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Introduction on Drug Repositioning (with examples)

November 10, 2012, 11:49 am
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Drug market is huge. There’s already ~95 000 publically available drugs. Drug development process is long (10-15 years) and it is expensive. A lot of potential candidates are eliminated in the preclinical stages. Even if drug enter clinical trial, it’s not a guarantee that it will be marketed (1 out of 10 drugs complete clinical investigation successfully). Despite having marketed a lot of drugs, pharmaceutical industry still needs to find solution for the long list of disorders.

Drug repositioning is relatively new field in pharmacology that is focused on discoveries of new indications and molecular targets that could be cured using already known drugs. Cases of drug repositioning were noted in the past when new drug indications were accidentally noted during clinical trials. Today, drug repositioning is completely new branch in pharmaceutical industry; large companies have separate divisions (known as “Indication discovery unit” in Pfizer and Novartis or “Common mechanism research” in Bayer) that are dealing with this issue.

Drug repositioning is popular for many reasons. It saves money (probably most important factor in pharmaceutical industry). Over billion dollars and a lot of time and effort are invested in drug development but there’s no guarantee that drug will reach market and be successfully used at the end. There’s always a chance that drug wouldn’t be safe or effective enough to bring invested money back. Also, drug could easily be excluded from the market if some unexpected side effects appear. Other negative aspect of drug development is that it lasts between 10 and 15 years - sick people don’t have that much time to wait for new medicine to appear. And finally, drugs that are proved ineffective for one disorder, but effective for some other, could be applied immediately because evaluation of drug's safety is already finished.

Viagra is one of the most famous examples of drug repositioning. Sildenafil (generic name) is initially developed for pulmonary hypertension and angina pectoris. Erection was side effect noted in all male participants in the study. Scientists recognized the values of the newly detected side effect and Sildenafil (under brand name Viagra) soon became first marketed drug indicated for erectile dysfunction in men. Viagra is on the market from 1998. and it’s still highly profitable (selling profit exceeded 1,9 million dollars in 2008). Successful switch of indications is made thanks to Viagra's mechanism of action. It is PDE5 (cGMP-specific phosphodiesterase type 5) inhibitor. PDE5 degrades cGMP in penile corpus cavernosum tissue. When PDE5 actions is prevented, increased cGMP level result in smooth vascular muscle relaxation and increased blood flow to the penile sponge tissue resulting in erection. Sildenafil was initially designed to prevent pulmonary arterial hypertension by increasing cGMP level that will decrease pulmonary arterial resistance and induce pulmonary arterial wall relaxation. Biochemical "collision" in two seemingly distant organic disorders lays in the fact that PDE5 is distributed within the arterial wall of the lungs and penis exclusively (vasodilatation is not induced in the rest of the body). It turned out that drug is more effective in penis than in lungs and soon enough it was repurposed.

Few more examples on drug repositioning:

Buprenorphine is developed as anti-analgesic in 1980s. In 2002, list of indications was expanded and today it’s almost exclusively used in treatment of drug addiction (for detoxification and long term replacement therapy).

Requip is initially designed as medicine for Parkinson disease. List of additional indications expanded later and now it’s used in treatment of restless leg syndrome and in the treatment of SSRI-induced erectile dysfunction.

Colesevelam was developed as LDL-C lowering agent for the patients with primary hyperlipidemia. Today, it can also be used in patients with type 2 diabetes to improve their glycemic control.

Gabapentin is initially designed as medicine that could treat epilepsy, but it was soon discovered that it’s more efficient in treatment of anxiety disorder.

Examples of drug repositioning are numerous. “Repositioning” approach has few advantages over classic drug discovery process. Whole idea behind drug repositioning is to find alternative indications for the already marketed drugs or drugs that are rejected due to low efficiency rate or more side effects than expected (dose reduction is also an option). Keeping in mind that the number of marketed drugs is incredibly high, it’s just a matter of time when new treatment options for the severe medical conditions will be “reinvented”.
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Connectomics - Study of Neuronal Connections in Brain and Nervous System

November 10, 2012, 10:10 pm
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Brain is the central organ in the nervous system of all animals except few invertebrates. Everything we do is under brain's control and that’s the reason why scientists are so eager to "decode" it.

Brain is extremely complex organ, anatomically divided in multiple functional units (visual cortex, olfactory cortex…). Signals coming from the brain are transported via synapses. Although much information about brain is already known, there are still a lot of gaps that need to be filled. Number of neurons in the brain is enormous; just in cortex, there are between 15 and 33 billion neurons. Each one of them is connected with at least one other neuron, resulting in huge number of created synapses. Discipline focused on the research in the area of neuronal connections is called connectomics. Map of all synapses in the brain is called connectome (just like genome is set of all genes that one organism possesses). Tricky thing about connectome is that brain is dynamic system that is undergoing changes all the time. Network between neurons that were present while we were young will inevitably be altered as we are growing old. Learning is process that creates novel connections between neurons, and on the other hand, neurons have limited lifespan and once they die, link with the neighboring cells will be lost. However, the biggest problem in connectome project is asscoiated with tracking and counting the large number of existing neuronal cells and their connections. Fully explored and mapped brain belongs to C. elegans. It’s relatively simple connectome consisting of 302 neurons and 7000 connections. Mouse brain (>10^8 neurons) is under investigation, but there are still some technical issues that need to be solved before complete and accurate map of mouse brain become available.

Brain networks could be obtained in the couple of ways. Connections between cells could be determined on the cellular level by tracking each neuron and its connections. This approach is the hardest and complete mapping would demand substantial amount of time considering that over billion neurons are normally present in the brain of the highly evolved animals (just human cortex consists of 10^10 neurons creating ~10^14 synapses). Mapping connectome on this level is called microscale connectome.

Mesoscale connectome is focused on revealing anatomical and functional connection between larger populations of neurons (hundreds and thousands of cells sharing the same function).

Macroscale connectome is dealing with larger brain systems that are divided in functional nodes. Main goal is to determine their connectivity.

Several techniques are applied in brain mapping. Tracing agents and different staining techniques are used for single cell tracking; visualization of the neuronal networks is achieved by light or electron microscope. Disadvantage: these methods can’t provide long-range neural projection and light microscope derived images have low resolution. Improvements in the field of mapping individual neurons are made after applying fluorescent proteins in the process named brainbow. Method is relatively simple. Red, green and blue derivatives of green fluorescent protein are inserted and randomly expressed in the neuronal DNA, resulting in over 100 color variations that could be tracked using confocal microscopy. Brainbow is tested in mice, and so far, this method proved to be successful, especially for mapping complicated neuronal circuits. To obtain complete view of neural networks in the brain – thousands of pictures had to be collected and aligned to match perfectly. Method used in brainbow tracking inspired another group of scientist to develop new technique for neuron labeling. Using DNA sequences that are acting like a barcodes, each neuron could be marked. Connections between neurons could be established using viral vectors that will transfer another barcode to the following postsynaptic neuron. At the end, each neuron will be a “bag full of barcodes” (bearing his and virally transferred DNA sequences), grouped in pairs. High-troughput sequencing could further provide information on the neuronal connectivity. Sequencing is not as expensive as it was couple years ago, and this approach has a great chance to be successfully implemented in the future connectomics projects.

Although, all currently used methods have some weak points, they are constantly improving and mapping of the mammalian connectome will probably happen soon. Due to dynamic structure of the brain - every map will be unique, but fundamental connections will be revealed.
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Couple of ways to improve vaccines…

November 11, 2012, 6:12 am
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Vaccines are providing resistance against certain infectious agents by stimulating adaptive immune response. Idea that immunity could be boosted using attenuated or dead viruses is very old. First notes about primitive vaccination are dating back in 18th century. Today, vaccination is common practice and number of available vaccines (for different purposes) on the market is growing rapidly. A lot of things changed and improved since first vaccine was invented.

Cancer vaccines are one of the relatively new inventions in the vaccination field. They are used either to prevent cancer development or as a part of cancer therapy. Typical examples are vaccines that protect against cervical (induced by human papiloma virus) and liver carcinoma (induced by hepatitis B virus). Antigens used to provoke immune system are usually proteins isolated from the cancer cells. Other type of anti-cancer vaccines could enhance immune response in situ. Modified herpes simplex virus in the OncoVEX GM-CSF vaccine is genetically engineered to carry GM-CSF gene. This gene is stimulator of the immune response. During viral replication (which is selective - tumor based), GM-CSF level will increase and boost immune system response. Main problem with vaccines that could be used in cancer treatment is associated with the antigen of choice. Tumor cells are bearing unique and shared antigens. Shared antigens are mutual for many cancer types and unique are tumor specific. Mutation that is characteristic for some viruses is making antigen selection process even harder. Success in cancer therapy is depending on couple of factors. Illness stage: the sooner treatment starts, the better outcome will be. Right antigen choice: perfect vaccine could induce immunization against couple of antigens, minimizing the chance on developing treatment resistance. Health status of the individual that is undergoing the treatment: healthier immune system will result in longer lifespan.

Adjuvants are tightly associated with vaccines. Those agents don’t induce immune response on their own but significantly increase immunization by enhancing body reaction to the ingested antigen. Adjuvants (like aluminium salts, some type of oils, virosomes…) are mimicking pathogen-associated molecular patterns and trigger whole set of immune cells that are typically seen during infection (dendritic cells, macrophages, lymphocytes…). Adjuvant needs to match antigen. Some combination induce weaker than expected immune response, can provoke local reaction or trigger IgE response. Recent study focused on finding new adjuvants that could be safely applied and provide efficient immune response. Nanotechnology offered solution. Nanoparticles as adjuvants for vaccine are currently under investigation. Couple of studies showed that nanoparticles carrying proteins, peptides or DNA could deliver it safely to the targeted cell (dendritic cells, for example); often more than one (encapsulated) antigen will be delivered; slower (controlled) release of the antigen could be achieved; being biodegradability and non toxic, nano-particles could be easily degraded and eliminated after antigen is released. Gamma-PGA nanoparticles will be tested soon for its potential use as next adjuvant in vaccine development.

Vaccines are usually delivered subcutaneously, using needle. Couple new options for vaccine delivery arose recently. Nanopatches could be used for transdermal delivery of the vaccine. They consist of 20,000 microscopic projections per square inch and unlike needles - they don’t penetrate to the muscle. Beside lack of pain, other advantage of nanopatch vaccine delivery is higher concentration of immune cells in the skin compared to a muscle. Application is very simple; once nanopatch is glued to the skin, it will release its content to the skin and induce immune response. New method that could improve vaccine delivery even more is associated with bacterial spores that could serve as carriers. Bacillus suptilis derived spores could last for million years, they are highly resistant to different environmental condition and germination will happen only under special circumstances. Spores carrying vaccines against tuberculosis, influenza and tetanus are already tested with promising results. Spores are stable and substantial amount of money could be saved by eliminating expensive preservation methods. Bacillus subtilis is easily grown in the laboratory (spore are inexpensively produced). Nasal or oral vaccine administration routes are much more convenient than classical needle approach. Also, they could be developed as biofilms for the sublingual delivery. Finally, adverse effects associated with different excipients could be avoided using spores as delivery system.

Vaccines are one of the most influential treatments ever designed with tremendous effect on the global health. They are associated with a lot of failures as well, but modern age and development of technology improved vaccine industry a lot.
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Endogenous Gene Expression in Plants

November 12, 2012, 5:59 am
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In plants, certain genes are responsible for certain characters. Plants processes are often modified so that they can be made of use commercially. In these cases, it is often seen that upon eliminating the gene expression of some of the endogenous genes, the required traits was improved. Several mechanisms responsible for these are:
(i) Anti sense gene approach: A DNA consists of two strands complementary to each other. The strand which run from 5’ to 3’ is known as sense strand and the genes in the same is known as sense gene. Similarly the complementary strand to the sense strand is known as anti sense strand and runs from 3’to 5’ end. The genes in the latter are known as antisense genes. The mRNA produced by transcribing the anti sense strand are therefore similar to the sense strand.

In order to produce an antisense gene, the protein encoding region of the gene is inverted so that it becomes oriented in 3’ -5’ direction. The RNA produced upon transcription of the same is therefore similar to the original anti sense strand and is known as anti sense RNA. In a nucleus which consists of normal endogenous gene as well as altered anti sense gene, the transcription of both yields mRNA molecule which are in turn complementary to each other. Thus, the two RNA molecule pairs with each other resulting in the formation of a double stranded RNA molecule. The double stranded RNA molecule so produced is not available for translation and the protein formation is altered. It may also be attacked by RNAases which are double stranded specific and hence attacks only the altered mRNA molecules. Another effect can be methylation of the promoter as well as coding regions of such genes resulting in the silencing of endogenous genes.

Production of Flavr Savr tomato:
The technique of anti sense gene approach has found an important application in improving the quality of tomatoes. In tomatoes, an enzyme called polygalacturonase is produced which is responsible for degrading pectin. The pectin is major component of cell wall and the degradation of the same results in ripening and deterioration of the fruit. In order to improve the shelf life of tomato, anti sense gene therapy is utilized wherein anti sense gene construct of the enzyme polygalacturonase is produced in such modified tomato so that the over ripening of the fruit is seen to reduce rapidly and thus the general quality of the tomato is increased.

(ii) Co suppression of endogenous genes
It is found that, over expression of sense RNA in some plants was responsible for drastically reducing the expression of the corresponding genes. This process of suppression of gene expression is known as co suppression.

This co suppression can be achieved by introduction of homologous sense construct into the nucleus so that it produces target mRNA along with the endogenous mRNA leading to an over expression and consecutive suppression of the gene expression.

It is proposed that, accumulation of RNA transcripts activates production of aberrant RNA from endogenous gene. These aberrant RNA produced activates RNA dependent RNA polymerase which inturn transcribes RNA leading to production of antisense RNA. This will form duplex of RNA molecule resulting in the degradation and reduction of expression of the corresponding RNA. An important example is that of ethylene in fruit ripening. Ethylene is a phytohormone actively involved in ripening of fruits. It is produced from methionine by an enzyme called as ACC synthase into aminocyclocarboxylic acid (ACC). This, when acted upon by ACC oxidase forms ethylene. The production of ethylene can be controlled by integration of antigenic constructs of any of the two enzymes ACC synthase or oxidase or co suppression of any of the enzymes. Thus a reduced ethylene production results in delayed petal senescence as well as delayed fruit over ripening.

(iii) RNA –mediated interference (RNA i):
This technique involves silencing of homologous gene expression activated by presence of double stranded RNA molecule. The double stranded molecule gets degraded by specific degradation mechanism. The long double stranded RNA molecule is cleaved by an enzyme called as dicer. This breaks up long double stranded DNA molecules into small molecules called as small interfering RNA (si RNA). These bind together to form a complex known as RNA-induced silencing complex. The antisense RNA molecule so generated pair with the target RNA molecules and hydrolyse it by initiating an endonuclease activity thus resulting in controlling the respective gene expression.
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Genetically modified pigs - Production and Application for medicine and agriculture

November 14, 2012, 11:15 pm
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The use of swine in biomedical research has gained much importance as they have always been considered excellent models for the studies related to atherosclerosis, various cardiovascular diseases, cutaneous pharmacology, diabetes, cancer biology, ophthalmology and toxicology, lipoprotein metabolism, pathobiology of intestinal transport, injury and repair, repair and healing of wounds, etc. They have also been considered for being potential source of different organs for the xenotransplantation as can be seen in the heart transplantation studies.

While the different biological systems of pig have similarities with the corresponding human biological systems, there are a number of advantages offered by the study of pig as research model such as short generation interval, short gestation period being around 114 days and the production of multiple offspring at a time. The complete sequencing of the genome of a particular species of animal is essential for basic research involving it, otherwise the species is considered second-class species. Phylogenetic approaches have shown the presence of high rate similarity between pig and human genome compared to that of mouse. Moreover, the availability of inbred strains of pig facilitates the use of pigs in different research studies.

There are a number of methods to carry out the genetic modification of the animals.

a) Injection of DNA construct directly into the pronuclei of zygotes, first tested in mice has been applied in mammals.

b) Oocyte transduction, whereby the replication defective retrovirus is integrated into the chromosomes of the oocyte after its injection between the zona pellucida and the plasma membrane of oocyte that is arrested in the metaphase II of meiosis. It was carried out in pigs after application in cattles.

c) Sperm-mediated gene transfer, a method highly efficient for the transgenic pig creation, whereby the in-vitro fertilization or insemination of the pigs was carried out with sperm previously mixed with DNA construct of interest.

As targeted integration is not achieved properly in other methods, it makes way for the development of homologous recombination method or use of Embryonic stem cells (ES cell technology). However, this method has been successfully applied only in mice and for other species, a true ES cell that goes with germline is yet to be developed. Presently, the method utilised for the introduction of targeted modifications, knock-ins or knock-outs, is via the cloning of modified somatic cells. Due to advancement of technology, cloning is being developed using different stem cell lines, which has been proved successful by the production of cloned pigs from the stem cell line developed from mesenchymal stem cells and skin-derived stem cells. The introduction of genetic modification using zinc finger nucleases in combination with the donor stem cells may prove to be a highly efficient method for the genetic modification of swine.

The different applications of genetically modified pigs in medical field can be summarized as follows:

1) The α-1, 3-galactose cell surface epitope produced by the GGTA1 gene in pigs causes the production of the anti-gal antibodies in humans leading to hyperacute rejection (HAR) of the transplanted organs. Hence, the GGTA1- gene knockout pigs would offer successful transplantation of the organs without evoking any immunological response within the human body.

2) The production of human haemoglobin in the blood of transgenic pigs for isolation and treatment of trauma patients is one of the interesting applications being studied. The production of Protein C, in-activator of certain human coagulation factors Va and VIIIa in the milk of pigs has been studied. It has been found that the mammary epithelial cells of the pigs are capable of making the coagulation factors VIII and IX biologically active due to post-translational modifications.

3) The transgenic pigs can be used as better models for different diseases such as Retinitis pigmentosa, cardiovascular diseases: Fat-1, Diabetes, Alzheimer’s disease, cystic fibrosis, Huntington’s disease by the introduction of different mutations in the genes involved in the pathophysiology of the diseases.

4) The transgenic pigs can be used for cell tracking with the introduction of genes expressing different fluorescent proteins into the pigs. The stem cells expressing fluorescent proteins isolated from these transgenic pigs can be used as molecular markers for the tracking of various biological mechanisms.

5) Production of human and pig hybrid organs is a very interesting application that needs further in-depth study. The production of human hepatocytes in transgenic pigs to help in the transplantation of the regenerated human hepatocytes to patients of liver failure from the transgenic pigs shows great promise.

6) Transgenic porcine livers expressing albumin gene are being studied for use as bio-artificial liver support system as a bridge to human liver transplantation.

Transgenic pigs also have application in agriculture in the production and growth of pigs whose meat are safe environmentally, lean and healthier for human consumption by the introduction of different genes expressing growth hormones and to reduce pollution by alteration in the composition of the carcass.

The in-depth study of genetically modified pigs has met with remarkable advancement in biotechnology and can be utilised for various therapeutic applications in near future for the benefit of mankind and community.
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Somatic Hybridisation for the Production of Hybrid Plants

November 15, 2012, 3:16 am
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The process by which protoplasts of two different plant species fuse together to form hybrids is known as somatic hybridisation and the hybrids so produced is known as somatic hybrids. The technique of somatic hybridisation involves the following steps.

(i) Isolation of protoplasts:
Plant cell consist of cell wall which has to be degraded if the protoplasts of the cell has to be obtained to be manipulated as required. For this purpose, the plant cell is treated with enzyme like pectinase, macerozyme, and cellulase etc., which hydrolysis the plant cell wall. The conditions are altered so that successful release of protoplast is aided. The osmotic pressure of the solution is controlled by addition of calcium chloride salts into it. This improves the plasma membrane activity. Since protoplasts are present in every plant cell it can be theoretically isolated from all the parts of plant. But most successful isolation was made possible from leaf of the plants. The leaf is surface sterilized and lower epidermis is removed, and treated with enzyme solution.

(ii) Fusion of different protoplasts:
Different protoplasts isolated are treated with different mechanisms so that they fuse together. The mechanisms followed:

High calcium high pH treatment:
Here, the different protoplasts in one solution are together treated with conditions like high calcium and high pH so that they fuse together. In some cases such extreme conditions has proved to be toxic to certain protoplasts.

Polyethlylene glycol (PEG) treatment:
This has proved to be one of the most effective methods for protoplast fusion. The cells are treated with a concentration of around 30% poly ethylene glycol which binds to plasma membrane. This is treated with calcium solution which being cationic binds to PEG. During washing, the PEG pulls out the plasmalemma leading to fusion of protoplasts in close proximity. This leads to fusion of protoplasts randomly and so is a non selective fusion process.

Electro fusion technique:
This process involves passing low voltage electric pulses in a solution of protoplasts to be fused so that they line up for fusion. The protoplast can be fused by subjecting it to brief exposure to high voltage electric current which leads to alteration of membrane so that the adjacent protoplast fuse. The protoplasts so lined up can be moved by the use of micromanipulator so that required protoplast can be fused. This is carried out in an electroporater.

Selection of hybrid cells
After a successful protoplasmic fusion experiments, a variety of structure are available like unfused protoplast and protoplast of same species fused together and protoplasts of different species fused together or hybrid cells. In order to separate the hybrid cells from other residue several techniques are followed. Mechanical isolation of fused protoplasts is done or taking advantage of natural properties exhibited by host cells so that the cells showing absence in that property indicate the hybrid cells. Another important method is by culturing all the residues and formation of calli which is then studied to identify the hybrids.
The hybrids formed are of different types like:

Symmetric hybrids: These contain the somatic chromosome of both the parental species. These are very significant as they show all the properties exhibited by parent species.

Asymmetric hybrids: These are those hybrids which preserve the genetic material of one parent organism. The chromosome content of other parent species is lost.

Cybrids: These consist of nucleus of one species and cytoplasm from both the species. They are produced by fusion of one species with another having enucleate protoplast or having inactivated nucleus or loss of chromosomes of one parent by repeated mitotic division. It can even be induced by inactivating nucleus of a protoplast prior to fusion. The cybrids produce many advantages like transfer of plasmagene of one species into the nuclear background of another species, formation of recombinants between mitochondrial or chloroplast genomes.

Thus somatic hybridization techniques help in forming wide variety of recombinants among the plasma gene of different species and plasmagenes and chloroplast genes. It also helps to form hybrid cells exhibiting chloroplast genome of one species and mitochondrial genome of another species which is not possible by ordinary means of hybridization of two plant species. These different levels of fusion and recombination helps in production of new species which has all the qualities of parent organisms or even better.
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Detection of Transgenic Animals

November 15, 2012, 11:25 pm
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Transgenic Animals Detection:

In recombinant DNA technology after the gene of interest has been isolated and transferred efficiently, the next major step is to ensure its proper and maximum expression in order to obtain proposed results. Thus in a group of transgenic animals it is important that the animal with proper transgenic integration is identified.
Identification of transgenic animals:
The identification of transgenic animals is done using different mechanism.

In animals, where the transgene produces a distinctive phenotypic effect, the transgenic successful animals can be noted easily. But the results of all the gene transfer does not result in such distinct effects therefore other techniques have to be employed. The dot blot technique and PCR technique are to name a few.

Dots blot technique:
This technique helps in detecting several samples in one experiment. The sample of DNA collected is fixed onto a support like nitro cellulose filter. This then undergoes denaturation so that the double helix separates. Such membrane containing denatured sample when treated with radioactively labelled probe of corresponding transgene, the sample DNA incorporated with transgene binds with the probe. Upon removal of free probes by washing and analysed by autoradiography, it reveals presence of transgene which can be detected by fluorescence produced by radioactive probes. The strength of radioactivity exhibited shows the strength of transgene integration.

PCR technique:
This is the most important and wide speed used technology to identify the transgenic successful animals. The primers corresponding to the integrated transgene is used to amplify the test DNA sequences isolated from the transgenic animals. This results in the amplification of the transgene. The amplified DNA when blotted and hybridized, the presence of transgene is confirmed. But the techniques adopted for identification of transgenic animals does not reveal the actual level and site of integration. For achieving these further steps has to be adopted like:

Analysis of transgene integration:
In order to analyse the transgene integrated, the isolated gene samples which are detected with transgene integration, undergo restriction digestion with known restriction endonucleases. The fragments are separated by agarose gel electrophoresis and these fragments are analysed by southern blotting procedure. The fragments on the gel are transferred to a nitrocellulose filter membrane and denatured and probed with radioactive probe to reveal the actual site of transgenic integration. The integration of transgene can be confirmed by choosing the appropriate restriction enzyme. The presence of single or multiple integration of transgene is indicated by southern blotting.

Analysis of mRNA production:
The mRNA produced from a transgene can be detected as it is different from the native mRNA of the organisms. These can be purified and hybridized with a radioactive probe to detect mRNA production.

Analysis of protein expression:
The final aim of transgenic integration is to produce proteins coded by the transgene and its efficient expression. Therefore the detection of transgene integration is possible by protein expression of the transgene. The analysis of transgenic proteins can be done by two methods: by identifying specific antibodies produced by transgenic proteins and by studying the enzymatic properties of the concerned proteins. In the case of antibody identification, various immunologic assays can be used. ELISA test is a classic example of the same. In this case, the antibody specific to the antigenic protein is added and allowed to react. Following the first reaction, this is further reacted with an antibody specific to the former antibody results in formation of a complex. This when treated with the substrate of corresponding enzyme, it produces colour proportional to the strength of the antibody indicating the amount of corresponding transgenic protein expressed. The other assays included are immunoblotting, radioimmunoprecipitation, immunohistochemical staining etc.

Enzyme activities can be detected by using transgenic genes which produces different enzymatic activities or different pathway which is not shown by host enzymes. Various procedures can be employed to detect this. The main procedure followed is by transfer of scorable marker genes. Scorable marker genes are those which produce a definite phenotype. Main examples for these are chloramphenicol acetyltransferase (CAT) genes used mainly in fish or mammal cells, betagalactocidase and luciferase gene used in fish.

All this analysis has to be repeated in two or more different potentially transgenic animals so as to determine the level of transgenic integration as, in different organisms it tend to differ. This occurs because the site of integration may differ among animals. The animals has to be studied to confirm which site integration yield maximum expression and further research must be forwarded to achieve the same.
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Organs Participating in Developing Immunity

November 15, 2012, 11:56 pm
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Like an army has group of several battalions, the defensive mechanism of the body called as the immune system is composed of several organs, each performing a significant role contributing to the overall protection of the body from pathogens. Based on the type of activity the organs provide they are classified into primary lymphoid organs, secondary lymphoid organs and tertiary lymphoid organs. The organs with specific role grouped under these categories are collectively called as the organs of the immune system.

The main function of the primary lymphoid organs is to develop matured lymphocytes and the secondary lymphoid organs acts as the ground for lymphocyte – antigen interaction. In an event of inflammatory reaction, the role of the tertiary lymphoid organ is to import the lymphoid cells.

Primary Lymphoid Organs: Bone marrow and thymus are the primary lymphoid organs acts as the sites for lymphocyte maturation. Lymphocytes are the major cells of immune system composed and classified into B-cells, T-cells and null cells based on their function. Bone marrow houses B-cell maturation whereas the T-cells mature in the thymus. The primary lymphoid organs outgrows in the fetal stage itself from the junction of ectoderm and endoderm or from the endoderm. The stem cells of the bone marrow are the source for all the cells involved in immune response. The soft tissue of the bone, the bone marrow is composed of two compartments called as the Haemopoietic compartment and the vascular compartment. The former enclosed by layer of reticular cells possess the precursors of all the blood cells, clusters of lymphocytes and macrophages and the later, the vascular compartment is lined by endothelial cells and crossed by reticular cells and macrophages.

Thymus, the flat bilobed structure located in the thoraxic cavity acts as the site for T-cell maturation. The bilobed structure has two sections called as the cortex and the medulla. Cortex, the outer compartment is crowded with Thymocytes, the immature T-cells and the medulla region is thinly populated with T-cells. The proliferation and maturation of thymocytes happens in the cortex region following which they pass through the medulla region for further maturation before leaving the thymus. The alpha and beta thymosine, thymopoietin, thymulin and thymostimulin are the hormonal factors of the thymus participating in the differentiation and maturation of the thymocytes. The Bursa of Fabricius is the bone marrow equivalent primary lymphoid organ present in birds.

Secondary Lymphoid organs: The secondary lymphoid organs develop from the cells of mesoderm in the later stages of fetal life. Antigenic response stimulates the growth of the secondary lymphoid organs. These organs are rich sources of B cells, T cells, macrophages and dendritic cells. The lymph node, spleen and the mucosa associated lymphoid tissue are the secondary lymphoid organs.

The bean or round shaped lymph nodes with reticular network packed with lymphocytes, macrophages and dendritic cells are the first tissues to encounter antigens entering the system. The morphological division of the lymph node has three sections namely the outer layer cortex followed by a second layer paracortex and the inner area medulla. Cortex is rich in B cells, macrophages and dendritic cells. The paracortex is populated with T cells and dendritic cells and the medulla region is sparsely populated with lymphocytes mainly B cells and fewer amount of macrophages. The fate of the antigen depends upon whether the person is already exposed to the antigen or not. An antigen entering the system for the first time is carried by the lymph to the lymph node, where it enters the medulla region and gets phagocytosed by the macrophages present in that region. The phagocytosed antigen travels to the paracortex region where they are encountered by the T cells which migrate to the cortex layer triggering the B cell division and the divided B cells (Antibody producing cells) travel back to the medulla and released into the efferent lymph of the lymph node allowing it to pass through the other lymph nodes. The entire process of antibody production in response to an antigen takes several days and if the person is exposed for the second time to the same antigen, the foreign molecules get trapped by the antibody coated dendritic cells present in the cortex region of the lymph node.

Spleen functions by filtering blood resulting in the removal of antigens and the old blood cells. Spleen is considered as the major site for antibody production and effector T cells. Spleen has two compartments called as the red pulp and the white pulp. Red pulp region forms and stores the red cells and also involves in antigen trapping whereas the white pulp, rich in lymphocytes is the site for immune response. Mucosa Associated lymphoid tissue (MALT) is the secondary lymphoid organ governing the mucosal lining of the digestive system, respiratory system and the urogenital system from infections.

The cutaneous associated lymphoid tissue is the tertiary lymphoid organ which is the skin. The external epidermal layer of the skin is composed of Keratinocytes, the epithelial cells secretes biologically active substance called as Cytokine which actively participate in local inflammatory reaction.
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Biotechnological Methods Of Disease Diagnosis

November 16, 2012, 1:24 am
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Disease diagnosis refers to identification of the cause of the disease. Conventional methods include microscopy, culture of specimen and testing for sensitiveness, several immunological assays etc. But these conventional mechanisms often have negative aspects like being tedious; taking longer time etc. In order to overcome this, various biotechnological approaches has been developed. These are:

Probes:
Small nucleotide sequences used in detection of complementary sequences in nucleic acid sample is known as probes. These probes can be radioactively or non radioactively labelled so that they can be used for detection purposes. The samples like blood fluids, tissues etc., can be analysed with probes for disease diagnosis. The main mechanism by which probes can be used is by:

(i)Hybridisation: The process by which DNA samples are allowed to bind to complementary probes for detection purposes is known as hybridisation. This may be by dot blot method, southern hybridisation. A probe hybridizes with a test sample only when the complementary sequences match. A sample preparation is done either on a solid support like nitro cellulose filter or it can be prepared in situ and used for in situ hybridisation. The probes which are used in diagnostic procedures are extremely sensitive to the causative organism. Hence a positive hybridization tests implies the presence of pathogen and thus the disease.

(ii) Ligase chain reaction: In the corresponding process, the DNA sample prepared is added to the reaction mixture having ligase enzyme and two oligonucleotide probes. The DNA sample pair with the complementary probes and a chain reaction is initiated. This when viewed under UV, bands corresponding to probes as well as those corresponding to target DNA is visible. The probes alone appear as a single band and the target DNA is flanked by two oligonucleotide probes and appear as a band which is equal in size to the sum of two probes.

The advantages of using a probe is that it is highly specific, and the procedure is relatively simpler and rapid. The results are obtained even when the amount of sample is less.

Monoclonal antibodies:
Monoclonal antibodies are a preparation of antibodies so that it is highly specific to a single epitope of an antigen. It is employed in immunological assays like ELISA, immuno PCR wherein monoclonal antibody specific for an antigen is attached with a marker and used for identification of specific antigen. This is also used in preparation of autoantibodies. Autoantibodies are produced by an organism in instances of auto immunity against its own organs. The antigenic specificities of such antibodies can be used in treatment of autoimmune disorders.

Detection of genetic diseases:
Genetic diseases are in born defects of a person. These are mostly caused due to single recessive mutation. Foetal cells are retrieved and diagnosed for any possible genetic diseases. The sample for such diagnosis is obtained from biopsies of trophoblastic villi which is an external part of human embryo. These can be used for detection of genetic diseases in many ways.

(i) Karyotyping of the cells helps in obtaining information about any chromosomal aberrations.

(ii) Assay of foetal cells reveals information about any defective enzymes produced relating to genetic diseases.

(iii) Modification of recognition site of restriction enzyme can occur as a result of genetic diseases. This can be detected by conducting RFLP analysis of foetal samples after southern hybridization process. For eg: in the case of sickle cell anemia, the defective and normal gene is analysed in this method revealing different band patterns in defective and normal individuals.

(iv) Oligonucleotide probes complimentary to mutated gene sequence caused as a result of genetic diseases and probes complementary to corresponding normal gene sequences are used to probe with the samples suspected of genetic diseases. The radioactive bands produced can be used to distinguish the former from the latter. A classic example is that of sickle cell anaemia. The sample is probed with probes complementary to the sequence altered by mutation and also with probes complementary to normal sequence. This is possible only in cases where, the bases of mutated sequence as a result of genetic disease as well as the normal unaltered sequence is known to allow synthesis of two probes for the technology.

These mechanisms have several advantages from conventional methods being comparatively powerful, and do not include risk of contamination as in the case of microbial culture method. It is even applicable in detection of pathogens which cannot be cultured. Another important application of these methods is that it has the capability to detect even latent viral infections.
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Biological agents as fuels: Bio fuels

November 16, 2012, 1:53 am
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Fuels obtained from biological sources are known as bio fuels.
The branch of biotechnology dealing with the exploitation of biological agents to convert it into sources of energy is known as fuel biotechnology. The bio fuels produced should be portable in large quantities in vehicles, should be able to burn in internal combustion engines of vehicles and should be approximately equivalent to petrol in energy content.

Biogas: It is one of the early and largely produced sources of energy. It is produced from biomass by simple burning or using sophisticated technologies of breakdown. It can be produced by small scale production units, recovery and conditions of production is not costly. But the product released is of low yield and some times pure gases are not evolved.

Bio ethanol: Bio ethanol is produced from biomass by the action of various microbes. The production of bio ethanol occurs from two different raw materials.

(i) From sugar and starch crops: The raw materials is acted upon by microbes like Saccharomyces cerevisia, Bacillus licheniformis, Zymomonas

(ii) From cellulosic materials: This involves two method by enzymatic hydrolyses and by chemical hydrolysis. Organisms like Trichoderma reisei, Saccharomyces cerevisiae, Clostridium etc., break down the biomass or rather the cellulose present in the biomass by the production of enzyme cellulase. The cellulose gets converted to sugars which are broken down by any S. cerevisiae into ethanol. In certain cases, the pentose is formed as an intermediate and only genetically modified E. coli can break down this into simple sugars.

The ethanol so produced is recovered from the water-ethanol mixture by distillation utilizing the difference in boiling points of water and ethanol.
Advantages of bio ethanol as a fuel include: The heat of vaporization is much higher than petrol resulting in less heating up of cylinders. The higher octane number than petrol results in higher power production and no pre-ignition of bio ethanol over commercial petrol. Since it is burnt completely, hydrocarbon residue is not released forming a much cleaner fuel. It has comparatively less chances of catching fire during accidents. In commercial market, petrol is mixed with ethanol to produce ‘gasohol’ which yields good energy and high octane number.
It also include negative aspects like it absorbs moisture, the downstream recovery is high, and the engines under ethanol utilizes more fuel than petrol.

Biobutanol:
This is produced from Clostridium acetobutylicum by anaerobic fermentation. The substrate used is molasses. The production has not met with much success as the cost incurred was too high and application of genetic engineering techniques by modifying the organism resulting in high expression; high substrate utilization is being considered.

Bio diesel
Bio diesel is produced mainly by two ways- from lipids and from hydrocarbons by plants and algae

Bio diesel from lipids:
Lipids are source of energy and can be utilized to release the same. Many plants store lipids in their seeds and this can be processed to produce esters of lipid fatty acids. The product seems to resemble diesel hence known as bio diesel. Bio diesel can be used in the natural form without much modification directly as a fuel.

Bio diesel from hydrocarbons:
Certain plants have the ability to accumulate hydrocarbons which can be utilized to produce fuels. The plants that accumulate hydrocarbons do it as latex. The plant species of family Euphorbiaceae, some milk weeds like Asclepias species, and a tree called as C. multijuga has the above said ability.

Plants of the family Euphorbiacae produce latex which has hydrocarbons emulsified in water. Separation or removal of water yields necessary hydrocarbons. The milk weeds also store latex which can be removed and utilized. The tree C. multijuga is a native of Brazil and it fixes nitrogen in its roots and produces a liquid large in volume and which is quite similar to diesel oil. This can be utilized as an efficient source of bio diesel production. Some freshwater and marine algae are also known to deposit hydrocarbons. This also acts as a source of diesel.

Positive features of bio fuel:
The source from which the bio fuel is produced is biological which is renewable in nature resulting in unlimited production without fear of depletion of resources as in the case of conventional sources like oil, petrol etc. The carbon dioxide emission by burning of bio fuels is much less comparatively. The other polluting gases like sulphur dioxide are not released helping in keeping environment clean. They burn completely and so the energy released is high. The left over residue can be used as manure.

Negative points:
Larger volumes of raw material are required to produce a good quantity of bio fuel and the cost of production is also high.
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Markers Involved in Creating Genome Maps

November 16, 2012, 4:31 am
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The whole genetic constitution of an organism is known as genome of an organism. A detailed description of positional, structural and functional properties of the entire genome is known as genome maps. It can be broadly classified as cytogenic and molecular genome maps depending on the position of the genes and the total information of the gene respectively.

An initiative was started by many countries to obtain entire information about the genome of humans. This is known as human genome project. This was started in the year of 1990 and a complete draft was submitted in 2003. The strategy involved in human genome project was:

(i) Preparing maps of each gene of the genome to decide the location of the gene

(ii) Sequencing the genes to determine the different gene elements and any variations in the same within or between two genomes

(iii) Functional analysis of genes to determine the role of each gene in an organism.

The major markers involved in the genome projects are RAPD, RFLP, VNTR, chromosome jumping etc. This helps in mapping and sequencing of unknown genes.

Restriction fragment length Polymorphism (RFLP): This employs a mechanism by which same genes of different organisms or related organisms can be studied. The mode of action involves restriction digestion with a specific restriction enzyme. Genomic DNA is isolated from different species or related organisms. This DNA is digested with the help of a selected restriction enzyme and the fragments produced are separated through gel electrophoresis. This gel is transferred onto a solid support and is followed by hybridization of the gel with appropriate radio-labelled DNA probes. The gel is then scanned by auto radiography. The pattern generated by different organisms differs in these patterns and mainly depends upon the DNA used for digestion, the restriction enzyme used and the DNA probe used. The difference in the genomic DNA accounting to various RFLP is the changes in the base recognition sequences of the restriction enzyme, or addition or deletions. The probes used is available from a variety of genomic library, chromosome specific library etc, based on the bands available on the gels, each organism has a specific RFLP loci for specific restriction enzyme and an RFLP map is created. This technique helps in mapping even very small segments of DNA and the process is very rapid but the disadvantages like high cost and the need of skilled personnel.

Random Amplified Polymorphic DNA (RAPD): This technique mainly employs PCR. In this, a genomic DNA is isolated and fed into a PCR. Here, the process of denaturation leads to unwinding of the two strands of DNA. The denatured strands when renatured is added with a short oligonucleotide sequence called primer which is of known sequence. Upon annealing this sequence pairs with the homologous sequences in the DNA which has similar sequences at random locations. Sequences in DNA complimentary to primer at the both ends are amplified during the amplification process. The amplified DNA is detected by gel electrophoresis followed by fluorescence detection.

Since this process does not utilize restriction enzymes and probes it is comparatively cost effective. But the reproducibility in comparison to RFLP is poor.

Chromosome walking:
This is a chromosome based technique and helps in obtaining detailed knowledge of chromosome. It requires knowledge of a genetic marker. This known gene marker is used to identify a clone that has a corresponding DNA insert. The DNA fragment consisting of such a known marker is isolated and a restriction map of this fragment is prepared. This is followed by isolation and sub cloning of a small segment of this fragment. This clone acts as a probe in identifying the presence of such segment in a genome library. The gene identified in this manner will have one end of the segment similar to the probe and an unknown fragment. A restriction map of this unknown fragment is prepared and similarly used to probe identical sequences in another library. The resulting probing will involve production of new fragment with one known and one unknown sequence. This process can be continued until the end of chromosome is reached. Thus it helps in ‘walking’ of the chromosome.

Variable number of tandem repeats (VNTR): This consists of regions in DNA which has variable number of repeats. Different individuals show different repeats at a given loci or position of a chromosome. This constitutes alleles of VNTR loci. They can be broadly classified as micro and mini satellite DNAs. These make up the hyper variable region of DNA. Minisatellite DNA consists of pro terminal regions of DNA. Micro satellite DNA consist of regions which are short sequences and more frequent hence represent an efficient marker system.

Thus it is possible, with the help of such markers to sequence and map efficiently genetic constitution of an organism as a whole resulting in developing genome map of the organism.
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Application of recombinant interferons in medical field

November 17, 2012, 3:35 am
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Cytokines belong to the group of proteins, which are usually regulatory in nature and maybe glycoprotein in some cases, produced by the body in minute amounts. Their main role can be seen in cell communication by the trigger of different signal transduction pathways within the cell after binding to the specific receptors on cell surface. They are usually produced by leukocytes and play major roles in the immune system such as haematopoiesis and inflammatory systems such as healing of wounds. Interferons belong to the class of cytokines that were discovered first. Wide range of interferons are secreted by different species, and in humans, 3 types of interferons have been studied- Interferon α, Interferon β and Interferon γ, which play important roles within the human body such as
a) Development of cellular resistance against viral attack,
b) Immune function regulation,
c) Growth and differentiation of different cell types, and
d) Sustenance of preliminary stages of pregnancy.

The interferons have potential medical applications due to their antiviral and anti-proliferatory activities as well as their ability in regulation of immune and inflammatory responses within the body. The production of interferons in minute quantities remained a drawback in their therapeutic applications. Hence, various techniques for their isolation from different sources were studied. The recombinant DNA technology has helped in the large-scale production of interferons for meeting the different medical needs by the recombinant expression of the interferons in the microbial organisms. However, various purification techniques are essential to remove the non-human substances from the produced interferons before the application of the recombinant interferons in medical purposes. Different types of interferons have been found to have different medical uses as given below:

a) Studies related to Interferon α (IFN- α) have shown its anti cancer properties. It has been associated with tumor regression in patients suffering from multiple myeloma, lymphoma as well as breast cancer. Recurrence of tumor growth after surgery was prevented by IFN- α in patients of osteogenic sarcoma. The development of recombinant IFN- α (rh IFN- α) by the cloning and expression of the genes encoding it has helped in the progress of the clinical studies related to IFN- α. The production of rh IFN- α is generally done in E.coli system. IFN- α have been shown to have anti-viral, immune-modulatory, and anti-tumour properties that has helped in its medical application. Various recombinant interferons have gained approval for marketing such as the PEGylated interferons (PEG IntronA and Viraferon Peg) and the interferon product, which is synthetic known as Infergen. rhIFN- αs have been found effective in the therapeutics for various viral conditions, of which viral hepatitis is one. IFN- α has been found potent to combat a number of diseases induced virally, AIDS being one of them. Hence, it is being appraised for different clinical trials.

b) Recombinant forms of IFN- β (rh IFN- β) have found successful medical application in the therapeutics of the disease affecting the nervous system known as multiple sclerosis that is relapsing and remitting in nature. Although the exact cause for the onset of the condition remains unknown, various factors have been implicated including genetic and environmental (possibly viral infection). Different rh IFN- β preparations have gained approval for medicinal purpose such as Betaferon, Betaseron produced in E.coli cells and Avonex, Rebif produced in the CHO cell lines. However, the exact mechanism by which the IFN- β induces its therapeutic effect remains unclear and it is hypothesised that the down-regulation of the pro-inflammatory response may be the responsible factor.

c) IFN- γs have found medical application in the treatment of a rare genetic condition known as the chronic granulomatous disease (CGD), in which the phagocytic cells are incapable of being effective against the foreign invaders such as bacteria and protozoa and hence make the body susceptible to various infections that may be life threatening. Rh IFN- γs have been produced in E.coli cells. IFN- γ have been found effective in the stimulation of the phagocytic activity in the patients suffering from various diseases such as cancer, lepromatous leprosy (caused by the bacterium Mycobacterium leprae) as well as AIDS.

Although, interferons have found successful application in medical field, their administration is also accompanied by some side effects like most drugs. The characteristic flu-like symptoms is observed in the administration of the interferons, however in some cases, their administration is accompanied by serious side effects like
a) autoimmune reactions or nervous and cardiac disturbances in IFN- α,
b) hypersensitivity, menstrual disorders, suicidal thoughts and depression in IFN- β, and
c) Heart failure, CNS complications, and metabolic complications in IFN- γ.

However, prediction of the possible side effects is impossible without administration. Hence, careful monitoring of the concerned patients after administration of the interferons is essential before the treatment can be suspended due to the development of unwarranted, serious side effects.
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Application of Nanorobotics-The future of molecular nanotechnology

November 17, 2012, 9:54 am
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Much advance has been made in the field of nanotechnology and nanomedicine, which has initiated the study of the use of robots in the nanometer scale known as nanorobots. The technology of nanorobots has become a raging topic and advanced research is being carried out for the use of robots in the therapeutics of various fatal diseases, for various biomedical applications and manipulations in nanomedicine. The building of biosensors and the nanokinetic devices are a major requirement in the operation and locomotion of nanorobots. Although, nanorobots remain to be a part of scientific fiction, they may have much clinical aspect in future medical diagnostics. Manipulation of nanorobots is a technology enabled by the NanoElectroMechanical Systems or NEMS. With various novel materials and structures in nanoscale, NEMS will help in the development of new nanosensors and nanoactuators.

The science of nanorobotics plays a vital role in the development of robots, whose structure is built by using nanoscale components and objects. The nature of the components being in the nano scale allows the researchers for the engineering of the mimic of human beings. The construction of the various complex parts, which constitute the robots have been possible due to nanorobotics. Nanobots, nanites, nanoids or nanomites are some of the hypothetical devices created with the knowledge of nanorobotics.

Various approaches have been used for the development of nanorobots such as
a) self-directed assembly as seen in the self-assembled monolayers, self-assembled lipidic micelles and vesicles, which follow the Brownian theory of self-assembly.

b) DNA-directed assembly using part of DNA for assembling, which works on the self-assembly principle of complementary base pairing and has application in the DNA based rotary motors.

c) Protein-directed assembly as is seen in genetically engineered chaperon proteins that help in the assembly of gold nanoparticles and CDSe semiconductor quantum dots into arrays in the nanoscale range. Ratchet action protein based molecular motors have also found much application in biology.

d) Microbes and virus directed assembly, which includes various bacteria that are incorporated into microelectromechanical systems (MEMS) and help in acting as living motors, pumps, etc. Viral capsid shells have also found application in acting as scaffolds for the assembly of the nanoparticles such as quantum dots.

The study of the nanorobots is creating wider applications in near future. A number of potential applications of the nanorobots have been brought forward such as
1. Transmigration of the WBC and other inflammatory cells to the inflamed tissues by attaching to them for accelerating the healing process.

2. Drug delivery nanorobots, known as ‘pharmacytes’ will be applied in future therapeutics related to cancer in chemotherapy for precise dosage administration of the chemicals as well as in the anti-HIV therapeutics.

3. Can be used as ancillary devices for processing different chemical reactions in the injured organs.

4. Can help in the control and monitor of glucose levels in diabetic patients.

5. They may be utilized for the targeting and destruction of kidney stones.

6. Can be applied in the therapeutics for atherosclerosis. The atherosclerotic plaques are localized mainly in the coronary arteries. The medical nanorobots may help in locating the atherosclerotic lesions in the stenosed blood vessels and help in their mechanical, chemical, or pharmacological treatment.

7. Nanodentistry is one of the unique applications, whereby nanorobots help in different processes involved in dentistry. They help in inducing oral anaesthesia, desensitization of tooth, manipulation of the tissue for the re-allignment and straightening of the irregular set of teeth and for the improvement of the teeth durability, major tooth repair, generation of nanofiller, improvement of appearance of teeth, etc.

8. Can help in surgery by using surgical nanorobots for nanomanipulation in the target site with programming and guidance from a surgeon.

9. Can find application in cryostasis i.e. reversal of freezing injury by introduction of cryoprotectants and other chemicals into the vascular system rapidly suing nanorobots.

10. Can help in the diagnosis and testing of different diseases and help in their monitoring by recording different biological variables such as temperature, pressure, activity of immune system, etc very rapidly at the target site after oral introduction of nanorobots.

11. Can help in gene therapy for different genetic diseases by helping in introducing different modifications and correction by editing in the right place in the DNA or the proteins attached to the DNA.

However, some disadvantages accompany the use of nanorobots. The complexity of the design and manufacture accompanied by high cost is a major drawback in its wide application. The other disadvantages are the possible anti-social applications that accompany every new discovery in science.

In spite of the drawbacks, the application of molecular nanotechnology may help in the development of therapeutics for different fatal diseases in future, thus creating a revolution in healthcare.


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Effect of Nanomaterials in the environment

November 18, 2012, 3:45 am
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Nanomaterials (NMs) have been defined as particles having one dimension less than 100nm. Among them, the materials with atleast two dimensions between 1 and 100 nm are known as nanoparticles (NPs). In the environment, nanoparticles always exist from different sources both natural as well as anthropogenic and are referred to by many names for traditional use. In air, they are referred to as ultra fine particles and as colloids with different range of size in soil and water systems. In the urban areas, different combustion sources including diesel and gasoline fueled vehicles have contributed a high percentage of different particulate matter including nanoparticles that amount to almost 36% of the total particulate number concentrations. The effect of the nanoscale particulate matter on health especially the respiratory system is being investigated. In comparison to the studies on the ecological systems, the research on human health has focused on various adverse effects that include inflammatory and fibrotic reactions as well as oxidative stress.

Apart from the natural sources of the nanomaterials present in the environment like colloids in soil and water, the manufacture of synthetic nanomaterials has also contributed to the increase in the amount of the nanoparticles present in the environment. The unique properties of the nanomaterials such as mechanical, optical, electrical conductivity, catalytic, etc due to their size in nanoscale has resulted in an exponential growth in the development of various engineered and manufactured nanomaterials for their exploitation in different fields. The development of a wide range of nanomaterials including carbon nanotubes, nanopolymers, quantum dots, dendrimers, nanofibers, nanowires, etc are constantly expanding the synthetic preparation of the NPs. Due to the tremendous increase in the production of NMs, their release into the environment affects the ecosystem health, which is an increasing concern for the regulatory authorities. It necessitates the setting of different guidelines that will give adequate environmental protection as well as help in the growth and development of nanotechnology.

The effect of the accumulation of NMs after uptake is not clearly known, as much research has not been conducted in this area. However, it is assumed that the different organisms living in the environments loaded with NPs would incorporate the NPs within their bodies mainly through the gut, which then gives rise to the possibility of their translocation within the body. Various ecotoxicological studies have been conducted on the different animal models e.g. daphnids. Uptake of the NPs and their translocation from the gut to the reserve fat droplets has been demonstrated successfully. However, the exact mechanism of the whole process is still under investigation. It has been suggested that the entry of NPs is also possible by diffusion through the plasma membranes as well as by endocytosis or adhesion processes.

The release of the NPs into the environment could be intentional or unintentional. The intentional release includes the release of iron NPs into the groundwater for remediation and is controlled in nature. However, the unintentional release of the NPs includes the emissions in atmosphere as well as the solid or liquid waste streams from the production facilities and is uncontrolled. The NPs present in fabric, health care products, cosmetics, etc also enter the environment proportional to the use of the products. Although, the toxicity mechanisms related to the NMs have not been completely elucidated, the different cellular mechanisms in the human body in which the NMs may have adverse effect include
a) the disruption of cell membranes leading to loss of membrane integrity;

b) protein oxidation and loss of structure and function of proteins;

c) genotoxicity due to damage in nucleic acids;

d) energy transduction interruption and disruption in intracellular communication;

e) reactive oxygen species (ROS) formation leading to cell damage; and

f) release of toxic components

Ecotoxicological studies on other aquatic organisms and microorganisms have shown the toxic effects of NPs. In case of microorganisms, they have been found to inhibit their growth acting as antibacterial agents and are toxic to other microorganisms due to the formation of ROS species. In case of other aquatic animals, they have been found to accelerate the lipid peroxidation in the brain acting as neurotoxins and causing changes in the gene expression as well as affecting the developmental stages of the animals post fertilization. The toxicity studies of NPs on soil faces a number of issues due to the presence of manufactured as well as natural NPs that limit the knowledge regarding the effect of different NPs on soil and terrestrial plants. Hence, in-depth study related to the interaction of the NPs with the soil components is essential. The repercussions of the interactions between the NPs and natural organic matter on the fate of ecosystem can be known by the study of the behaviour, bioavailability and characterization of the NPs.
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Gene Therapy And Cancer Treatment

November 18, 2012, 4:36 pm
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Altering the genes as a treatment against a disease is known as gene therapy. A variety of diseases has been known to be caused as a result of defective genes in humans such as Parkinson’s disease, cystic fibrosis, haemophilia, cancer etc. Thus a change in the genes can be used as a treatment mechanism for treating the diseases.

Several modes of mechanism are followed in gene therapy:
(i) Replacement: In this therapy, the defective gene is replaced with a properly functioning one. In certain cases the diseases may be caused by loss of certain genes as a result of mutation, or the diseased condition can arise due to the genes being permanently turned off.

(ii) Regulation: Certain regulations or alterations in genes leading to decline of certain important functions or activation of some defective function can be the cause of the disease. Appropriate regulations of gene expression can lead to proper gene expression and treatment of the disease.

(iii) Enhancement of defective cell appearance: Certain disease can be caused as a result of the defective cell being not recognised by the immune system. The gene therapy is targeted so that these cells become distinct and the same is recognised by the immune system and acted upon them.

In gene therapy, a gene cannot be inserted directly into a human cell. It needs specific carriers which are known as ‘vectors’ which carry these genes into the cell. In gene therapy, viruses are mainly used as vectors.

Depending on the target cell, the gene therapy can be divided into two main types:-
Germline gene therapy and Somatic cell gene therapy.

In germline gene therapy, the gene transfer is targeted to germ cells and the modification of genes in the same is acquired. This results in transfer of modified genes into the future generations. This can help in eradicating certain diseases from a family or from a population as a whole. But this process has been so far possible only theoretically. The dangerous implications of the proposed methodology have inhibited it from being acquiring acceptance.

In somatic gene therapy, the gene is introduced into somatic cells of the diseased. The gene is introduced into the somatic cells where the expression of critical genes is important for restoration of specific cellular activity. Since somatic cells are non reproductive this change in the genes are not transferred into the next generations and remain in the same species.

Mode of action:
In gene therapy, mostly a normal gene is replaced in the position of a defective gene. This transfer of corrected gene is done with the help of carriers known as vectors. The most common vector used in these cases is viruses which have been genetically altered so that it does not actually affect the person negatively but rather improves the diseased condition of the person. The vectors are directed to infect target cells that are cells where a defective gene is present and the corrected genetic material is unloaded into the defective cell. The correction of the defects helps in restoration of the defective condition.

Cancer treatment by gene therapy:-
Gene therapy has been applied most successfully in the treatment of cancer. The property of selective targeting and tumour destruction is the most prominent accounting to its use in cancer cure. A main example is defective P53 gene in tumour cell. The P53 gene is a tumour suppressor gene. It is seen that in persons with tumour, the P53 gene has been affected with mutations and is non functional. Introduction of wild type gene by gene therapy into the affected persons restores the functional gene and results in death of tumour cells.

Another major example in gene therapy in cancer cure is regulation of K-RAS. This is an oncogene known for causing cancer. In cancerous condition, the over expression of the corresponding gene is reported. Gene therapy facilitates introducing antisense gene into the cell over expressing this gene. This causes silencing of the corresponding gene by formation of double stranded RNA affecting the protein production and expression.

The process also involves several risk factors the main one being instability of viruses. The viruses which are used as vectors may develop its infectious property and lead to toxicity, immune responses from the body or the accidental integration into some other site will lead to lethal conditions.
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Somatic Embryogenesis and Artificial Seeds

November 18, 2012, 6:14 pm
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An embryo derived from a somatic cell of a plant rather than the zygote is known as somatic embryo. The process of production of somatic embryo is known as somatic embryogenesis. Micro propagation refers to the technique by which different meristem such as root, shoot, somatic embryos are utilized to produce new plants from them under controlled environmental conditions or in vitro. The regeneration refers to development of organised structures like root or shoot from culture cells or tissues. Somatic embryos when used in micropropagation and differentiation leads to formation of a whole new plant in vitro.

Mechanism of development of somatic embryos:
The development of somatic embryo by somatic embryogenesis occurs from a single cell through micropropagation of meristematic cell also known as explant. This single cell undergoes rapid division and differentiation to form a cluster of cells. This gets isolated by breaking of cytoplasmic connections between different cells of the cell mass. The highly active mass of meristematic cells of somatic embryos undergoes rapid changes by differentiation. It develops through different phases like globular, round shaped, heart shaped, torpedo shaped and finally cotyledonary stages to form a somatic embryo.

A somatic embryo consists of a shoot plumule and a root radicle. Often it is seen that, in a developing somatic embryo the shoot plumule is seen propagating outwards and the radicle towards the center of callus or cell mass. Thus in most cases, the developing embryo develops only shoot and undergo shoot regeneration. In order to regenerate root, this has to be induced with growth factors which promote root regeneration. The somatic embryos which are produced by the following were seen to be associated with abnormal developmental features like cotyledons which are more in number, abnormally shaped. This problem can be prevented by the addition of abscisic acid in the culture medium used to propagate somatic embryos.

The development of somatic embryos goes through various processes like somatic embryo induction phase where the induction is initiated in auxin medium to produce a mass of cells. This is then transferred into developmental medium low in auxin concentration where the cell develops into cotyledons. The cotyledons so produced enter a somatic embryo conversion phase to form embryos. These are mostly subjected to a maturation phase so that the somatic embryo formed gets stable.
In certain cases, the micro propagation of plants is possible only from somatic embryos such as oil palm, date palms etc. It is also recommended to micro propagate plants affected with virus of plant body by somatic embryos only.

The production of somatic embryos gets affected by factors like:
Growth regulators:

The presence of certain growth regulators is seen to influence the growth of somatic embryos. In most cases, the growth medium is added with an auxin which promotes development of somatic embryos. The presence of auxins promotes hypermethylation of DNA leading to the totipotency of the cell. In certain plants the presence of auxin is known to trigger the development of cells so that they divide asymmetrically and the daughter cells produced by each division sticks together to form a clump of cells known as proembryogenic masses or embryogenic clumps. These can be differentiated and each cell can be developed to produce a somatic embryo. Totipotent cells, which have the ability to divide and differentiate, release some glycoproteins into the medium when they differentiate. This glycoprotein when isolated and added to medium of cell cluster was found to initiate differentiation leading to somatic embryogenesis. These glycoproteins produced are known as arabinogalactan proteins.

Several other factors are also known to influence the growth of somatic embryos like nitrogen source, genotype of explant used, high potassium levels, dissolved oxygen level and even the presence of cytokinin in certain species.

Production of artificial seed:
Somatic embryo can be used to produce artificial seeds. It consists of a bead of gel containing somatic embryo along with nutrients, growth regulators, pesticides, antibiotics, all the necessary requirements needed by the embryo to develop into a new plantlet. It is produce by mainly two systems

Desiccated system: This involves hardening of the somatic embryo in the maturation phase by adding polymer or treating them with abscisic acid. This is followed by drying or desiccation to produce a desiccated system.

Hydrated system: This involves coating the embryos in a gel with materials similar to sodium alginate. The corresponding process involves allowing sodium alginate to fall into a solution of calcium chloride. The drop before falling is inserted with embryos thus it falls into the solution forming a gel coat around the embryo.

The hydrated system is less stable and has to be planted soon. It also undergoes hydration when it comes in contact with atmosphere thus by this process it has made possible to produce seeds which can be transported , stored and even planted to produce plants when required.
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November 19, 2012, 2:28 am
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Biotechnological Applications In Disease Treatment

November 19, 2012, 4:08 am
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A wide variety of compounds obtained from organisms is used in treatment of diseases. These compounds may be obtained from recombinant and non recombinant organisms.
A large number of products from non recombinant organisms serve as pharmaceuticals. In certain cases, the micro organisms as such serve as cure for certain diseases like, for eg: the lactobacillus species. Various products like antibiotics, vitamins, enzymes, organics acids etc., play an inevitable role in disease treatment. Pharmaceutically important biochemical is also produced from plant cell cultures. Cultured animal cells too play their role in prevention and treatment of diseases.
The major drawback in using products from non recombinant organisms is the lower availability the products and the comparatively limited ability of products to cure diseases. They have to be used in its natural form and hence the spectrum of diseases covered is not wide spread. The advent of genetic engineering has enabled large scale production of existing and new products in disease treatment. The products from genetic engineering include:
Genetically engineered micro organisms:
Human gene has been known to encode a large number of pharmaceutically important proteins. These are cloned and expressed in micro organisms for increased production. Microbes are used as hosts in cloning purposes. E. coli, yeast are some of the most common examples of microbes which are used as hosts. In yeast many recombinant proteins having pharmaceutical significance has been produced. Eg: recombinant insulin used in the treatment of diabetes, human growth hormone for dwarfism, interferon, interleukin, granulocyte macrophage colony stimulating factor, etc.
The most extensive protein developed in such a manner is human insulin. It consists of two chains known as A and B which are interlinked by two disulphide bridges. The gene coding was integrated separately to a host cell, expressed and modified to produce functional insulin.
Animal cell cultures are also used for expression of human genes encoding pharmaceutically valuable proteins. The main proteins produced by this way are erythropoietin and blood clotting factor VIII. Plant cells producing recombinant proteins are high in demand as many ethical issues related to animal cell culture do not exist with the transgenic plants. In the case of transgenic plants, the process of retrieval of recombinant proteins from the parts of plant cells is comparatively easy. The most relevant example of transgenic plants aiding in the disease treatment is production of a polypeptide called hirudin. This is produced by a synthetic gene expressed in the plant Brassica napus. This is produced as a fusion protein with oleisn which is an oil body protein. Upon successful integration and extraction, the hirudin is extracted with water and later centrifuged to separate our protein from the rest of the proteins produced with the help of oil body. The pure hirudin is separated from olesin by subjecting the obtained oil moisture to proteolytic cleavage.
Antisense nucleotides:
A novel approach of disease treatment is the production of antisense oligonucleotide. It involves production and use of oligonucleotides complementary to the 5’ end of the parasite mRNAs. This antisense oligonucleotide is often linked with acridine for increasing the effectiveness of the same. The application of such oligonucleotides is in the case of cancer.
Monoclonal antibodies:
These have been known to exhibit several therapeutic applications like providing passive immunity, treatment of certain diseases like leprosy, deliver of immunotoxins specifically to cancer cells etc.
Drug designing:
This involves designing special drugs for special requirements of disease or the patient. These are designed to specifically bind to the critical site of target molecules thus inactivating the latter. These are so designed so that they do not exhibit any side effects rather than conventional drugs. Important examples are propanolol used in treatment of hypertension or heart attacks. Another example is cimetidine which blocks the hydrogen receptor in the stomach so that it effects the formation of ulcers in the stomach curing it. This has served as an important mechanism in treatment of cancer, gout, malaria, etc. Thos has resulted in developing a drug called azidothymidine (AZT) for treatment against HIV.
Drug delivery and targeting:
An effective improvement in the disease treatment is the introduction of technique of drug targeting. Often it is seen that conventional medicines loose their activity or sometimes part of their activity as they follow general distribution pattern. In drug targeting, the drugs are so targeted so that it affects only the required tissues and does not act upon anything else. This greatly enhances the drug effects and limits the amount of dosage required. Immunotoxins are the main example of such site directed delivery of drugs.
Gene therapy is yet another novel approach in the field of disease treatment with many successful examples in treating disease like cancer. Studies are being conducted on improving and developing the procedure for large scale use.


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Risk Assessment Of Biotechnological Activities And Its Corresponding Products

November 19, 2012, 4:28 am
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Since time unknown, man has been involved in exploiting the nature around him for the benefit of his own. With the advent of biotechnology the tendency has increased and attained the thresh hold level. The level of interference by man is to a great extent arising thoughts of concern within the human population themselves. The genetically modified organisms and their products may disturb the equilibrium of ecosystem and can even pose as a threat to the stability of the planet even.
It was in the early 1970s that the discussions regarding ethical and non ethical issues concerning recombinant DNA technology was initiated. The concern is about non stability of such experiments conducted and their effects upon nature. A committee called as National Institute of Health, USA was formed which look into the issues which needs attention. The committee has put up certain guidelines for the experiments which have to be strictly adhered to by any experiment conducted in any of the nations. The objectives laid down mainly include practices which consist of conducting experiments on any living organism and the techniques applied for the same.
Risk assessment:
Risk assessment of the experiment involves determining the amount of uncertainty and the possible consequences involved in the same. The prime objectives included are determining the kind and level of experiments to be conducted on the living beings. The identification and evaluation of potential threats by the planned introduction of living organisms is done by risk assessment. This assessment is done by two steps:
(i) Initial assessment: These involve determining the risk involved based on the organism which is used for the experiments. Depending on the effect of organisms they are classified in to different Risk Groups (RG) -1, 2, 3, and 4.
RG 1- involves organisms which are not associated with any disease occurring in humans.
RG2- involves organisms which are involved in causing non lethal ailments in humans and for which treatment is easily and effectively available.
RG3- includes organisms which are linked with serious diseased condition in humans and the cure may be available.
RG4- consists of organisms which can cause highly dangerous health issues for which prevention and treatment is not developed.
(ii) Comprehensive assessment: This involves factors such as features of organism included like pathogenicity, virulence etc. and the type of manipulation involved.
Following risk assessment, the experiments are grouped under different categories which require clearance from different organisations like, Institutional Bio Safety Committee (IBC), Recombinant DNA Advisory Committee (RAC), Office of Biotechnology Activities, and many more. After risk assessment, the concerned group of persons involved will be directed to get approval from these committees. The risk assessment takes into consideration the following factors:
(i) The physical and biological nature of both donor and recipient organisms.
(ii) The properties of vector involved
(iii) The characteristics of DNA insert involved
(iv) Different techniques involved like detection, isolation, transfer and expression.
(v) Differences in the native and genetically modified organisms (GMO), the use of such organisms for several benefits and impact of such organisms in the environment and the impact of experiments on the organisms.

Potential threats involved in biotechnology:
The transgenic organism produced may affect other organisms negatively such as development of transgenic weeds or parasites. Often, the transfer is intended to be expressed by organisms lacking some property or by organisms which can provide products of economical importance. The chance of transfer of the transgene into a similar organism resulting in production of a hazardous organism or conditions may also arise wherein the transgene can also be overly expressed disturbing the well being of other organisms. Developments of insects or pathogens which are resistant to such transfers are also possible. The existence of the GMO s may affect the environment in which it thrives upsetting the equilibrium as they do not actually belong to the environment. Finally the product produced can also cause health hazards among organisms and especially humans.
Following the assessment of organisms involved in biotechnology, the products obtained from such organisms is also assessed to confirm that they do not pose any kind of threat to the humans when consumed. The assessment is based on the product that is modified, safety of the introduced transgene and the corresponding products formed .It essentially takes into consideration the genetics of organism involved, the technical procedures followed and composition of product formed.
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chromosome number of sepal leaves

November 19, 2012, 4:29 am
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Hey everyone,

Is it possible for sepal leaves to get chromosome number n, or are they always 2n?
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