While others were being counseled

I was captivated by the beauty of winter. I saw these awesome shaped crystals of water on the windshields of cars in the morning. They're like the snowflakes I used to see on Cartoon Network as a child. I also took a second picture of the quad in front of Earle Hall on which I saw that the part of the lawn that was in the shadow of Earle hall still had frost, but the part that the sun could reach was defrosted.
I like the picture because it tells the story and I think it might be a good cure for the aforementioned pathetic people who require peer counseling for winter-blues.

A future full of whiners

I saw a singularly distressing poster in the UC this morning. It advertised peer-counseling for winter-blues. What the hell? Is there a single thing that we don't need counseling for? Are we breeding a society of wimps who need to be cheered up by their peers for the mildest unpleasnt occurrings in their lives? Whatever happened to "Stop whining, old chap; pick up your socks and move on"? Next we'll have counseling for late trains, lost chewing gum, bad-tasting coffee, and other ridiculous trifles. Bah!

An ecosystem of my own

As I was ascending the steps to my room today, I noticed something almost...heartwarming.

I have missed two trash days in succession. Consequently, I had tonnes of rubbish at home which had begun to attract the attention of flies. In an attempt to avoid being busted by health and safety officers, I moved the rubbish bags outside and kept them on the landing of the stairs. The stairs overlook the garden. I did this three days ago.

Today, I noticed a spider web adjacent to the rubbish bags (it is indicated by the arrowin the picture to the left). It struck me that I had created an awesome bait for the spiders. Sensing the trash bags, some moronic fly would probably zoom towards it. Unfortunately for it, his velocity would be abruptly brought down to zero by the subtly woven spider web between the two bars of the railing right in front of the garbage. The aftermath needn't be mentioned.

I must say, however, that I did that old spider a bit of a favour when I put the rubbish out. To me, this is another instance of the chaos theory; I hadn't even dreamed that I'd create a sort of micro-ecosystem through my simple act of putting the garbage out. I love the complexity of our world.

Hmm...I don't suppose my actions were particularly heart warming for the moronic flies, though.

Indian Genomic Variation Database

The government of India is doing something really fantastic for once. You can see it yourself at http://www.igvdb.res.in/.

Basically, they're mapping genes of pharmacological and medical significance and keeping track of the different haplotypes and Single NucleotidePolymorphisms (SNPs) in the Indian population. They find pretty vital things about the population's response to drugs. For example, they found that 13% of the North Indian population is unresponsive to 30 of the major drugs used by doctors.

Such discoveries could pave the path to tailor-made treatments and should, probably, lower the medical expenses for the Indian people. Obviously, if they know that they're resistant to a particular expensive drug, they won't waste their time and money trying it out in ignorance.

This project shall have an impact on things ranging from Asthma to Malaria. I'm really quite excited by this. For some reason, I used to harbour the idea that molecular biology doesn't have much of an application in improving rural health in India. I suppose I'll have to rethink my beliefs now.

ICAR

ICAR. No, that’s not a new Apple product. It is the acronym for the international conference on Arabidopsis Research. I have been attending it since Wednesday (23^rd July 2008). Tomorrow (Sunday) will be the last date. It’s really been quite an experience. Anyway, I’ll try to begin from the beginning.

We drove for seven hours to Montreal. It surprisingly close. I thought it would take several days to get here from NY by car. lol! Shows you how smart I am. Anyway, the customs didn't really pose a problem and I was really delighted to be amongst signs that featured extra U''ss and re's instead of er in the word centre. Yes, Canada is part of the British commonwealth and I began to feel absolutely at home, despite the fact that the car still drove on the right side of the road. That fact disappointed me, but Dr. Hobie heaved a sigh of relief. I don't know why he did that because he was here previously and knew that already. He did it anyway. Oops, I forgot to introduce the cast of characters. There' s Dr. Hobbie, me, and this sri lankan graduate student called Mederian Kotuwale Achala Jayasen. I believe she has a fifth name as well, but it escapes me. She dearly seeks to reduce her names to a manageable two or three. I pointed out that she was bloody lucky to have only five names. A very very famous sri lankan bowler's name is Chaminda Vaas. However, that isn't his whole name. There are 46 other names in between the Chaminda and the Vass ("Hmm....what was my name again?").

When we got into montreal proper, I had to become responsible. Dr. Hobbie handed me the directions and I had to help him navigate to the Hyatt Regency Hotel where the conference is being held. I had some fun with the french names, but I was happy to see that my pronounciation wasn't as spectacularly off as it was in the german class. In fact, I think my seventh grade french teacher would be positively proud of me. Whether it be Jeanne-mance or renee- levesque boulevard. I kept my end up! Anyhow, we reached the hotel and I saw lots of chappies with banners with the letters CSN on them. I naturally assumed that they were part of some sport fan group. However, on arrival we were informed that they were, in fact, hotel staff that were on strike and were being negotiated with. Consequently, there was no parking service and we had to sort through the sorties (exits) and the entrees (entrys) to the Stationment des Jardins (parking space of the Garden mall within the hotel). I was quite happy to see that I could decipher the bloody french signs after a fashion. I suggested to Dr. Hobbie that Canada could save tonnes of money by adopting English as its sole official language and print boards only in English. However, I don't think that's happening since Canadians are like the Nepalese of North America. They're dependent on the US in many ways, but will never admit and insist on maintaining a disparate identity.

For some bloody weird reason, the hotel had 6 floors and from the 6th floor, you could take another escalator to floors 1-17, completely different from the ones we had just encountered. Anyhow, we managed to find our way around and after an ice-cream (for which we paid US dollars and got canadian dollars instead)., we went in for the first talk. This talk is by a guy who you consider to have no life since he established my weed as a model genetic organism. It was spectacular. He talked about how ethanol is not the future due to its inefficiency and how we can use other crops and processes to manage our energy requirements. It was fascinating, particularly, because if I do a Ph.D, then this is one of the fields I would consider working in. Anyway, we have set up our posters and found our way to the McGill residence hall. Honestly, this hall looks amazingly like a hotel. My room-mate is an asian post-doctoral chap called Mingje Chen. I didn't want to ask if he was from China, but I think he is. He seems to know a little bit about my work so that's good. I always thought I did very obscure work in the lab, but most of the posters I saw today are on Auxin and I actually met some of the people whose scientific papers I had read last year during my independent study. It was a great pleasure to shale their hands. However, if I meet a particular guy called Benfey, I'll make sure I wring his hand hard for all the torture his paper inflicted on my brain cells. It was horribly complex and neither Dr. Hobbie nor I could make head or tail of his work.

The highlight of the conference, for me, was a meeting with Dr. Malcolm Bennett who works in the University of Nottingham and is also working on the AXR4 gene. I had seen him speak earlier on and he actually gave one of the best presentations in the conference. At least, that’s the way it appeared to me. Having read the paper he presented might have helped my understanding and enhanced my enjoyment of his talk.We talked for a few minutes during the poster sessions and we discussed auxin transport modelling. Last year, there had been a paper, by Dr. Ben Scheres of Utrecht, in which auxin import proteins were considered of negligible importance. That paper outraged my sensibilities (no surprise because I spend a lot of time working on an auxin-import related gene) and I had protested to the author directly. Malcolm agreed with me and explained why Dr. Scheres’s model worked despite this royally flawed assumption. Apparently, physiological parameters for auxin permittivity had not been adhered to. It was heartening to be vindicated.!!!

All in all, it was a fantastic trip. I learned tonnes and it was nice to add another country to my “visited list”. I think I am also in a better position, now, to decide whether I want to do a pure Ph.D or not.

Ganga Mata (Mother Ganges)

I was quite appalled when I read the Economist's article about the crappy state of the Ganga (no pun intended). Sewage is being discharged into it without treatment and corpses are flung into the river with religious fervour. Basically, we Indians (esp. Hindus) manage to mess up the river throughout our lives and even after death. The image to the left is from an organisation called the Eco-Friends. They monitor the pollution in the Ganga and have written an admirable report about it. I applaud their efforts.

Do allow me to shock you. The coliform density (density of E.coli bacteria from human intestines) is 12000 times above the safe limit. Last year, 3000 corpses were floating around in the Ganga. This is an illegal practice. Electric crematoria have been set up to avoid this source of pollution. and there are stretches in which the river is actually Black! Millions of Indians are dependent upon this river for their domestic and their spiritual needs. Yes, the situation is indeed ghastly.

The government of India has put in 600 million dollars into the Ganga and the Yamuna, but nothing seems to have transpired yet. The irony is that the government is intent on using electric sewage treatment units. That's a joke in our country of power-cuts. Luckily, no matter how messed up a country is, its gravity usually functions well enough.

That's Mr. Veer Bhadra Mishra's plan. Units that use gravity treatment techniques are cheaper and better options for our country. The scientific Mr. Mishra drinks a glass of turbid, fetid water from the Ganga out of devotion every day. He knows that the water is dangerous and has already suffered from many water-bourne diseases. Less scientific Indians along the Ganga's coast do the same. Doctors tell them to boil the water to make it safer. Think about it, these chaps don't have enough fuel (read cow dung cakes) to make one meal a day. Do you suppose they'll waster their fuel on boiling water? This is why diarrhoea, jaundice, and cholera wreak havoc in our country. Something needs to be done.

One of the few good things that has happened is that the centre has given a directive to the Uttar Pradesh (one of the states of India through which the Ganga flows) govt. to check out Mr. Mishra's proposal. His work had been disrupted previously in 1998 when the sanctions by the US put paid to funding for his experimental gravitational sewage treatment plants. I hope India rallies and supports him. There seems to be light at the end of the tunnel and it's hopefully not the fiery breath of a dragon.



I found a few websites that you may want to look at.

http://www.abc.net.au/rn/science/earth/stories/s183185.htm

http://www.serverhostingmantra.com/eco/reports/default.htm

http://www.serverhostingmantra.com/eco/reports/05217foreigners.htm
(It's a shame when this happens. Are we Indians as powerless as the electric sewage treatment plants without electricity?)

Nitrite and Ischaemia

Embarrassingly, I didn't know the precise meaning of the word Ischaemia (written Ischemia by Americans) until Early March this year. That's quite a confession for the son of two doctors. Basically, ischaemia occurs when blood supply is cut off in a blood vessel. For instance, a clot could occlude the vessel and lead to ischaemia. Anyhow, this is a massive problem in today's world. Millions of chaps suffer from this and quite a few die from it as well. Kumar et al. wrote an interesting paper in the Proceedings of the National Academy of Sciences in which they prescribe the systemic administration of Nitrites as a possible treatment for ischaemia.

Now, Nitrites can be reduced to Nitric Oxide (NO). NO is a very important signalling molecule in the body and it is involved in things ranging from immune response to angiogenesis (formation of new blood vessels). Incidentally, Viagra functions by modulating NO production in the body. Mazzone and Carmeliet, who reported Kumar et al.'s findings in the 26th June 2008 issue of Nature, point out that NO modulates Vascular Endothelial Growth Factor (VEGF) which may result in angiogenesis. Also, VEGF helps in the increased synthesis of NO, presumably by upregulating Nitric Oxide Synthetase. Thus, a positive feedback loop is created.

Now you might be wondering how this is safe. After all, as Mazzone and Carmeliet point out, NO in large quantities is dreadful. For instance, No messes with haemoglobin's oxygen carrying capacity and also disrupts cytochrome c oxidase. Furthermore, reactive oxygen species (such as free radical superoxides) are known to react with NO to make peroxynitrite (ONOO-). Read the article by Pál Pacher, Joseph S. Beckman and Lucas Liaudet at http://physrev.physiology.org/cgi/content/full/87/1/315.
They talk about how peroxynitrites are involved in aging, hypertension, and Atherosclerosis. Basically, this species can cause tremendous oxidative damage, particularly to the cell's DNA and proteins.

However, NO doesn't work indiscriminately. This is the beauty of the thing. In vessels with a high oxygen concentration, Nitrites gets oxidised to nitrates. However, in occluded vessels, where the oxygen levels are low, nitrites get reduced to NO and angiogenesis gets initiated, thereby helping ease the ischaemic conditions. This makes it possible to administer the NO non-specifically, perhaps even orally. The challenge is to find the perfect NO concentration that avoids high peroxynitrite concentrations and is sufficient to promote angiogenesis at the same time.

How much time do you give it?

Superman flew away too soon: recent advances in stem cell research

I had written this article two semesters back. Basically, it talk about this new technology called induced pluripotent (iPS) cells. These can be made from adult somatic cells. Basically, the iPS cells are almost exactly like the immensely controversial embryonic stem cells. This may herald the end of the stem cell controversy because iPS cells are made by expressing 4 genes within the cells. This means no embryos are harmed in their construction. Hopefully, this technology will help US scientists come up to speed with the rest of the world in terms of stem cell work. It may also be another step closer to the cure for neurodegenerative diseases such as Parkinson's and ALzheimer's.

-------------------------------------------------------------------------------------

The horse changed its mind about jumping over the fence and the burly rider was thrown violently to the ground. The helmet saved his brains, but the impact shattered the first and the second vertebrae; His spinal cord was disconnected from his brain. Unlike skin cells that are replaced everyday, cells of the nervous system don’t mend themselves once damaged. Superman doesn’t fly off-screen, after all.

Christopher Reeves went on to campaign for Human Embryonic Stem Cell research. His death in 2004 didn’t decelerate the race to develop stem cell technology to a point where a miracle cure for people like him would exist. In addition, stem cell mediated regenerative therapies have frequently been considered the way to cure degenerative diseases such as Alzheimer’s disease, Parkinson’s disease, etc.

Recently, some exciting events have taken place in the world of stem cells. This review paper recounts some of the recent advances in this marvellously promising, but frightfully controversial, field of science. Indeed, The Economist has dubbed this field “the most operatic in science.” However, it is imperative to establish some basic facts regarding stem cells first.

Stem cells are pluripotent cells; undifferentiated cells that have the potential to become any cell type. They can be classified into two types: adult stem cells and embryonic stem cells. Adult stem cells are used to repair and replace cells that are lost during wear and tear. These can be found in minute quantities within the bone marrow and the umbilical cord. Embryonic stem (ES) cells have the potential to mature into any cell type. They are collected from the inner cell mass of embryos in the blastocyst or morula stage of development. ES cells have significant potential in regenerative therapies.

However, it is distinctly possible for the body of a stem cell recipient to reject it due to histo-compatibility issues. Therefore, it was proposed to prepare embryonic stem cells using the DNA of the recipients themselves, thereby reducing the probability of a rejection. Hence, a technique called Somatic Cell Nuclear Transfer (SCNT) was developed. In this procedure, the nucleus from a somatic cell (like a skin cell) is transferred to an enucleated egg cell. Next, cell division is simulated using an electric shock. The cell now develops into an embryo that can be harvested for stem cells. The stem cells are extracted and grown in separate Petri-dishes to keep them away from chemical signals from other cell types that might prompt their differentiation; robbing them of their pluripotent status. These isolated stem cells divide to form “cell lines” and can be used for research or therapy.

Unfortunately, stem cells can’t be extracted from embryos without destroying the latter. This aspect of stem cell research offends the moral sensibilities of those who oppose it. Political ideologues have deftly entangled the stem cell extraction process with the abortion controversy. Consequently, the moral outrage of a minority of tax-payers has brought stem cell research in the USA to a shuddering halt. Today, a limited number of stem cell lines are available for research purposes the USA. These lines, are contaminated possibly contain mutations due to repeated division. Many scientists question why the destruction of embryos during the extraction of stem cells draws such vociferous protests when fertility clinics routinely dispose of extra embryos as medical waste. Stringent laws have resulted in countries like Japan and South Korea have racing past the US in stem cell technology. However, there may be light at the end of the tunnel (and it’s not an LIRR train).

The success of SCNT and the subsequent cloning of Dolly⁴ made the scientific community aware of the existence of cytoplasmic trans-acting factors that were capable of reprogramming somatic cells into reverting to an embryonic stem cell like state. This realisation prompted a hunt for these transcription factors.

In November 2007 --10 years after Wilmut’s critical breakthrough-- Takahashi et al. reported their success in synthesising pluripotent cells without using the controversial SCNT protocol. They reported that induced pluripotent cells (iPS) could be produced from human dermal fibroblasts (HDF) by transducing four transcription factors- Oct3/4, Sox2, Klf4, and c-Myc.¹

It seems almost amazing that the transduction of four transcription factors can lead to such an amazing transition from an HDF to a pluripotent cell. Prior to Takahashi et al., Wernig et al. had shown that Oct3/4, Sox2, Klf4, and c-Myc can epigenetically reprogramme a mouse somatic cell into an embryonic stem cell like state². These seemed to be some of the aforementioned trans-acting factors involved in converting somatic cells into embryonic stem cells. Oct3/4 and Sox2 have been shown by previous studies to act as the central pluripotency generating transcription factors. Takahashi et al. speculate that c-Myc and Klf4 make the chromatin structure more conducive to the binding of Oct3/4 and Sox2. This speculation is strengthened by the fact that Klf4 is known to regulate the acetylation of histones¹.

Wernig et al. transduced the four transcription factors into mouse fibroblast cells. iPS cells were selected for by looking for the activation of a gene downstream of Oct4- Fbx15. However, the cells, though pluripotent, were dissimilar to ES cells in some regards. For instance, some differences exist in the methylation patterns and gene expressions of ES cells and iPS cells selected using Fbx15. Furthermore, these iPS cells do not contribute to viable chimaeras².

Okita et al. showed that selecting for Nanog instead of Fbx 15 produces cells that resemble the ES cells even more with regard to epigenetics and gene expression³. Nanog is more closely associated with pluripotency than Fbx 15 since it is known that disrupting Nanog in mice results in a loss of epiblast pluripotency. Conversely, disrupting Fbx15 doesn’t produce a readily observable effect. The researchers, isolated a bacterial artificial chromosome (BAC) containing the mouse Nanog gene and inserted a GFP- internal ribosomal entry site(IRES)- puromycin resistance gene (Puro^r) cassette into the 5’ untranslated region. These BACs were introduced within ES cells. The ES cells that incorporated the modified BAC were GFP-positive. However, they became negative for GFP when they differentiated because the Nanog gene is highly expressed in pluripotent cells, but not in differentiated cells. Thus, this cassette enabled the researchers to spot cells that expressed Nanog strongly and to select for such cells using puromycin.

The ES cells that stably incorporated the GFP were, then, introduced into mouse blastocysts to obtain chimaeric mice that were used to produce transgenic mice containing the aforementioned reporter construct (Nanog-GFP-IRES-Puro^r). The blastocysts from the transgenic mice contained the reporter in the inner cell mass. The primordial germ cells were found to be GFP-positive 9.5 days post coitum (d.pc.) and by the 13.5 d.p.c, the genital ridges were also GFP positive.

Okita et al. extracted MEF (mouse embryonic fibroblasts) cells (which were GFP negative) and introduced the aforementioned transcription factors into the mouse cells using retroviral vectors. The researchers used a mutant of c-Myc (T58A) instead of the wt gene. The MEF cells were cultured on feeder cells. After giving the cells time to heal from the retroviral infection, puromycin selection was commenced. Twelve days post retroviral infection, hundreds of GFP positive colonies were observed. Some GFP-negative colonies were also present. The researchers are unsure about the reason for their occurrence. By contrast, no GFP positive colonies were observed when mock DNA was used instead of the four transcription factors. Remarkably, raising the puromycin concentration during the selection process lowered the number of GFP-negative colonies. Significantly, if any of the four transcription factors were not added, GFP-positive colonies weren’t produced. This shows that all four transcription factors are necessary for the production of iPS cells.

Bisulfite sequencing was used to show a congruence between Nanog selected iPS cells and ES cells in their methylation patterns. Methylation patterns reveal the pattern of gene silencing in a genome. DNA methytransferases mark cytosines with methyl groups. This results in the formation of 5-methylcytosine that has normal base-pairing abilities, but has an altered structure. Methylation results in the formation of heterochromatin which forms DNA that cannot be transcribed. Treating DNA with bisulphite results in the conversion of normal cytosine bases into uracil. However, the existing methylcytosine is not altered. The conversion of cytosine to uracil is almost like inducing Single nucleotide polymorphisms (SNPs) within the DNA. These SNPs can then be studied to produce a high resolution picture of the DNA methylation in the cells using numerous sequencing techniques.

Furthermore, these iPS cells resembled ES cells in their morphology (ES cells have flat colonies), teratoma formation, and proliferation pattern. It was shown that Nanog gets downregulated when a cell begins to differentiate, thus further strengthening the case for using Nanog as a selection criterion for pluripotent cells. However, the induction efficiency was very low. Now that the induction of pluripotency using these four transcription factors has been established, it would be profitable to scrutinize the work of Takahashi et al. in greater detail.

The first step taken was to increase the transduction efficiency of the HDFs they were working with. This was achieved by the introduction of a mouse retroviral receptor (Slc7a1) using a lentivirus as a vector. These HDF-Slc7a1 cells had increased transduction efficiency from 20% to 60%. This was assayed by using a gene construct with GFP.

Subsequently, retroviruses containing Oct3/4, Sox2, Klf4, and c-Myc were introduced into the HDF-Slc7a1 cells. Six days after transduction, the cells were harvested and plated onto feeder cells. The next day the medium was replaced with a medium for primate embryonic stem (ES) cell culture (supplemented with basic fibroblast growth factor). At first, granulated colonies that did not resemble ES cell colonies were seen. However, by the 25^th day after transduction, some flat ES cell-like colonies were seen. They were called human induced Pluripotent (iPS) Cells.¹

It was found that the human iPS cells resemble human ES cells in numerous ways. Takashi et al. carried out reverse transcriptase polymerase chain reaction analysis (RT-PCR) to show that the human iPS cells expressed many genes normally found in undifferentiated ES cells. The list included OCT3/4, SOX2, NANOG, GDF3 (growth and differentiation factor 3), REX1 (reduced expression 1), FGF4 (fibroblast growth factor 4) etc. RT-PCR is an excellent method of assaying transcription levels. It contains two steps: first strand reaction and second stand reaction. Essentially, the first strand reaction involves the treatment of mRNA with reverse transcriptase to convert it into cDNA. The primers are targeted to the 3’ polyadenylated sequences (Poly A tail) of the mRNAs being processed. Subsequently, the cDNA is amplified using DNA polymerase. This is the second stand reaction. The RT-PCR results can be seen in figure 1.

Moreover, western blot analysis reported that the iPS cells resembled ES cells in their OCT3/4, SOX2, NANOG, SALL4, E-CAD-HERIN, and hTERT protein levels. Western blot is a technique used to assay the expression of a specific protein. Essentially, the protein is extracted and electrophorosed. After the electrophoresis, the proteins are transferred onto a nitrocellulose (or PVDF) membrane where they are probed with antibodies. Subsequently, a secondary antibody is added. This antibody attaches to the primary antibody. Usually, the secondary antibody is fluorescent or chemo-luminescent. By monitoring the abundance of the secondary antibody, it is possible to estimate the level of protein expression. By comparing the displacement of the band to a molecular marker, it becomes possible to identify the protein on the basis of its mass.

Furthermore, DNA microarray analysis confirmed that iPS cells and ES cells are similar (though not exactly equal) in terms of gene expression. When compared with HDF cells, it was found that HDF cells had methylated loci for OCT3/4, NANOG and REX1 while iPS cells had unmethylated loci for the same genes (probably since these are required for maintaining pluripotency). This was confirmed using the chromatin immunoprecipitation (ChIP) analysis.

Proteinaceous trans-acting factors bind to specific DNA domains. However, they don’t bind to all possible domains at the same time. It is possible to infer DNA methylation and gene silencing patters based on information regarding the DNA sequences that a particular regulatory is binding to in a cell. This can be done using ChIP analysis. Basically, the DNA is extracted and broken down mechanically. Subsequently, the broken up DNA is probed with the antibody of the regulatory protein. This yields a collection of DNA sites that the regulatory protein was bound to. This methodology can be, and was, employed to detect histones modified by methylation. The ChIP analysis showed that the histones in the promoter sites of Oct 3/4, Sox2, and Nanog were demethylated (unlike the HDF cells that were highly methylated at these loci).

In addition, iPS resembles ES cells in their high telomerase activity, exponential proliferation, and teratoma formation. Furthermore, Takahashi et al. were also able to demonstrate the ability of the iPS cells to differentiate into dopaminergic neurons by co-culturing them with PA6 cells. They demonstrated using PCR analysis that OCT3/4, SOX 2 and NANOG were downregulated (showing reduction of pluripotency) while dopaminergic neuron markers (assumption of dopaminergic neuron properties) were upregulated; showing that the cells were capable of differentiation. Perhaps bisulfite sequencing could have been done to ascertain whether the pluripotency related genes were methylated and the neuron genes were unmethylated in the iPS cells co-cultured with PA6 cells. Alternatively, a serial analysis of gene expression (SAGE) could have been done as a confirmatory to monitor the transcription levels of the known dopaminergic neuron genes within the cocultured iPS cells.

This characteristic of the iPS cells creates hope for people suffering from degenerative diseases such as Parkinson’s and Alzheimer’s. Even more phenomenally, the iPS cells were induced, using Activin A and bone morphogenic protein (BMP), to turn (in-vitro) into cardiomyocytes. Within 12 days of induction, the cells began beating. RT-PCR showed the presence of cardiomyocyte markers. Clearly, the iPS cell technology may, once perfected and fine tuned, be the much-awaited knight-in-shining-armour of the field of regenerative medicine, especially since histo-compatibility issues can be swept away by using somatic cells from the patients themselves.

In addition to the HDF cells (taken from a 36 year old Caucasian woman) that were converted into iPS, human-fibroblast-like-synovocytes (HFLS) from the synovial joint of a 69 ear old man were also successfully turned into iPS cells. For an encore, Takahashi et al. also turned BJ cells from neonate fibroblasts into iPS cells. All the iPS cells resembled the HDF iPS cells in the aforementioned ways.

There is, however, a small drawback to this procedure. It was found that each iPS clone receives three to six retroviral integrations per transcription factor. This increases the probability of tumour formation. Indeed, 20% of mice derived from iPS cells possessed tumours. This has been blamed on the reactivation of the c-Myc retrovirus¹. c-Myc is a known oncogene. Indubitably, the utilization of c-Myc is a risky proposition. The usage of retroviruses is controversial as well and the authors propose to either begin using adenoviruses instead, or to find molecules small enough to induce gene transfer sans gene transfer.

However, it seems the, somewhat hazardous, transduction of –Myc is not imperative for the creation of iPS cells. Yu et al. made a list of genes that were enriched in ES cells relative to myeloid precursors. OCT 4 is known to be expressed greatly in pluripotent cells and is also known to confer geneticin resistance to cells. Hence, geneticin selection was used to isolate pluripotent cells. Initially, a combination of 14 genes was used to reprogramme CD45+ haematopoetic cells into OCT4 positive, geneticin resistant cells that resembled ES cells in the morphology and cells surface markers. Re-testing was carried out. Finally, using this approach, Yu et al. showed that OCT4, SOX2, NANOG, and LIN28 genes were ample for making iPS cells. Once again, DNA microarray analysis showed that the gene expression in the reprogrammed cells resembled ES-cell gene expression. Furthermore, these cells were shown to form teratomas like ES-cells. Additional tests verified the similarities between ES cells and the iPS cells. The STR (short tandem repeat) pattern of the iPS cells was checked to ensure that they were reprogrammed cells and not contaminating ES-cells⁵. These results, by eliminating the use of the dangerous c-Myc transcription factor, enhance the prospects of the usage of stem cells in regenerative medicine.

In other news, Byrne et al. report the production of primate ES-cells using a modified SCNT protocol. Byrne had previously reported that the removal of lamin A/C was essential for the remodeling of the oocyte into an ES cell. This required the functioning of a maturation-promoting factor (MPF) which might have been degraded in the previous protocol (due to the usage of Hoechst stains and UV light) resulting in non-removal of lamin A/C. The old protocol also tended to damage mitochondrial DNA, thereby reducing the success rate. In their new protocol, Byrne et al. used a system called the oosight imaging system that utilises polarized light to view the chromosomes; allowing them to manipulate the oocyte without damaging MPF. The researchers attribute much of their success to this system. In addition, they modified calcium and magnesium levels in the oocyte media to decelerate the degradation of MPF. Whatever they did obviously worked! Indeed, the “most operatic field in science” seems to be turning into the most dynamic one as well.

Why should we care about all this? Stem cells are a fascinating resource. They can be used extensively for research purposes. For instance, if it is difficult to obtain diseased tissues to study, patient-specific cells can be concocted to study the disease (using ES cells) since these cells are identical to the diseased cells. Drug trials could be expedited with the use of these iPS cells as well. Obviously, regenerative medicine is one of the most touted, as well as the most famous, applications of stem cells.

Some concerns do exist about the long time delays and the difficulties in producing the differentiated treatments required for regenerative medicine. However, one thing that has been established by these recent developments in the stem cell world is that technology and science are sprinting rapidly. It is very likely that in a very short amount of time, regenerative medicine will go from just being a distinct theoretical possibility to a routine medical procedure. As this paper was being finished, the media announced that a putative stem cell treatment for Duchenne’s muscular dystrophy has been developed. Excitingly, the DMD treatment technique can find applications in other genetic diseases as well. Things like neuronal regeneration are right around the corner. Superman flew away too soon.

1. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131, 1-12.

2. Wernig, M., Meissner, A., Foreman, R., Brambnik, T., Ku, M., Hochedlinger, K., Bernstein, B.E., and Jaenisch, R. (2007). In Vitro Reprogramming of fibroblasts into a pluripotent ES-cell like state. Nature 448, 318-329.

3. Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Generation of germline-competent induced pluripotent stem cells. Nature 448, 313-317.

4. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS. (1997). Viable offspring derived from fetal and adult mammalian cells. Nature 385, 810-813.

5. Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosieqicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. (20 November, 2007). Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. (Epub prior to print) Science [DOI: 10.1126/science.1151526]

The Epstein Barr Virus

I have another blog. It is comprehensiblecomplexity.blogspot.com. Yeah, I actually expected people to remember that. Anyway, I have begun transferring some of my articles from that blog to this one. xiphus is a nice name, isn't it? It's easy to remember at any rate.

Epstein Barr Virus (family: Herpesviridae) is an enveloped, ds-DNA (approx. 185kbp genome) virus that is the causative agent of numerous diseases such as Infectious Mononucleosis and Multiple Sclerosis. However, it is also known to cause Burkitt’s Lymphoma in developing countries (esp. Africa). Indeed, EBV was the first oncogenic virus to be discovered.
Primary infections occur through oral secretions (kissing, etc.). The infection is initially limited to the oral mucosa. It subsequently infects the B-cells using the CD-21 receptor (a member of the Immunoglobulin family highly expressed in B cells) and transforms them to replicate repeatedly. Indeed, the assay used for EBV is to check its ability to immortalise B lymphocytes from EBV- individuals. Immortalising B-lymphocytes is a function of the EBNA-2 gene.
Thus, EBNA-2 transforms the B-cells and makes them reproduce in an uncontrolled manner. This is the reason, we find that mononuclear lymphocytes predominate in Infectious mononucleosis. The gene also regulates the size of the antigenic site. Consequently, “Ebnotyping” has been considered as a method of classifying EBV. In fact, this has been used to show that the bone marrow is the initial site of infection. This is supported by the observation that people, who received a bone marrow transplant, lost their own ebnotype and gained the donor’s.
Following infection, most cells don’t express the entire genome to replicate. Instead, the origin of plasmid replication (oriP) gene (1.7 kbp long) enables autonomous, extrachromosomal replication. The EBNA-1 gene has also been implicated in the maintenance of the episome because in EBNA-1 deficient mutants, the virus gets integrated in the host’s genome. This gene shares the same promoter as EBNA-2 and helps to recruit the cell’s DNA replication machinery. Most cells replicate once per host cell division. A small proportion, however, does enter the lytic cycle and produce multiple copies.
The lytic cycle is controllef by a protein called oriLyt. It is aided in this function by BHLF-1 and BZLF-1. In latently infected cells, the cell division occurs at the rate of one division per 30 minutes. On the other hand, a cell in the lytic cycle produces 1000 copies per cell. All herpes viruses produce DNA concatemers. EBV is no exception.
Epstein Barr virus was first isolated from a Burkitt’s Lymphoma tumour. This disease is the fastest progressing Non-Hodgkin’s lymphoma that occurs in children due to the translocation of the myc gene (a protooncogene; normally codes for transcription factors) from chromosome 8 to chromosome 14. It has been widely accepted that this may be due to a weakened immune system created by a simultaneous chronic malarial infection.
Interestingly, EBV may mimic cellular functions to evade the scrutiny of the marauding immunosurveillance system. Indeed, stunning similarities have been noted between Interluken-10 (a mouse cytokine) and a viral protein called BRCF-1. This is a most remarkable aspect of the EBV. It is possibly the most successful parasite in the world since it stays latent in 90% of the human population throughout their entire life. You have it in you right now!

Well, I am going to try to make this blog the spot for regular rumination on every topic under the sun. Mostly, though, I'll be talking about the interesting new things in science. I am usually not this boring. It is, however, 1.44 am and I am feeling the first pangs of sleep. I just finished destroying China in Command and Conquer Generals. I did it four times. A little excessive perhaps. Oh well.