Sunday, July 20, 2008
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.
(It's a shame when this happens. Are we Indians as powerless as the electric sewage treatment plants without electricity?)
Monday, July 7, 2008
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
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?
Sunday, July 6, 2008
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
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
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 (
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-Puror). 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
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.
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 25th 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-
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.
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
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-
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
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
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. (
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!