consists of three phases: a long inspiratory phase, a brief acme, and finally rapid expiration. This is accompanied by many physiological and neurological changes, such as muscular stretching, increase of blood flow and a sense of pleasure with dopaminergic activity in certain regions of the brain (Daquin et al., 2001). Yawning does not, as such, affect arousal in a physiological sense (Guggisberg et al., 2007), but it is obviously associated with boredom, waking up and feeling sleepy. As everyone knows very well, yawning is also contagious. This is not true just for humans, this is true for all social animals who have a sense of self-awareness (i.e. can recognize themselves in the mirror) (Perriol et al., 2006).

Why do we yawn? There are so many different changes associated with yawning that it could really be anything. Hippocrates considered it to be a mechanism for exhaustion of the fumes preceding fever. For the largest part of the 20th century, it was commonly thought that it was a mechanism for "balancing" oxygen and carbon dioxide levels, until this was shown to be untrue in 1987 (Provine et al.). There is obviously some connection with the states of sleep and wakefulness, but is that all? Also, why is it contagious? Is it just a mirror neuron driven response, or does it have a useful function?

The evidence for the connection between yawning and the sleep-wake rhythm is quite convincing. Even though this is generally common knowledge, Zilli et al. (2007) showed that yawning occurs more frequently in the early morning and late evening. Also, it was shown that evening-types (people who tend to stay up late, like myself) yawn more frequently than morning-types, showing that there is a connection between changes in the sleep-wake rhythm and yawning.

Another interesting connection is that between yawning frequency and REM sleep as observed throughout life. Humans can yawn from as early as 12 weeks after conception. Yawning frequency then very slowly declines with age. There is a very similar pattern in the amount of REM sleep, along with some physiological connections, such as the muscle stretching in yawning counteracting the muscular atonia seen during REM sleep (Walusinski, 2006).

The contagiousness of yawning is, for most people, its most interesting aspect. Anderson et al. (2004) showed that yawning is contagious in chimps, while Paukner et al. (2006) showed that the same is true in stumptail macaques. Infant chimps exhibited no yawning in response to the same experiments, which is in line with the evidence that human children under the age of 5 do not respond to seeing a person yawn like older humans do. Considering this and the fact that only animals with a sense of self-awareness exhibit contagious yawning, could contagious yawning have some connection with intelligence or social interaction?

Another aspect of contagious yawning is that it is correlated with interoception (sensitivity to stimuli arising within the body), self-awareness and empathy, as well as mental state attribution (the ability to inferentially model the mental states of others) (Platek et al., 2003). Functional MRI shows that brain activity correlated with contagious yawning is significant in the bilateral precuneus and posterior cingulate, regions highly associated with self-processing but, surprisingly, there is no activity in regions associated with mirror neurons (Platek et al., 2004). Yawning seems to circumvent the mirror neuron system, suggesting that yawning is an automatically released behavioural act and not an imitated motor pattern requiring detailed understanding (Schurmann et al., 2004).

The most surprising data comes from a 2007 study by Gallup et al. which suggests that yawning is a mechanism for thermoregulation of the brain. The response of human subjects to videos of people yawning was observed. Subjects breathing through the mouth exhibited 48% contagious yawning, while subjects instructed to breathe nasally did not yawn at all. Nasal breathing is associated with brain thermoregulation by the cooling of the vertebral artery and the forebrain. Also, subjects with a cool compress applied to their foreheads yawned significantly less than subjects with no compress or a warm compress applied. This leads to the formulation of an interesting theory with testable predictions about the thermoregulatory function of yawning based on the rapid intake of cool air. The authors predict that as ambient temperature approaches body temperature, yawning should diminish, and when ambient temperature exceeds that of the body, yawning should stop. This might also explain why subjects with a warm compress applied to their forehead did not yawn more, since the body might detect the high temperature compress and interpret it as higher ambient temperature.

We are getting quite close to a Unified Theory of Yawning. Even though the physiology and neurology behind it is very complex, we seem to be moving in the right direction. We can even make some evolutionary hypotheses about yawning without sounding entirely speculative. For example, we might say that a population in which yawning is contagious will be more synchronised and better able to maintain its function as a unit, whether that involves moving around at specific times of day, making sure no-one wanders off after bedtime or keeping safe from enemies. Hopefully all the yawning you just did while reading this article was only because just thinking about it makes you yawn.

References

Andrew C. Gallup, Gordon G. Gallup Jr. (2007). Yawning as a Brain Cooling Mechanism: Nasal Breathing and Forehead Cooling Diminish the Incidence of Contagious Yawning Evolutionary Psychology, 5 (1), 92-101

Also, a website entirely dedicated to yawning called Baillement, which is French for "yawn". Lots of papers to read with some comments by the website authors themselves.

Cell death, is it all about apoptosis?

2007-06-11T16:56:00.001+01:00

Where would you be without cell death, well dead I reckon. Cell death is needed in development to shape you body, such as give you fingers by killing the webbing between them. Not only that, but cell death is needed to shape the development of the brain. These things are done by apoptosis, what I would describe and the clean way of dealing with dead cells. They get broken down by nice signals outside the cell or if something internal goes wrong they order themselves to die (like by p53 to stop the cell becoming a cancerous cell and killing you). When they break down they do it in such an ordered way you wouldn’t really notice it, the dead parts get eaten up by near by cells and no nasty lysomal enzymes get released to damage the rest of the tissue. However not all cells die in this lovely way. Necrosis is where the cell is damaged and really just bursts open and releases lysomal enzymes that damage the tissue and it causes an inflammatory response by releasing HMGB1 from the nucleus into the extracellular environment to clear away the corpses of the cells.

However the two may not be so distinct. The same injury can cause either and if the apoptosis mediators are not functioning through mutations then necrosis can take over. If ATP levels drop in a cell going through apoptosis necrosis kindly steps in and does the job. Necrosis seems to have a controlled cellular pathway involving ROS, calcium signalling and proteases being activated. Some of the necrosis program appears to work in the background of apoptosis and parts of apoptosis keep the death ordered. Necrosis also (sometimes) is beneficial to an organism. In rabbits it has been reported that excess cells in theur growth plates down by necrosis.

So necrosis may be damaging and causes a lot of brain damage in strokes and Alzheimer's disease, but it is probably a backup program for when apoptosis fails to handle the situation rather than simply the cell bursting open!

The figure is from: Leist M. and Jäättelä M. (2001) Four deaths and a funeral: From caspases to alternative mechanisms. Nature Reviews Molecular Cell Biology 2: 1-10.

Dosage compensation- not the textbook version

2007-06-02T22:28:00.001+01:00

As usual (or as necessary) we were taught the textbook version of something... in this case dosage compensation. Yes, it is true that in mammals one of the X chromosomes is randomly inactivated to form a mass of heterochromatin known as a Barr body... but is this the end of the story?

Dosage compensation is a necessity in any organism with morphologically different sex chromosomes. If no compensation occurred, then the homogametic sex, e.g. XX females in humans, would show twice as much X-linked genes expression than males. It just happens that inactivation of one of the X chromosomes is only one of the possible strategies.

In Drosophila there will be a two-fold increase in X-linked expression in males. This is possible through a protein complex called MSL (male-specific lethal) which includes enzymes involved in acetylation and phosphorylation of specific histone amino acids (characteristic marks of active chromatin). The complex is targetted to the X chromosome through hundreds of binding sites with variable affinities for the complex.

C.elegans shows a strategy more similar to that of mammals. However, instead of inactivating completely one of the Xs, it will partially repress both Xs on hermaphrodites (XX). It also uses a protein complex, DCC (dosage compensation complex), but its characteristics are quite different from that of MSL- it includes no enzymes, and no non-coding RNAs. Due to its homology and shared components with the 13S condensin complex, responsible for chromatin compaction during meiosis and mitosis, DCC is thought to repress transcription by positively coiling DNA.

In mammals we already know what happens... However there is no primary protein complex- it is a non-coding RNA, Xist, that will spread from XIC, a 1 Mb region in the X, and coat one of the X chromosomes. It will then allegedly recruit other proteins that mediate the epigenetic changes characteristic of the inactive Barr body. How the choice is made is not known, but a recent study suggests that the two XIC (one in each X chromosomes in females) can physically pair, which may be involved in determining which of the two is inactivated.

To finish off, and off course after reiterating that the story is even more complicated than that, it is important to say that even if we consider only mammals, not everything has been told to us. In fact, X inactivation does not always occur at random. It happens that in certain mammals, namely in mice, at the early embryo stages there is imprinted X inactivation of the paternal X only. This is, however, reverted in the cells of the inner cell mass, which will then undergo normal random inactivation... Of course we can't be told everything in lectures... but is interesting nevertheless to find these things out later on!

Two references:

Ng K., Pullirsch D., Leeb M., Wutz A. (2007). Xist and the order of silencing, EMBO Reports, 8. pp 34-49

Straub T., Becker P. (2007).Dosage compensation: the beginning and end of generalization, Nature Reviews Genetics, 8. pp 47-57

Your brain is weird

2007-05-31T03:10:00.001+01:00

If you thought quantum mechanics was weird, you haven't seen weird yet.

It's common knowledge that the human brain is probably the most complex system that evolution has produced to date. For (as far as we can tell) the first time in evolutionary history, life is able to turn its curiosity right back at itself and ask the most ridiculous questions ever asked on this planet: "How did I get here?", "How can I make green pigs?", and most importantly, "Why am I even asking questions?". The very endeavour seems almost paradoxical; we are trying to use brains to figure out how brains work.

Absurdity aside, the weirdness goes far beyond just boring philosophy, and well into the realm of useful things (i.e. science). Neuroscientists have been peering into the brains of unsuspecting mice, monkeys, and even humans, and as with pretty much any hard scientific problem, all they have come up with is just more and harder questions. Here are some of the weird things going on inside your brain:

There are an estimated 10,000 different types (or sub-types) of neuron. We, of course, have no clue how the diversity arises, but our best guess is that the mechanism is something analogous to the V(D)J mechanism in antigen receptor diversity of the immune system. Also, there appears to be some sort of selection mechanism, once again much like in the immune system, whose function we have very little clue about.

Your brain is a mosaic of euploid and aneuploid neurons (aneuploid cells have more or less chromosomes than the "normal" 23 pairs). Depending on the region of the brain, aneuploidy varies from 10% up to 50%. Aneuploidy has generally been associated with diseases such as Down's syndrome and cri-du-chat syndrome, but since studies on mice brains in 2001 by Rehen et al., all evidence has shown that there are aneuploid neurons in healthy mammalian brains. Only vague speculation is provided as to why this is the case, and it is generally that differential levels of gene expression between cells increases the complexity of the brain, contributing to behavioural variability.

LINE-1 retrotransposons seem to play a significant role in neuronal differentiation. L1 activity is kept at bay in neural stem cells by Sox2, but its downregulation seems to trigger several events that bring about differentiation. Subsequently, L1 insertion sites seem to be most commonly found near or in genes related to neuronal function, even though insertions are generally uncommon. This suggests that L1 insertion may be regulated and that neuronal development may lean heavily on insertion events.

These are just a few of the extremely unusual properties of brain cells. You can read about the above in more detail and discover even more neuronal weirdness in Muotri & Gage (2006). We still, of course, have no clue whatsoever where that bloody engram is.

'Green-turning-red spots' movie- world premiere

2007-05-30T18:46:00.000+01:00

Once upon a time scientists didn't know how proteins where trafficked and changed along the Golgi apparatus. Some argued they were transported in vesicles from one type of cisterna to the other, where they were differently altered by different resident proteins. Others argued that the cisternae themselves were mobile and could become the next cisterna in sequence by the retrograde trafficking of the required enzymes... There was much pain and confusion, and the scientists were not happy. Until one day one group of scientists decided to see if little green dots on cells could become red. And they could. Everyone was happy, and they lived happily ever after...

--> The moment of truth- when green becomes red- and our understanding of life, the universe and everything changes...

As Dr Gareth Evans has put it, on the email that kindly provided the link to this movie:

'You can then get the popcorn in and enjoy the show! (obviously you'll also enjoy the fantastic educational benefits of a green spot changing to a red spot) '

http://www.nature.com/nature/journal/v441/n7096/extref/nature04717-s14.mov

For all of you poor children who have not been in his lectures (or who may have fallen asleep):

Basically a protein specific of cis cisterna (Man-6-P receptor) was labelled with GFP while one from trans cisterna (ARF proteins) was labelled with dsRed. The fact that a single cisterna changes colour indicates that are the enzymes that trafick between cisterna changing proteins, rather than proteins to be changed that trafick to different cisterna.

The original reference: Losev et al., (2006). Golgi maturation visualized in living yeast, Nature, 441. 1002-6

What is an ORF?

2007-05-29T23:58:00.001+01:00

An open reading frame (ORF) is the DNA sequence between a start codon (ATG) and a stop codon (TAA, TGA and TAG). A long ORF suggests it could be a protein coding gene. In prokaryotes, like everyone’s fav bacteria E. coli, they are usually 1000base pairs so making a protein of about 350 amino acids. Many short ORFs are present and even some long ones may not make a protein. Even when quiet positive that an ORF makes a protein (codon bias is correct etc) we may not know what it does. It could only be expressed in a situation that cannot be replicated in the lab. Of course everything becomes more complicated in eukaryotes like us! Our ORFs are interrupted by introns so between the start and stop codon extra start and stop codons may be present in the intron so finding genes are a lot more difficult. So sequencing cDNAs (mRNA made into DNA) can show us where genes are in the genome. We can match up sequences from cDNAs (like ESTs) to the genome and find genes. This could show up pseudogenes so we need to check the gene is expressible.

So this blog is a place for us to talk about allsorts but all we know anything is about is biology so I suppose it will be mainly about that. It is an ‘open’ place to talk about it (get the pun, open…yeah I know I’m funny).

Osteoclasts are cool

2007-05-29T20:32:00.001+01:00

Right, so this blog was needing a post... but as I should be writing an essay rather than playing with the computer it is a small one. Basically in a tutorial last week one of the guys talked about osteoclasts and ostebolasts. As you clever folks probably know, the osteoblasts are responsible by forming the bone while the osteoclasts eat it up. An equilibrium between the two is required, as excess of osteoclasts means less bone and an excess of osteoblasts means an extra-calcified bone- in any case, the bone becames brittle and more easily breaks. Any therapy to prevent osteoporosis is complicated by the fact that they normally tend to target osteoclasts... which besides eating up the bone also secreted components that promote osteoblast development, in order to guarantee the normal equilibrium (that allows bone renovation).

Really interesting, however, are the photographs below.

In this photo you can actually see the contact between an osteoclast. Osteoclasts produce hydrogen ions that acidify and dissolve the bone surface, as well as hydrolytic enzymes

My favourite one, which I cheesily baptized of osteoclast at breakfast, basically shows an osteoclast with some similarities to a snail- leaving behind not a trail of mucus but rather of eaten bone.

Finally, there is a movie of an osteoclast. Not extremelly interesting (it cannot beat the green-turning-to-red spots of Gareth Evans) but at least it shows that osteoclasts are motile (note that it has been speed up 100 times) and have multiple nuclei: http://www.brsoc.org.uk/gallery/movies/arnett_osteoclast2.mov