Renewable sources are the future of the energy production since conventional sources are limited. Power of the wind and water or geothermal and solar energy…are just few examples of the alternative sources of energy. Biofuel is relatively new type of liquid or gaseous energy, derived from biological conversion of carbohydrates. Biofuels are gaining popularity as they are more eco-friendly and cheaper than conventional fuels. Most popular plants used in biofuel production include sugar cane, sugar beet, oil palm, soybeans, wheat, and the desert-grown. Ecological crisis and high price of oil are increasing the number of farms that are producing biomass for fuel production.

Bioethanol is produced by fermentation of the carbohydrates from corn and sugarcane. Cellulosic biomass can be used in bioethanol production also. It’s mostly used in USA and Brazil as a vehicle fuel (in its pure form) or to improve vehicle emission (as gasoline additive).

Biodiesel is the most common biofuel in Europe and it’s produced in the process of transesterification of the animal fats and vegetable oils. It can be used as vehicle fuel or as additive that could reduce level of carbon-monoxide and hydrocarbons in the diesel-powered vehicles.
In 2010, biofuel production reached 105 billion liters on the global scale, contributing to the fuels used in transportation by 2.7%. USA and Brazil are responsible for 90% of total world bioethanol production. Biggest manufacturer of biodiesel in the world is European Union (55% of all globally produced biodiesel in 2010). It’s estimated that biofuels have potential to reach 25% of world demand for transportation fuels.

Here are some of the world’s most popular biofuel plants that are currently operating or that are planned for the future:

1. Dynoil LLC is developing a biodiesel production company near Houston, Texas. Opening date is not determined yet. Goal of this plant is to produce 100,000 barrels of vegetable oil on a daily basis and 5.5 billion liters of biodiesel annually.

2. Dominion Energy Services, LLC started its business in 2006 and today this facility is producing 54 million liters of bioethanol annually. Plans for the future are big: millions of gallons of ethanol and biodiesel derived from canola, wheat and corn.

3. Brazil EcoEnergia could become one of the largest biodiesel plants in the world. Soybean oil is used in biodiesel production and goal is to produce 220.5 million gallons of biofuel a year.

4. Energen Development Limited (EDL) is using sweet sorghum for bioethanol production. Plant extracted juices are fermented to bioethanol. With 40 000 acres in cultivation, 300 million gallons of biofuel could be produced annually.

5. Agri-Source Fuels plant in Dade City, Florida. This facility produces 12 million gallons of B100 biodiesel per year and refines the crude glycerin byproduct to USP grade. It has potential to expand production up to 60 million gallons of B100 per year if public demand for biodiesel increases.

6. Imperium Renewables plant is located in Grays Harbor, Washington. Raw product for biodiesel production is oil derived from canola and soy grown in USA and Canada. This plant can produce up to 100 million gallons annually.

7. Louis Dreyfus plant is located near Claypool in Indiana. It uses soybean for biodiesel product resulting in 250,000 gallons of biofuel per day and over 80 million gallons annually.

8. Greenline Industries is located in Larkspur, CA. Biodiesel is produced from vegetable oil and animal fats. Current capacities of the plant are 1,400-120,000 gallons per day.

9. North Prairie Productions is biodiesel plant located in Evansville, Wisconsin. Soybean is starting point in biodiesel production. Current capacities: 45 million gallons of produced fuel annually.

10. Cargill plant in Iowa Falls, Iowa is current producing 37.5 million gallons of biodiesel per year. Cargill is an international producer and marketer of food and agricultural products. Company is planning to modernize and expand their soybean crashing facility to increase production capacities and ensure profit of ~ 60 million dollars a year.

Besides being eco-friendly and cheaper than conventional source of energy, biofuels have couple of negative aspects. Turning eatable crops into fuels when there are so many hungry people in the world is probably the biggest issue in biofuel production today. Biofuels still need to overcome some manufacturing obstacles, but with new fuel sources and improved techniques, it will definitely become more available in everyday life.

Ongoing research involving the study of the various causes and possible therapeutics for HIV infection has helped in the detailed study of the life cycle of the HIV retrovirus, as well as the progression of the HIV infection within the body leading to the most dreaded disease of AIDS (Autoimmune Deficiency Syndrome) in most of the cases. However, the study has revealed that in some HIV positive patients, the infection has delayed progression or in some cases non-progression leading to increased mortality of such patients.

Research to find out the main reason for the long-term survival of such patients has shown the role of the innate defense mechanism of the patients, which helps them survive for a longer time in spite of being infected with the HIV virus even without undergoing any sort of treatment. The presence of a particular key enzyme in the white blood cells of some patients has helped in their long-term survival. The study of nature and properties of this enzyme has become a raging topic of research, which may help in the development of new area of therapeutics for the HIV positive patients.

Among the HIV positive patients, a very small percentage around 5% almost do not develop full-fledged AIDS or develop in a delayed manner. The presence of the enzyme APOBEC-3G (A3G) in higher levels in the white blood cells of such patients has helped the patients in preventing the progression of the infection. The recent studies with human cells have confirmed the role of A3G in non-progression of the infection in some patients.

Researchers have proved the role of A3G in editing the genetic code of HIV[b] introducing changes with every replication cycle of the retrovirus. These changes introduced within the virus causes [b]corruption of its genetic code, thereby preventing its reproduction. However, the HIV has also evolved to produce a protein known as the Viral infectivity factor (Vif), to counter the attack of A3G. The Vif grabs the A3G and initiates the destruction of A3G by the body itself by tricking it. The destruction of A3G, the editing enzyme, helps the progression of the HIV infection, which ultimately attacks the immune system of the patient making them vulnerable to the development of AIDS resulting in death.

The absence of sufficient levels of A3G in most of the patients causes progression of the HIV infection, as the Vif secreted by the virus does not have enough A3G to overcome its effects and prevent its progression. Hence, studies are being conducted to devise methods to prevent the Vif from destroying the A3G produced, even if they are of low levels.

Extensive research is going on to unleash the mechanism involved in the mechanism of A3G and to prove its role in the progression of the infection in the HIV positive patients. It has been elucidated that in the absence of any sort of antiviral therapy, the A3G does help in causing rapid changes in the viral genetic code preventing the production of the viral proteins and ultimately making the virus unable for reproduction. It was also validated that presence of higher levels of A3G in some of the HIV positive patients studied helped in the delayed progression of the infection in them, while low levels of A3G in other patients caused normal progression of the infection. It was found that higher A3G levels corresponded to lower viral levels of HIV.

It was also reported that higher A3G levels were associated closely with higher counts of CD4 helper T cell. In normal cases, the helper T cells with CD-4 receptors helped the immune system by targeting the bodily invaders; however, the spread of HIV infection resulted in the destruction of the CD-4 helper T cells.

The discovery of A3G has helped in various ways in the HIV related research. It has helped in the development of a prognostic marker in the diagnosis of AIDS with the measurement of A3G in the HIV- infected individuals. It has helped in the study of the underlying mechanism involved in the non-progression or delayed progression of the HIV infection in some patients. Moreover, it has helped in the development of new therapeutics for the HIV infection by devising new methods for the protection of A3G from the viral attack to treat AIDS and other infectious diseases.

The new approach for HIV infection involves exploiting the 14editing enzymes having the property of editing the genetic code of the virus, as novel targets for the development of pharmaceuticals. The main focus is in disabling the action of Vif on A3G. The Vif is a dimer that grabs the A3G with its two arms and causes its destruction. The main aim in is development of drugs that can prevent the dimerization of the Vif and prevent the grabbing of A3G thereby preventing its destruction. A drug candidate, a Vif Dimerization Antagonist (VDA) has been developed by a biotech organization called Oyagen, which has been found to be successful in reducing HIV infectivity in different experiments. However, pre-clinical studies on human trials are essential before the drug candidate can be used in HIV therapeutics in future.

Man made chemicals present in the nature at high concentrations polluting the environment is known as Xenobiotic compounds. These compounds are not commonly produced by nature. Some microbes have been seen to be capable of breaking down of xenobiotics to some extent. But most of the xenobiotic compounds are non degradable in nature. Such compounds are known to be recalcitrant in nature.

The properties of xenobiotic compounds attributing to its recalcitrant properties are:

(i) Non recognizable as substrate by microbes to act upon and degrade it.
(ii) It does not contain permease which is needed for transport into microbial cell.
(iii) Large molecular nature makes it difficult to enter microbial cell.
(iv) They are highly stable and insolubility to water adds to this property.
(v) Mostly toxic in nature.
The recalcitrant xenobiotic compounds can be divided into different groups depending on their chemical composition

Halocarbons: They consist of halogen group in their structure. Mainly used in solvents, pesticides, propellants etc. They are highly volatile and escape into nature leading to destruction of ozone layer of atmosphere. The compounds present in insecticides, pesticides etc,. leach into soil where they accumulate and result in biomagnification.
Polychlorinated biphenyls (PCBs): They consist of a halogen group and benzene ring. They are mainly used in plasticisers, insulator coolants in transformers etc. They are chemically and biologically inert adding on to its recalcitrant nature.

Synthetic polymers: These are mainly used to form plastics like polyester, polyvinyl chloride etc. They are insoluble in water and of high molecular weight explaining the recalcitrant property.

Alkylbenzyl Sulphonates: They consist of a sulphonate group which resists break down by microbes. They are mostly found in detergents.

Oil mixtures: When oil spills occur covering a huge area the break down by action of microbes becomes non effective. They become recalcitrant as they are insoluble in water and some components of certain oils are toxic in higher concentrations.

The recalcitrant property of xenobiotic compound is directly linked to its complexity so that the higher the complexity the stronger recalcitrant property.

Hazards posed by xenobiotic compounds
The hazards posed by xenobiotics are huge. These compounds are highly toxic in nature and can affect survival of lower as well as higher eukaryotes. It also poses health hazards to humans like various skin problems, reproductively and even known as a trigger for causing cancer. These compounds are persistent and remain in the environment for many years leading to bioaccumulation or biomagnification. They also find a way into the food chains and the concentrations of such compounds was found to be high even in organisms that do not come in contact with xenobiotics directly.

Mechanisms involved in biodegradation of xenobiotics
Xenobiotic compounds, owing to its recalcitrant nature, is hard to break down and degrade. The complexity of its chemical composition adds to this. For breaking down such compounds the enzymes act on certain groups present in the compound. For eg: in the halocarbons the halogen group is targeted. Enzymes like oxygenases play a major role. The bonds like ester-, amide-, or ether bonds present in the compounds are first attacked leading to breaking down of compounds. In some cases the aliphatic chains and in aromatic compounds the aromatic components may be targeted. The site and mode of attack depends on the action of enzyme, its concentration and the favourable conditions. Often it is seen that the xenobiotics do not act as a source of energy to microbes and as a result they are not degraded. The presence of a suitable substrate induces its breakdown. This substrate is known as co – metabolite and the process of degradation are known as co metabolism. In another process, the xenobiotics serve as substrates and are acted upon to release energy. This is called gratuitous metabolism.

Biodegradation:
Certain microbes on continuous exposure to xenobiotics develop the ability to degrade the same as a result of mutations. Mutations resulted in modification of gene of microbes so that the active site of enzymes is modified to show increased affinity to xenobiotics. Certain mutations also resulted in developing new enzymatic pathway for xenobiotic degradation. Use of mixed population of microbes is usually recommended as it has been seen to yield faster results as the two different microbes attack different parts through different mechanisms resulting in effective break down. It also creates a condition of co metabolism. The modification of certain genes of microbes to break down xenobiotics is also recommended and is seen to produce high level of accuracy.

The presence of a mutant gene, chromosomal aberration and the complexity of the relationship of group of genes with the environmental factors (mutagens) are all well exhibited by various common and rare kind of diseases. The diseases developed as a result of mutation are classified under genetic diseases. A defective single gene (mutant) is powerful enough to develop a disease which may be even fatal to the affected person. The genetic disease is either inherent or found to develop in the offspring alone as a result of mutant gene present in the sex chromosomes.

Inheritance is the passing of disease from one generation to the next which usually occurs in three ways. They are Autosomal dominant inheritance, Autosomal recessive inheritance and X-linked inheritance.

Autosomal dominant inheritance: The presence of mutated allele in either one of the parents causes disease in the offspring. For example, combination of a male parent with one mutant allele (dD) with a normal female parent (DD) produces two mutated offspring (dD) and two normal offspring (DD). The individual with one normal allele (D) and one mutated allele (d) is called as a heterozygous individual (dD).

Autosomal recessive inheritance: The disease is developed in the offspring by the inheritance of mutant alleles one each from both the parents. For example, combination of a carrier male parent (Dd) with a carrier female parent (Dd) results in two carriers (Dd), a normal offspring (DD) and an affected offspring (dd) also called as homozygous individual.

X-linked inheritance: The presence of mutant allele in X – chromosome causes X-linked inheritance. The male population is the affected group because of the presence of only one X chromosome (XY) whereas the female with two X chromosomes (XX) may usually be carriers.

The point mutation and the gross mutation are to be blamed for the defective single gene disorders. Missense/silent mutations, nonsense mutation, frameshift mutation, splice site mutation and promoter mutation falls under one roof called point mutation. The insertion and deletion mutation, gene rearrangement and trinucleotide repeat mutation are classified as gross mutation. Some of the single gene mutation diseases are Hemophilia A and B, Thalassemia, sickle cell anemia, Duchenne muscular dystrophy, Becker muscular dystrophy, Fragile X syndrome, Huntington’s disease, Neurofibromatosis, Phenylketonuria and cystic fibrosis.

Hemophilia A: A condition of excess bleeding developed due to the mutation of Factor VIII gene and the mutation type is either frame shift or insertion or deletion mutation and it is a X-linked inherited disease.

Hemophilia B: Excess unusual bleeding due to the mutation of the promoter gene responsible for protein Factor IX which stimulates clotting of blood. Mutation of the gene arrests the blood clotting property of Factor IX causing unusual bleeding in the affected person and it is an X-linked inherited disorder.

Thalassemia: The anemic condition due to either splice site mutation or nonsense mutation of the gene responsible for β-globin resulting in the termination of β globin synthesis causes β Thalassemia. This is Autosomal recessive inherited disorder.

Sickle cell Anemia: The Autosomal recessive inherited disease occurs as a result of misense mutation to the gene coding β globin. Sickle cell anemia is represented by the presence of short lived sickle shaped red blood cell causing anemia and ischemia.

Huntington’s disease: The repetition of trinucleotide sequence causing mutation in the gene sequence coding for huntingtin causes Huntington disease, a Autosomal dominant inherited disorder. The disease is characterized by dementia.

Cystic Fibrosis: The Autosomal recessive disorder associated with lung damage symptoms due to the deletion mutation of the CFTR gene.

Neurofibromatosis: Autosomal dominant disorder due to the mutation of the NF-1(Type 1 disease) and NF-2 (type 2 disease) gene causing tumor of the nerve tissues.

If we see the Autosomal recessive type disorder, both the parents acts as carriers and the product receiving both the mutant allele (one from the father and the other from the mother) becomes subject for the genetic disease. The parents may be informed about the possibility of genetic disease in their child in advance if both the parents are identified as carriers. Gene Tracking is the technique employed to detect the carriers in a family. This involves the detection of restriction fragment length polymorphisms in a genomic DNA sample by southern blotting technique and also by using DNA repeat sequences like minisatellite DNA and microsatellite DNA.

Want to look pretty? Stem Cells may be your answer.

Looks have become a major issue in both men and women alike in the modern day and age. Hence, studies related to enhancing looks by adopting different methods and techniques is gaining popularity as people are rarely satisfied with their natural looks and forever look for various ways to improve and enhance their appearance.

The advent of stem cell technology, in general, has helped in providing solutions to a number of problems such as helping in the therapeutics for various fatal diseases, bone repair, etc. However, the research in the field of stem cells gives rise to new use of stem cells everyday and it has gone a long way in helping the mankind in different ways, enhancing appearance being one of them.

Stem cells have potential use in the skin grafting process that is used as a treatment for the burn victims. The transplantation of tissues and skin from the donors to the burn victims is a painful procedure requiring long period for healing and the patients are compelled to undergo enormous pain and mental frustration due to the wounds as well as the healing challenges. Hence, stem cells have provided a much better alternative for the patients, which could be used for the production of healthy, new tissues instead of relying on the donated tissues, the procedure being similar to the bone marrow transplants in Leukaemia patients, whereby the stem cells are transplanted for the creation of specialised white blood cells. The scientists follow the procedure of triggering the differentiation of the isolated stem cells from a tissue outside the body followed by the transplantation of the differentiated stem cells back into the patient for the replacement of the damaged tissues. The small piece of skin that is grafted into the patients can progressively grow back and cover the burn within a short period. Thus, the use of stem cells in the burn victims is one of the most successful developments for the benefit of humanity.

Stem cells have also offered solution in helping to re-attach the lost teeth thus creating a revolution in the field of dentistry. A smile with all the teeth intact is always considered a sign of beauty and health. Hence, the loss of teeth due to gum disease or any type of accident becomes a big issue. However, new research studies show that this can be treated with the stem cells, which can not only help in the reattachment of the lost teeth but also in growing back new teeth in place of the lost one.

Hair loss has become one of the major factors affecting the appearance of men and women and the association of head full of hair with beauty, youth and vitality has further helped in creating frustration and embarrassment besides creating stress in people losing hair. New research studies proving the use of stem cells in restoring hair growth is creating favourable response among people, who would like to avoid the claims of oral and other treatments having numerous side effects. Hair follicles are actually associated with hair growth and it has been found that the damage in the hair follicles as in case of balding halts the formation of hair from them. Scientists have studied the role of stem cells in restoring hair growth in mice. They found the role of Wnt gene in the hair follicle formation as well as healing of wounds. The studies in mice showed that the creation of wounds helped in the activation of this gene, which helped in the wound healing process and also in the formation of new hair follicles followed by hair growth. The blocking of this gene caused halt in the hair growth process, while increase in its activity increased the hair growth. Although, the studies of this gene proved successful in mice, it is in its infant stage and further research is essential along with its translation in the human studies. However, the role of stem cells in re-growth of hair shows great promise.

New researches in stem cells have showed a possible use of stem cells in reducing or removing the wrinkles in face caused due to old age. Some scientists have used plant stem cells for use in the topical creams for this purpose due to ethical issues related to the use of human stem cell in cosmetics. The stem cells isolated from a type of apple with long shelf life were shown to be responsible for the generation of protective shield on its surface. Culture of these cells was found to enhance the production of human stem cells. However, there was no validation of the results when replication of the study was done through a topical cream. Moreover, some of the scientists working on plant stem cells have stressed on the impossibility of the presence of such interaction of plant stem cells with the human stem cells. Hence, the possible use of stem cells in reduction or removal of wrinkles remains to be studied in-depth to be used for commercial purpose.

Thus, the use of stem cells in unique and innovative ways shows great promise in the research and development of stem cells. The technology has a long way to go and in near future may offer solutions to every type of problem faced by the humanity.

Recent talk at the Open Science Summit in silicon valley. Should biotechnology be made open source? Pros and cons and is it a sustainable model?





From OpenBiotech Website / FAQ: http://www.openbiotech.com/category_s/1821.htm


Aren’t you going to completely destroy closed-source companies’ business model?
We do think that the proprietary reagent industry managed to come into existence largely because so far, there were no open source players. After all, why would anyone buy products that they can’t make more of, with severe use restrictions, and pay extra for the privilege? When similar products are available in equally good or superior quality, in a form that can be copied, with no restrictions, and cost much less, then the proprietary business model will simply stop making sense.

It is not our intention to harm anyone’s business. We hope that these companies will adapt, ultimately acquire our open source DNA sequences and begin competing on manufacturing the same products. This will result in a business landscape where the key competitive trait stops being how well you keep your secrets, and starts being how efficiently you can make quality products. In the end, the main beneficiaries will be the world’s scientists and therapy developers, and with them, all of humankind.

Isn’t open source unprofitable?
Open source principles did indeed build a reputation of being unprofitable, largely due to the experience in software development. Any given piece of software can be copied indefinitely many times, for free, with zero skill required. Why would I pay for a piece of software, when its creator encourages me to legally copy it? Thus, it is at least a legitimate question how to make money in open source software.
In biotechnology, it is different. With access to the necessary DNA, biological sequences and objects can be copied, and doing so is much cheaper than, say developing a new sequence. However, it still does require some work, and significant technical expertise, to scale production, purify, characterize and fully validate the resulting biological products.

We find the situation more than sufficient to build a sustainable, innovative organization.

Why are you doing it?
We started primarily because we want open source biotechnology tools to use ourselves. We are a team of young scientists with a background in regenerative medicine. In trying to start our first regenerative medicine company, we perceived the use restrictions and, for lack of a better word, obscene profit margins seen in contemporary research tools products as extremely frustrating. So we set out to make our own research tools. In doing so, we were able to free ourselves from many types of use restrictions, remove most of the cost of doing our own research, and continue our work in regenerative medicine. But we also realized that we cannot make regenerative medicine happen all by ourselves. We decided to make our tools fully open source, and offer them to the world, so that other groups can join in the same benefits.

Like a library is a place sheltering large collection of books on different subjects for public use, a DNA library involves collection of DNA sequences from various organisms, serving different purposes. These DNA sequences are stored as recombinant molecules by ligating them with suitable vectors. The two types of gene libraries are genomic library and cDNA library, categorized based on the source from where they were constructed. A genomic library holds the DNA sequences derived from genomic DNA whereas the cDNA library represents the DNA sequences generated from mRNA. The library that represents the source DNA as such can be considered as an ideal gene library.

Construction of genomic DNA library: Preparation of the genomic DNA library involves isolation of genomic DNA, purification of the genomic DNA and fragmentation of genomic DNA into desired size and then cloning of the fragmented DNA using suitable vector. The eukaryotic cell nuclei are purified by adopting digestion by protease and phenol-chloroform applied phase extraction. The derived genomic DNA is long and needs to be cut into desirable fragment sizes. Fragmentation of DNA is achieved by physical method and enzymatic method.

Physical method includes pipeting the DNA molecule or applying intensified ultrasound waves (sonication). The enzymatic method involves the use of restriction enzyme to fragment the purified DNA. The desirable DNA size suitable for the cloning vector decides the method to be used for fragmenting the DNA and the length of exposure of the DNA molecule. The distribution probability of site prone to the action of restriction enzymes limits the use of the enzymatic method which will produce shorter DNA fragments than the desired size. To overcome this problem partial digestion of the DNA molecule is done using known quantity of restriction enzyme which yields fragments of desired size. The two factors governing the selection of the restriction enzymes are the type of ends they generate by their action, like blunt end or sticky end and the susceptibility of the enzyme to the chemical modification of bases like methylation which inhibits the enzyme activity. The exact sized fragments are recovered by using either agarose gel purification technique or sucrose gradient technique which is then ligated to suitable vectors. λ phage, yeast artificial chromosome are considered as suitable vectors for larger DNA and λ replacement vectors like λDASH and EMBL3 are the preferred vectors in constructing genomic DNA library. T4 DNA ligase is used to ligate the selected DNA sequence into the vector.

Construction of cDNA library: Developing cDNA library involves 4 steps. 1. Initial extraction and purification of mRNA, 2. Production of cDNA, 3.Treating the ends of cDNA and 4. Ligation of the cDNA to the vector. The polyadenylated nature of the eukaryotic mRNA enables easy isolation of mRNA by using oligo(dT). Magnetic beads with oligo(dT) is added to the cell lysate which enables the binding of the poly A tail of the mRNA to the oligo(dT) and using strong magnetic force the binded mRNA is isolated from the total RNA. The recovered mRNA is checked for its integrity by using gel electrophoresis technique. Also translation of isolated mRNA is carried out as a step to check its integrity.

Following mRNA extraction begins the production of cDNA. Single strand is produced using mRNA as template by the action of reverse transcriptase enzyme along with addition of 4 dNTPs with oligo(dT) as primer. The enzyme Terminal transferase functions by adding nucleotides to the 3’ end, following which is the elimination of the mRNA strand by alkali treatment. Then the synthesis of second strand begins by the action of reverse transcriptase or klenow polymerase using oligo(dG) primer. This results in the formation of duplex cDNA with extended 3’ end of the strand 1 beyond the 5’ end of the strand 2. Single strand specific nucleases are used to treat the extended 3’ end and as a result any missing nucleotide at the 3’ end is filled by using Klenow polymerase I and dNTPs. cDNA is methylated to ensure the protection from the action of restriction enzymes before adding linkers to the 5’ phosphorylated ends with the help of T4 DNA ligase and ends are made sticky with help of EcoRI enabling the cDNA to ligate with the suitable vector. The frequently used vectors are the plasmids and T4 DNA ligase is used to ligate the cDNA with the vector.

The decreasing reserve of petroleum and the ever-increasing demand for energy by the developing industrial countries is initiating the development of alternative sources of energy to meet the demands. The economy and the national security of a country, both are threatened due to the increased dependence on imported petroleum. Moreover, the countries all over the world are affected due to the negative impact of the extraction, refining, transportation, and utility of petroleum on the environment. Hence, the alternative sources of fuel are being researched upon to provide better options to meet energy demands and biofuel is the most preferred alternative source of energy.

Tobacco was not included among the various plant resources for the alternative biofuel production as it was considered a plant grown especially for smoking and hence was expensive. However, closer analysis regarding the nature of the crop revealed that tobacco produced large amount of biomass or green tissues compared to many other crops when it was specially grown to produce biomass instead of its usual utility for smoking, so could be included among the industrial biomass crops producing outstanding levels of biomass. Like some of the trees with hardwood capable of being coppiced, i.e. regenerate from the stumps after being pruned to ground level for the harvesting of the stems, tobacco can also be coppiced for regeneration thus providing the possibility of multiple harvests in a year increasing the biomass production to great extents. Moreover, biofuel could be generated more efficiently from tobacco compared to other agricultural crops thus making it a better resource for biofuel production.

Usually, only the seeds of the crops are associated with the production of the biofuel oil as when compared to the other green tissues of the crops like leaves, stems, etc only the seeds are capable of accumulating triacyglycerols, the storage reserves, which constitute a form of biofuel oil. The tobacco seeds have the biosynthetic machinery of producing oil constituting almost 40% of the dry weight of the seeds thus are potent biofuel oil producers. According to Usta, the seed oil of tobacco has been tested successfully for the production of diesel engine fuel. However, the yield of tobacco seeds is very low compared to the other biofuel oil producers like the rapeseeds, soybean, etc and constitutes roughly about 600kg seeds per acre. Studies suggest that though the primary synthesis of oil takes place in the photosynthetic tissues of the plants, the accumulation of the biofuel oil takes place in the seeds, though in some plants, the leaves deposit oil in the form of oil bodies. According to Vincent, in case of tobacco, the leaves contain about 1.7-4% of oil per dry weight, which can be extracted as the esters of fatty acids (FA) that are the important constituents of biofuel oil and the green biomass like stem contains very low percentage of oil compared to the seeds. However, the potential of tobacco to produce huge amounts of biomass and the ease of performing genetic engineering on it makes it a promising biofuel producing plant.

Genetic engineering technology has played a major role in increasing and relocating the oil content by the gene manipulation of the parts of plant other than seeds such as root, stem, etc making the green biomass a good biodiesel manufacturing system. Studies have shown that increase in the oil accumulation in the alternative plant organs was possible by the enhancement in the expression of some lipid metabolizing enzymes.
a)The over-expression of the enzyme diacylglycerol acyltransferase (DGAT), an important enzyme in the TAG biosynthesis has been shown to help in the triacylglycerol accumulation in the tobacco seeds, leaves, and tubers. An increase to about two-fold almost 5.8% was observed in the tobacco leaves by the genetic modification of DGAT.

b)The Leafy Cotyledon genes (LEC1 and LEC2) regulate the development and maturation of the seeds. The constitutive expression of these genes in the leaves exhibit the transcription of the mRNAs specific to the seeds and it helps to channel the accumulation of oil by inducing the seed-like structure formation in the green vegetative tissues as shown in the transgenic Arabidopsis plants. Thus, the constitutive over-expression of DGAT and the induced expression of LEC2 genes have favoured the utility of tobacco as an alternative, renewable resource of biofuel increasing the oil content thereby shifting the FA composition in the green biomass.

Thus, it can be seen that tobacco is emerging as a good source of biofuel and can help in meeting the increasing demands of energy to a great extent with the development of genetic engineering technology and can also provide an attractive Energy plant platform for other high biomass plants to be used in the biofuel production.

I prepare my thesis, I just want to know if there is a database on the stability of DNA as a function of pH??.

Whole set of genetic material or genes present in an organism are called as the genome of the organism. Study involving genome of an organism or different genomes of different organisms is termed as genomics. The determination of function of all the genes of a genome of an organism is known as functional genomics. This mainly involves study of location of gene expressions, the functions of the proteins produced and the interactions of corresponding proteins with any other biological molecules.

Expression profiling: The process of determination of the location of expression of each gene and the conditions required for the successful expression of the genes is known as expression profiling. Study of expression patterns of a whole genome of an organism is known as global expression profiling. This can be done by conducting study at either RNA level –involving direct sampling or micro arrays; or at protein level by mass spectrometry or protein arrays. Complete set of RNA molecules produced from the genome of the cell is known as transcriptome. A mature eukaryotic genome is so advanced that it has the capability to produce multiple mRNA from a single gene. The process by which the transcripts produced from genes undergo removal of introns and combining of exons to yield a functional mRNA is known as splicing. In alternative splicing, a single primary transcript RNA is spliced in different patterns. Each pattern of splicing results in different functional mRNA. The expression pattern in different tissues of same eukaryotic organisms differs. Thus, a set of genes expressed in one kind of tissue may not be expressed in some other. This wide study of different expression mechanism in the organism is done by expression profiling and the results are recorded.
Determination of gene function:
In functional genomics, it is important that the function of each and every gene be analysed. Several strategies have been applied for this; the most important one being mutational genomics. In mutational genomics, the function of a single gene can be noted by creating a mutation in the gene leading to loss or disruption of the gene function. The method involves isolating the particular gene, the function of which has to be determined, production of clone of the gene and inducing mutation leading to loss of function. This when re- introduced into the host organism, the loss of function of gene can be noted by analysing the different samples. Thus it has been made possible to analyse the function of each and every gene by inducing mutation to a single gene one at a time. The mutant strains during the experiments are collected to produce mutant genome libraries. One of the different methods adopted for the creation of such libraries are- creation of mutation in a single gene to produce single mutant which can be recorded to form a library. Another method involves induction of random mutations of several genes in a genome. Each mutation is then studied and the mutants are isolated forming the library. This is mostly conducted by insertional mutagenesis where mutation is caused by introduction of DNA into random sites of gene causing loss of function. The introduced DNA also serves as a tag aiding in isolation of specific gene. In yet another approach, the expression of a group of specific or random genes gets mutated in one impact.
Study of protein interactions:
Functional genomics also involves study of products of genes – that is proteins. Different interactions involving different proteins or that involving proteins and other molecules are studied. If behaviour of a protein is unknown, studying the proteins interacting with the corresponding protein reveals the properties of unknown proteins. The technique of protein interactions are studied by high throughput methods. Protein mapping made possible by use of library loaded with protein information, allows studying a large number of proteins at a time by screening methods. Screening of such interactions may be done by in vitro or invivo interactions. The data available from different screening methods are put together to form protein data bases. Different bio informatics tools have been developed to extract information from such data bases as and when necessary.
Thus, different techniques involved in functional genomics enable vast data accumulation, provides insights about the biological mechanisms like differentiation, response to diseases, etc. and has found applications in many fields like drug designing.

Placebo is any pharmaceutically inert substance or medical procedure applied with the purpose to deceive the recipient. Even without appropriate treatment, patients could feel better simply because they believe that given pill will improve their condition. This phenomenon is called placebo effect. Brain’s effect on physical health is very important and medical researchers are using placebo as control in many experiments. However, using placebo in clinical practice is unethical due to deceiving patients with false information and ineffective treatments.

Placebo is not working with everybody. It’s believed that 35% of people respond to placebo. Effect is individual, such as the reaction to the active drug (some people don’t respond to pharmaceutically active pills). Dark side of the placebo is “nocebo”. If patient don’t believe that treatment could improve his health, nocebo effect will result in deteriorated medical condition. Negative attitude patient has toward the therapy could result in serious worsening of the symptoms. Patient could also feel the “side effects” of the investigated drug while on placebo. Withdrawal symptoms are noted as well. Study on hormone replacement therapy in women in menopause lasted for more than 5 years. When completed, > 40% of the women in placebo group reported same withdrawal symptoms as the women receiving hormone replacement therapy.

Despite all positive and negative effects of placebo, it’s still inevitable part of the clinical trials. Placebo can produce same beneficial effect to the health like the investigated drug during the clinical trial. That’s a problem. Cheap and medically inert substitute of a drug shouldn’t be effective like brand new, billion dollars worth pill (cost of typical drug development process). Reason is high susceptible to placebo. To overcome this problem, researchers are recruiting more people to get statistically significant results. Placebo is necessary, but it is not cost effective. Average study spends more than 1 billion dollars due to placebo.

To address this issue, scientists need to discover biological effect responsible for high placebo susceptibility. For the first time, it looks like that they finally have the right answer.

Genetic variations are responsible for the effect of placebo or drug on human organism. Previous experiments showed that dopamine level in the brain is higher in people responding to placebo (dopamine is associated with both sensation of pain and reward). Latest study focused on dopamine pathway, more specifically on catechol-O-methyltransferase (COMT) gene. COMT can be present in couple of forms. Person bearing two copies of a variant methionine allele will have met/met type of COMT gene, those with two copies of valine allele will have val/val type of a gene or, person can have both types of alleles resulting in met/val type of a gene. People with met/met variant have 3-4 higher level of dopamine in their prefrontal cortex (associated with cognition, decision making, social behavior and personality expression) compared to people bearing val/val variant of a gene. Since elevated dopamine level has already been linked with high placebo response, scientists proposed that people bearing different variant of COMT gene (met/met, val/val or met/val) will show different response to placebo. Study of irritable bowel syndrome conducted in 2008 proved that COMT gene proposed hypothesis was correct. During the study, patients were divided in three control groups: one that was on the waiting list, second that received placebo acupuncture treatment in a cold, business manner and third where placebo acupuncture treatment was delivered in a warm and supportive manner. Using the patient’s blood, genotypes were easily determined. People on the waiting list didn’t show any difference in the response, no matter what type of COMT gene was present. In the second group, where placebo was provided in a cold, business manner – met/met carriers showed slightly higher responding rate compared to val/val and met/val genotypes. Striking difference in the placebo response was noted in the third treatment group where met/met carriers showed exceptional difference in response to the placebo delivered in a warm manner, compared with other two COMT variants. This experiment confirmed that met/met genotype is typical placebo “responder” while val/val is not.

Genetics behind the placebo should be investigated further, but even this discovery could reduce the cost, duration and efficiency of the clinical trials by selecting placebo “non-responding” genotypes for the future experiments.

Study of RNAi based therapy for HIV:

The trigger of RNA interference in response to the double stranded RNA has become one of the vastly studied areas of molecular biology and this RNA interference effect has become a genetic tool in the gene function studies as well as the development of therapeutics for various diseases by manipulating genes and their related functions. The control of the gene function helps in the regulation of the different developmental stages in the life cycle of an organism as well as in the progression of the various stages of a particular disease.

The first target of the RNAi was the infectious agent responsible for HIV infection known as the Human Immunodeficiency virus. The reason behind this development maybe due to extensive research in this area leading to accumulation of knowledge regarding the life cycle of the virus and its pattern of gene expression. Further research has proved the role of RNAi in targeting other diseases such as Hepatitis B, Hepatitis C, Polio, etc.

Several HIV-encoded RNAs both early and late have been targeted in cell lines as well as in the primary haematopoietic cells including the tat, gag, pol, env, nif, rev, vpr, TAR element, reverse transcriptase, Long terminal repeat (LTR), etc by the expressed shRNAs (short hairpin RNAs) and the siRNAs prepared synthetically. In certain cases, RNAi has been illustrated in the prevention of HIV infection in cells i.e. before they are infected by the retrovirus. However, in cases where the cells are infected by the HIV retrovirus, the RNAi mechanism proceeds via the following steps:
a) The release of the RNA genome of the retrovirus transcribes with the help of HIV reverse transcriptase to form HIV-DNA, also known as the provirus that incorporates into the cell genome and gives rise to mRNA transcripts.
b) Artificially synthesised siRNA specific to the HIV mRNA in a particular stage of the virus life cycle are introduced into the cell by injection or lentivirus vectors called siRNA vectors.
c) These siRNAs inserts into the RISC (RNA-Induced Silencing complex) whereby only a single strand of siRNA remains, that binds to the specific HIV mRNA and cleaves it. RNases within the cell then remove these fragments.

Thus, the siRNAs help in the neutralization of the HIV mRNAs thereby reducing the chance of synthesis of HIV proteins within the cell thus preventing the progression of the infection.

Although, the inhibition of HIV-encoded RNAs mediated by RNAi has been made possible, the direct targeting of the HIV virus faces numerous challenges for clinical application due to an increased rate of mutation in the virus leading to the formation of mutants that escape from being targeted. This incidence of mutation is observed in not only HIV viral RNAs but also other RNA viruses encoding RNA Polymerases or reverse transcriptase, which also have a tendency of formation of mutants with every replication cycle. Hence, complementary approach of targeting of cellular transcripts that encode for functions such as the entry and replication of HIV virus i.e. the down-regulation of the various cofactors present within the cell necessary for the progress of HIV infection was studied.


Down-regulation of various cellular cofactors such as the HIV receptor CD4, NFκB, and the co-receptors CCR5 and CXCR4 have been studied, which proved successful in the replication of the virus as well as its cell entry. However, the CXCR4 receptor was found to be essential for the hematopoietic stem cell formation in the bone marrow as well as subsequent T cell differentiation and the CD4 was also found to be an essential cellular receptor. Hence, although the targeting of these cofactors was found to help in complete stoppage of viral replication, it created several problems within the body. Hence, viral targets became essential for the successful study of RNAi based therapy for HIV. The mixture of single shRNAs with several antiviral genes have become a potent alternative anti-HIV approach, which is becoming an active area of research.


The introduction of the siRNAs have posed great challenge as the vectors used for the delivery initiated immunological reactions within the body. It was overcome partly by an approach involving the isolation of the T-cells of patients, its transduction with lentiviral vector carrying the anti-HIV antisense RNAs and expansion, followed by re-infusion into the patient’s blood stream. Another significant progress in this area is the use of the hematopoietic progenitor stem cells, which are isolated, transduced with the vector carrying the therapeutic genes, followed by reinfusion.


In this way, it can be noted that compared to the ribozymes or the antisense approaches, the RNAi based therapy is more potent. The pre-clinical study of this approach on the human trials in near future will mark a revolution in the therapeutics for the HIV based infection.

The techniques of gene transfer have reflected many successful examples in variety of fields. Some of the examples in the field of agricultural biotechnology are:

Resistance to herbicides: It is important that plants do not get affected on the application of herbicides. In order to install this property of resistance, different methods has been approached through means of gene transfer:
Production of target molecules: The target molecules which are resistant to action of herbidicdes are identified. The gene responsible for such target molecules are incorporated into the plant genome so that the plants produce such target molecules expressing resistance to herbicides. Eg: the gene aroA isolated from Salmonella typhimurium or E.coli is known to produce an enzyme EPSPS which is resistant to the glyphospate herbicide. Transgenic tomato and tobacco plants produced by transfer of this gene were seen to exhibit herbicide resistance.
Detoxification of herbicides: Certain plants produce enzymes which has the ability to detoxify the herbicide action. The introduction of the respective genes can induce herbicide resistance in plants. Main example for this is detoxification of atrazine herbicide by maize plants producing enzyme glutathione-S-transferase.

Resistance to insects:
In the case of developing insect resistant plants, the process involves transfer of insect resistant genes. The major breakthrough in developing resistance to insects was obtained from Bacillus thuringiensis. A gene called as ‘cry genes’ produces a protein called as cry protein which are largely responsible for insect resistance. In the bacterium, these proteins produced exhibit resistance to insects. The cry protein ingested by insects undergoes modification to release toxins in the gut region resulting in the lysis of insects. These genes have been successfully transferred to the genome of plants like tobacco, potato and tomato. Such transgenic plants are found to exhibit resistance to insects like Manduca sexta and Heliothis virescens. Over a period of time, it was found that insects were developing resistance against such proteins. Combating to this, development of transgenic plants expressing alternate form of cry protein but maintaining its toxicity was introduced. The modified cry genes showed greater levels of expression and toxicity.

Virus resistance:
Several strategies have been followed for developing virus resistance in plants. It includes expression of coat proteins, developing cDNA from satellite RNA, degradation of viral genome, antisense RNA approach, and production of viral specific RNAase.
Of these, the technique involving expression of coat protein has met with high level of efficiency. The transgenic plants produced were integrated with genes responsible for producing viral coat proteins. This helped to reduce viral replication thus developing viral resistance.

Bacterial resistance:
In developing resistance against bacteria, different approaches targeting the growth and development of bacteria or bringing about degradation of toxins released have been applied. Of this the most successful phenomenon was that of artificial cell death. In response to certain stimuli the cell of certain organisms produces proteins which bring about the death of cells. Such mechanism is known as programmed cell death. In transgenic plants this capability of cell death was modified so that the infected cells gets targeted and trigger a response of artificial cell death leading to the death of infectious agent. This phenomenon is found to be active against fungi infection also.

Drought resistance:
Several genes has been identified which show increased response to abiotic stress like abscisic acid, osmotic stress etc. Some of the genes involved are:
Rab which respond to abscisic acid; SalT responsive against salt stress and proBA and proC involved in proline biosynthesis.
The genes responsible for proline biosynthesis when isolated and expressed in transgenic plants resulted in high expression and consequent development of resistance to osmotic pressure was indicated. This resistance was possible due to the deposition of proline in the cells which helps in regulating water activity of the cell. Certain other genes like mtl1D from E.coli have been seen to express mannitol accumulation in cells which aid in plant growth under non favourable conditions.

Improving seeds quality:
Certain seeds produced by native plants have been found to be deficient in some important nutrient lowering the seed quality. With the help of genetic engineering, the seed quality can be increased so as to produce economically important seeds. The basis of this is, introduction of genes into the genome corresponding to the production of missing nutrient or modification of endogenous genes of plants to produce seeds of high quality. Eg:- In peas, the seed produced has lesser sulphur containing amino acids. Such amino acids are rich in sunflower seeds. The integration of gene encoding for such amino acids in peas seeds and expression in transgenic tobacco, led to the production of seeds with the respective amino acids in adequate amounts.

Stem cells have shown great promise in the treatment of a number of major diseases. Their unique properties of differentiation have made their studies a widely researched area. Although, there are some ethical issues surrounding the subject, the development in this area can offer solutions and therapeutics for many fatal diseases with minimal complications. However, in-depth research and scientific testing on human trials is essential before it can have wide application for treatment throughout the world.

Heart diseases are one of the major diseases affecting majority of the population all over the world. The inability of the heart cells to regenerate and repair themselves after the occurrence of a heart attack prevents the application of further treatments on such patients. In such cases, stem cells have helped in regenerating damaged heart muscle cells by the introduction of adult stem cells from non-heart tissues into the damaged heart. Hence, they play an important role in the treatment of heart diseases.

Neuronal diseases and spinal cord injuries have always been a cause of fear for people as the treatment of these diseases are quite complicated and their complete cure is quite difficult to attain. In this area also, stem cells have made a major mark with proof of successful studies in rodent models. The condition of paralysis or impairment of motor function due to spinal cord injury was found to be reversible to perfectly normal condition with the introduction of embryonic stem cells. In a motor neuron degenerative disease called Lou Gehrig’s disease also, it was found that the introduction of embryonic stem cells helped in the restoration of the function of motor nerve cells. However, all these studies were found to be successful in the rodent models; hence, they remain to be translated into the human studies. Stem cells have proved successful in the treatment of Parkinson’s disease, in which they were able to restore the secretion of neurotransmitter, dopamine, with the transplantation of the stem cells. However, there were possibilities of hypersensitivity to increased dopamine levels. Hence, detailed study is necessary before it can be translated into potential treatment.

Stem cells have provided a path for the treatment of the degenerative retinal diseases. The successful differentiation of the stem cells into photoreceptor cells and the introduction of these photoreceptor cells into the retina of the patients helped in restoring the vision of the patients, thereby preventing complete blindness. The differentiation of the stem cells into specialised pancreatic cells with the ability to secrete insulin has provided a gateway to the treatment of diabetes associated with impaired insulin secretion due to the destruction of the pancreatic β-cells by the immune system.

The stem cells have played a major role in the treatment of cancer like leukaemia and this is one of the successful applications of stem cell based therapies. The disease of Leukaemia affects the white blood cells, which become cancerous and lose their normal functioning. The ability of the normal white blood cells to fight against different infections and protect the body is lost. This results in the vulnerability of the body to develop infections as the immune system is compromised and the functionality of the different vital organs within the body is lost. The bone marrow transplant along with chemotherapy helps in replacing the damaged blood cells with fresh blood cells. The patient’s damaged cells are removed by chemotherapy and the new stem cells introduced by transplantation give rise to new blood cells, thereby restoring the functions of the blood cells. The introduction of new refinements in the technique has removed the necessity of a match between the donor and the patient’s bone marrow. The multiplication of the stem cells in the donor blood to high concentrations has removed the need for matching the donor and patient, thereby improving the patient outcome, and helping in the eradication of cancer in future.

New research studies have shown the reattachment of the lost teeth and the growth of new teeth in place of lost ones with the help of stem cells within the patient’s body, thus opening a new area of research in the field of dentistry. The ability of the stem cells to repair bones and also regenerate lost bones within the body has demonstrated the remarkable progress in the stem cell research. Thus, stem cells research has become the future for different therapeutics and can be the ultimate solution for decreasing the suffering and increasing the mortality of the mankind.

Many a time people lose their bones due to different types of trauma, accident, etc; hence need bones for proper functioning and in certain cases, people need bones to replace the lost ones due to bone cancer or war injuries, etc. In such cases, bone transplant was the only type of treatment available in olden days. The potential of the stem cells to replace heart tissue and even create a trachea to replace a damaged trachea, has initiated the study and research involving stem cells in other areas too. Recent studies have showed the potential of using stem cells for the repair of bones and also their use in growing certain bones within the body. Thus, extensive ongoing research studies in the field of stem cells extend the boundaries for the use and application of stem cells in different types of therapeutics.

The substitution of bones was done by the surgeons using different types of metals, plastic, etc and the procedure had the possibility of rejection of the transplant by the body. All these initiated the development of a better approach for bone repair and substitution, in which stem cells showed great promise. Recent most study has showed one of the greatest challenges met by the stem cells in growing bones. Stem cells were used to grow back the facial bones in a boy with no cheekbones. Due to the use of stem cells in growing back bones, the possibility of rejection due to a transplant was automatically removed, which helped to a great extent.

The transplant of a bone from another part of a person’s body to the site where bones need to be replaced was not very efficient in form and function. Moreover, it still faced the possibility of rejection. Hence, the use of stem cells to regenerate bones found application. Due to the formation of functional bone cells from the stem cells and thus the formation of a bone with proper function in the target site proved the importance and use of stem cells in successful bone repair and regeneration.

The stem cells used for the replacement of bones and also for growing bones were mainly extracted from the fat cells or the adipose tissue of the affected individual. Hence, this removed the necessity of donors for the transplants. The use of stem cells isolated from the fat of the affected individual also removes the possibility of any sort of immunological reaction against the stem cells as the body recognises them as its own. In most of the treatments, the immunological reaction causes hindrance, which is avoided in this case of stem cell treatment. Fat or adipose tissues are mainly selected for the purpose of stem cell isolation, as it is completely accessible besides being far more convenient, cheaper, as well as less painful for the patient. The procedure of stem cell isolation from other areas within the patient body is very much painful and time consuming. Even the recovery of the patient after the isolation procedure takes long time thereby delaying the application of the isolated stem cells. Previously, the introduction of the isolated stem cells within the body was done directly to the target site of injury, which indicated the movement of the stem cells throughout the body. This movement lead to the failure of the procedure. Hence, the isolated stem cells were then introduced within the body using a gel like substance that helped in localising the stem cells in the bone injury site thus helping in the repair of the bones by healing them.

The use of stem cells in the regeneration of bones was seen in a study involving a teenaged boy, who suffered from a rare type of inborn genetic defect, in which he had no cheekbones in his face. Replacement of cheekbones became necessary not just to improve appearance, which was a minor requirement but mainly to restore the important functions of the cheekbones such as protection of the eyes. The procedure proved successful with the regeneration of the facial bones with the stem cells extracted from the adipose tissue. Although, the procedure proved successful, the scientists have some doubt regarding the practical applicability of this procedure in all types of patients. The young age of the boy was one of the important factors that lead to the success of the procedure due to better ability of regeneration and healing at young age. The application of the procedure in an elderly individual may not meet with same success as in this case. Moreover, the functionality of the bones regenerated even after five years of the procedure need to be analysed to assess the effectiveness of the procedure entirely. Hence, in-depth research regarding the procedure is essential before it can be used for the practical application in bone repair and regeneration.

The technique of utilization of biological agents for degradation of pollutants is known as bioremediation. Phytoremediation is a branch of bioremediation wherein plants are employed for the purpose of removal of pollutants from a specific area.

Plants aid in phytoremediation by absorption, assimilation of compounds, vaporization of pollutants, metabolic digestion, or by microbial degradation by plant associated microbes. The plants which can accumulate and degrade the contaminants are known as ‘hyperaccumulaters’ which play a major role in phytoremediation.

Different modes of Phytoremediation
Depending on the technique applied phytoremediation can be subdivided into many kinds:

(i) Phytoextraction: it refers to a process in which plants breakdown contaminants and adsorbs the same into its tissues. After adsorption, plants can be removed from site and disposed or incinerated. Different plant types have different ability of phytoextraction and the plants with most effectiveness are chosen usually. This process is mostly used for treatment of metal pollutants in soil.

(ii) Rhizofiltrtion: in this process the pollutants gets adsorbed and deposited, not on the shoot tissues but in this case, in the root or rather in the rhizosphere of plants. This technique is specifically employed for waste removal from contaminated water sources.

(iii) Phytostabilisation: this concept involves immobilization of the pollutants following absorption and adsorption of it by plant roots and finally precipitation of the pollutant in the root so that it does not migrate from soil into air or other sources.

(iv) Phytotransformation: it deals with transformation or degradation of pollutants as a result of various metabolic processes by plants. Thus it is also known as phytodegradation.

(v) Phytovolatilization : the contaminants are absorbed by plants, undergo many changes and finally gets volatized from leaf surface during transpiration process of plants.

(vi) Phytostimulation: the microbial population near the root system of plants gets induced by the presence of rhizosphere which initiates them to break down the pollutants. This process is also known as rhizosphere degradation.

Role of biotechnology in phytoremediation:
Off late, biotechnology has been found to increase its wide spectrum of applications into phytoremediation as well. Plants adopted for phytoremediation are usually found to exhibit the specific property due to the presence the special genes coding for it. These plants are usually seen in area where metal ores exist. The genes responsible for this resistance by such plants are isolated and expressed in wide variety of transgenic plants so that they can be made resistant as well. This increases the number of plant species that can be used for such purpose. It is also possible with the help of biotechnology to increase the gene expression for maximum resistance.

Certain plants are seen to show increased resistance under the presence of certain microbes. Biotechnology makes it possible to isolate such microbes and enrich the soil so as to enhance the phytoremediation by respective plants.

Examples of application of biotechnological aspects in phytoremediation

Selenium: The micronutrient selenium is known to induce toxicity in the soil where the concentration of the same is found to be high. It is found that methylation of amino acids at specific site can result in volatilization of selenium compound. Thus a transgenic plant is constructed which has the ability to volatize the same by following the guidelines of genetic engineering and utilizing the information obtained by studying hyperaccumulators of selenium.

Mercury: Mercury can be degraded by certain bacterium due to the presence of merA and merB genes. Thus integration of these genes into certain plant genomes has seen effective mercury degradation by such transgenic plants. The genes are targeted to be expressed in chloroplasts so that after degradation into relatively less toxic form, it is volatized. Thus transgenic tobacco produced by this phenomenon was shown to exhibit mercuric resistance.

Arsenic: Certain bacterial genes present in E. coli, such as ArsC is responsible for reduction of arsenic and formation of a complex in the presence of glutathione(GSH). An increased amount of GSH can be produced by expression of glutamyl cysteine synthetase enzyme. These genes are isolated and transferred to form a transgenic plant which can effectively absorb arsenic and accumulate the same in its vacuoles resulting in phytoremediation.

Thus it can be concluded that biotechnological tools can be utilized to improvise many existing phytoremediation systems yielding more effective and faster results.

Viruses are smaller in size to bacteria and can also be described as nano particle stating its small size. Viruses are parasitic in nature as they always depend on a host cell or organism for replication. Viruses are known for its infectious nature, infecting right from bacteria to humans.

Virus grouping: Viruses are divided and grouped into various types using the key factors like morphology (structure), biological role, type of genetic material and mode of multiplication. The unique feature of virus is that their genetic material is covered by a protective layer termed as capsid made up of units of protein encoded by the virus itself. The association of the capsid and the genetic material (nucleocapsid) describes the structure of the virus. The viral structure has an outer envelope made up of lipoproteins and the space between the capsid and the envelope is called as Matrix which acts as a bridge between the inner nucleocapsid and the outer envelope. The matrix region is composed of proteins again.

Viruses are classified based on the type of genetic material present into DNA virus and RNA virus. DNA virus, as the name indicates have DNA as their genetic material and the DNA is either linear or circular and double stranded or single stranded. Based on the length of the genetic material present, these viruses are again divided into big and small DNA viruses. Herpes virus and pox virus are examples of double stranded DNA virus and parvo virus is single stranded DNA virus. The virus with RNA as genetic material are grouped under RNA viruses and the RNA present may be double stranded or single stranded. Also the single stranded RNA virus may have either cationic or anionic strand. Reo virus is an example of double stranded RNA virus and picorna virus is a positive single stranded RNA virus and Rhabdo virus falls under the group of negative single stranded RNA virus.

Viral Life Cycle: Virus is host specific and this nature enables them to bind themselves to the host cell. Once attached it penetrates and enters the host cell environment. The outer envelope is shed inside the host cell and mRNA is synthesized through transcription which is followed by translation into proteins. Following this is the glycosylation process and the replication begins resulting in multiple viral copies, which then assemble and exits the host cell as mature viruses.

Viral Infections: The potential of a virus to cause infection to the host cell is termed as virulence. Viruses infect bacteria, plants, animals and humans causing various reversible and irreversible diseases.
Humans: Eye infection caused by Herpes simplex virus, cytomegalo virus, encephalitis by LCM virus, Rabies virus and the occurrence of common cold is due to para influenza virus, respiratory syncytial virus. The Hepatitis virus of different groups A, B, C, D & E in causing hepatitis, a liver disease and Coxsackie B virus is identified in pancreatitis. Infection by rota virus, adeno virus and corona virus causes GI tract related diseases. HIV, Herpes simplex 2 and Human Papilloma virus are the major causative agents for the sexually transmitted diseases.

Plants: Plant viruses have different shapes like icosahedral, rod, filament or isometric. Some of the plant viruses are Tobacco Mosaic virus, cucumber mosaic virus, Lettuce mosaic virus and citrus psorosis virus. The viruses are named based on the type of disease they cause in plants.

Birds and Animals: The H1N1 episode due to the infection of birds by Influenza virus A and the infection of pigs by influenza virus (B or C) spreading swine flu are the classic examples of bird and animal viruses and their transmission to humans.

Bacteria: virus with a potential to infect bacteria is called as a bacteriophage. T4, T5, T7 phages, MS2 phage and Qβ phage are examples of the bacteriophages. The structure of a bactriophage is unique with three regions like icosahedral shape as head with shaft like middle region and tail like structures at the base.

Cancer and virus: Few viruses are detected with their ability to cause cancer in humans. The cancer causing oncogenes were first identified and studied in retrovirus. The human Papilloma virus causes cervical cancer and the chronic liver disease due to the infection by Hepatitis B virus activates the liver cells to become cancerous.

The structure, function and mode of replication enabled scientists to make use of viruses in different field of biological science. The ability of the virus to deliver the genetic material into host made them as suitable vectors in genetic engineering. The association of the virus with the immune system enabled to develop vaccines for various viral diseases. The use of virus in the field of nanotechnology is cited by the use of cowpea mosaic virus as signal amplifiers in DNA microarray technique by the researchers of Naval Research laboratory, Washington, D.C. The different perspective on virus as a tool in cancer therapy and gene therapy will be beneficial. The ability of the virus to infect bacterial cell is used to kill pathogenic bacteria (Phage therapy). Besides all this beneficial applications of virus, the threat lies in the fact that they can be used as bioweapons.

A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component.

An analytical device which functions to analyse a sample for the presence of a specific compound is known as sensor. A sensor which utilizes biological material to specifically interact with an analyte is known as biosensor. An analyte refers to the compound which has to be ‘sensed’ or the presence of which has to be determined. The interaction of analyte and biosensor is measured and converted to signals, which are again amplified and displayed. A biosensor thus involves converting a chemical flow of information into electrical signals. The biological materials used in biosensors are mostly enzymes, antibodies, nucleic acids, lectins, a cell as a whole etc.

According to the mode of interaction biosensors are of two types:
Catalytic biosensor: The interaction of biological material in the biosensor and the analyte result in modification of analyte into new chemical molecule. The biological material used is mainly enzymes.
Affinity biosensor: Here, upon interaction, the analyte binds to the biomolecule on the biosensor. These are mainly composed of antibodies, nucleic acids etc.

Essential properties of a biosensor:
(i) Specificity: a biosensor should be specific to the analyte which it interact.
(ii) Durability: it should withstand repeated usage.
(iii) Independent nature: It should not be affected by variations in the environment like temperature, pH etc.
(iv) Stability in results: the results produced by interaction should be corresponding to the concentration of analyte.
(v) Ease of use and transport: it should be small in size so that it can be easily carried and used.

Components and mechanism of a biosensor:
A biosensor mainly consists of two parts
(i) a biological part: this constitutes of enzymes antibodies etc., which mainly interacts with the analyte particles and induce a physical change in these particles.
(ii) a transducer part: which collects information from the biological part, converts, amplifies and display them. In order to form a biosensor, the biological particles are immobilized on the transducer surface which acts as a point of contact between the transducer and analyte.
When a biosensor is used to analyse a sample, the biological part specific to the analyte molecules, interacts specifically and efficiently. This produces a physicochemical change of the transducer surface. This change is picked up by the transducer and gets converted into electric signals. These then undergo amplification, interpretation and finally display of these electric units accounting to the amount of analyte present in the sample.

Types of biosensors:
(i) Calorimetric biosensor: some enzyme- analyte reactions are exothermic and releases heat into the sample. This change in temperature is detected by the transducer. The amount of heat generated is proportional to the analyte concentration present and is processed likewise.
(ii) Potentiometric biosensor: an electric potential is produced as a result of interaction which is detected by the transducer
(iii) Amperometric biosensor: analyte when comes in contact with biological material induces a redox reaction. This results in movement of electrons which is picked up by transducer.
(iv) Optical biosensors: in this, a biosensor reacts with analyte to absorb or release light which is identified by the transducer and interpreted.
(v) Acoustic wave biosensors: biological component of biosensor undergoes a biomass change ascertained by transducer.
The advantages of biosensors include accuracy in results, minute detection capability, ease of use, versatile and continuous monitoring available.

Applications:
A biosensor has a wide range of applications in different fields.
Medicinal Application: biosensors have been used in various diagnostic procedures to determine various tests.
Industrial application: various manufacturing processes can be monitored by biosensors to provide assistance with regard to increase the quality and quantity of product obtained.
Environmental application: it helps in measuring the toxicity of water bodies, microbial contamination of natural resources helping in developing steps towards a cleaner environment.
Military application: it helps to detect explosives, drugs etc., aiding in defence of the people. Another breakthrough in the field of biosensors was the production of a product called ‘smart skin’. It is a kind of biosensor which detects any chemical or biological attack nearby and warns the person using the same.
Drug development: a biosensor called ‘nano sensors’ has been developed which detects and analyse the binding of proteins to its targets which has proved very useful in drug designing. This also helps to monitor certain side effects caused by some medicines.

RNA interference (RNAi) as a therapeutic strategy.

Forward genetics has proved to be useful in the detection of the function of the genes in the early days with the help of the knowledge about the phenotype of the mutant gene. However, with the development of technology, reverse genetics has proved to be a better and effective method for the discovery of gene function due to the development of genome sequencing technology, which helped in the discovery of a number of genes without knowing their function. The general method of homologous recombination mediated gene targeting for reverse genetics proved costly, which led to the development of other approaches such as Antisense technology using antisense oligonucleotides as well as the use of ribozyme technology, which also has only limited utilities. Research related to the development of other methods for reverse genetics led to the discovery of the small interfering RNA technology, which showed great promise in revolutionalizing the approach of reverse genetics by knocking down the expression of a particular gene in the vertebrate cells. This technology of using double stranded RNA (dsRNA) to silence specific genes is known as the RNAi technology.

RNA interference involves two types of RNA molecules-micro RNA (miRNA) and small interfering RNA (siRNA). The RNAs are direct products of genes, which produce specific mRNAs by transcription. The siRNAs are duplex RNA molecules 20-25 nucleotides long, which are formed from the long dsRNA by enzymatic cleavage catalysed by Dicer, cytoplasmic RNaseIII enzyme. One of the strands of this siRNA known as the passenger strand is degraded, while the other strand known as the guide strand becomes a part of the RISC (RNA-induced silencing complex) or iRNP complex. It then base pairs with the complementary sequence of the mRNA molecule within the cell and induces its cleavage by the catalytic part of the complex, Argonaute proteins that are actually endonucleases. In this way, the siRNAs prevent the translation of the mRNAs by initiating their degradation. This process is known as RNA interference, which is a posttranscriptional event.

The Pathway of RNAi has some salient features such as the involvement of dsRNA and highly efficient and potent silencing of specific genes with minimum effort that can be introduced in various developmental stages. It has also found application in the identification of different components of various cellular pathways by systemic gene silencing, hence can be used for the development of various targeted and personal therapeutics. RNAi approach has also been useful in cancer therapy as it helps in the knocking down the expression of the anti-apoptotic genes or the cell cycle genes. Research studies have proved the presence of miRNAs in the fragile areas of genome associated with cancer, hence miRNAs may have role in tumor suppression. In some cases miRNAs may function as oncogenes as some studies have found the association of miRNA mutation with various cancers. Studies have shown that some miRNAs may bind to complementary regions in the promoter and may upregulate the expression of some genes, though it has not been clearly illustrated. Hence, miRNAs are also known as Oncomirs, due to their role in cancer.

RNAi has important role in generating immune response against different viruses and has found application in the prevention of self-propagation of the transposons in plants. The novelty of RNAi approach using endogenous mechanism shows great promise in the field of functional genomics that is spreading into the therapeutics. In future, it may offer therapeutics for different metabolic diseases including diabetes, various neurodegenerative diseases, and cardiovascular diseases, which originate from the faulty expression of tissue-specific genes. Hence, it has become a valuable and effective research tool for the biotechnological studies of both living organisms and cell cultures.

However, there are two main challenges in the development of RNAi as therapy. Firstly, the off target effects of RNA interference must be avoided for which in-depth study of the different mechanisms leading to non-specific effects of siRNA is needed and secondly, the efficient delivery of the synthetic siRNAs or iRNA into the specific cell or tissue is very essential for proper development of therapeutic utility of the RNA interference. The use of viral vectors in the delivery of the therapeutic siRNAs into the specific tissues or cells has some safety concerns, which has prompted detailed delivery-related research. RNAi has also found application in the genome wide high-throughput screening of the genes responsible for loss of function as well as genes responsible for specific biological phenotypes. Hence, due to great potential of RNAi in therapeutics, pre-clinical study using this technology is becoming essential.