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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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”.

Application of knowledge of DNA based information for resolving crimes and knowing the identity of criminals is known as forensic science. DNA fingerprinting is a highly accurate and sensitive mode of approach of resolution of crimes.

The technique of DNA fingerprinting was first demonstrated by Alec Jeffreys in 1986.

Principle behind the DNA fingerprinting or profiling process is to determine and indicate the presence of specific allele in a series of polymorphic loci or locus in his/her genome. A polymorphic locus refers to a region in the genome of an individual different from others. The presence of different alleles in such a locus is studied by using restricted fragment length polymorphism (RFLP) or by PCR amplification. Thus, the whole number of polymorphism existing in the mini, micro satellite and mini variant repeats constitute the variable number of tandem repeats(VNTR) and are analysed for the DNA fingerprinting process. The site of presence of VNTR is known as VNTR locus. The different allele consists of different number of repeating units of VNTR sequence. The sequence of unique DNA elements flanking the repeats are known and primers corresponding to the same is produced and used for amplification in PCR of mini and micro satellite DNA. The final product of such amplification tends to be different in different individual and can be separated by gel electrophoresis technique.

The DNA is extracted from several sources like hair, semen, solid tissues, blood tissues, blood stains, buccal cells of saliva etc. The extracted DNA is stored in a clean, cool, dry place. In the cells extracted, it may be digested by sodium do decyl sulphate, and the proteins digested by some proteinase enzyme. This is followed by repeated extraction of these with phenol and chloroform to remove cell debris. The obtained DNA is dissolved in a solution. This can be precipitated by addition of alcohol. DNA degradation is specified by staining DNA with ethidium bromide and undergoing agarose gel electrophoresis. The denatured DNA appears as smear whereas those not digested appears as a single band.

DNA profiling
The DNA obtained is studied by mainly two mechanisms –by RFLP and by PCR analysis.

RFLP: - These analyses are of two types: multi locus polymorphism (MLP) and single locus polymorphism (SLP) .The DNA digested and run on agarose is transferred to a membrane and probed with highly specified probe. Thus probe also hybridize with fragments of similar sequences. The hybridized probe is detected by autoradiography or in the case of alkaline phosphatase present in the probe by chemiluminescence. It is not possible to differentiate fragments differing by few repeats by this process.

PCR: This analysis are of two types STR (short tandem repeats) and MVR (minisatellite variant repeat) analyses. The most common type is to analyse STR. In this methods two probes complementary to the sequences flanking an STR is used to hybridize and each STR obtained is amplified by PCR. The resulting amplified product appears as distinct bands in agarose gel. Each STR has as many as 9 to 10 different alleles in human. Each individual has two loci for STR. Therefore it is necessary to analyse 9 in 10 repeats to attain a definite result. This process has several advantages like the procedure is very sensitive and DNA can be retrieved from very minute specimens available, the result is obtained very quickly and even the fragmented DNA can be used as primary sample for this purpose. Thus a single PCR cycle provides us with multiple copies to work on.

The information of the DNA obtained by such methods is used to interpret the results of the crime. For eg: the band of DNA is compared with those of the DNA obtained at the crime spot. If the bands are similar then the accused can be confirmed as the culprit. Thus it enables a high sensitive technique for identification of the criminals.

The advantages of DNA profiling include
Identification of criminals: The main and most widely used application of DNA profiling is use of the technology to identify criminal. This helps in determining whether the accused is the real culprit or not.

Kinship analysis: This method can be use to determine where two or more individual are members of the same family. It is also an important evidence for paternity testing in confirming the parents of a child.

Sex identification: This can also be used for identification of sex by amplifying the sequence for Y chromosome.

Stem cell science and therapies related to stem cell are making remarkable progress. Menstrual blood has always been a subject of study in different researches due to its easy availability. The possibility of isolation of stem cells from the menstrual blood and the umbilical cord blood cells has opened a new channel in stem cell research that can offer future therapeutic benefits to mankind. Several successful researches have shown the use of the stem cells derived from the endometrial blood for the treatments of different fatal diseases. The current breakthrough discovery that the menstrual blood contains stem cells that are proliferative and are capable of differentiating into different types of cells including cardiac cells, neural cells and into almost 9 types of tissues including heart, liver and lung, has opened a new field for therapeutic possibilities.

Since the stem cells can be easily obtained from the sources of umbilical cord blood and menstrual blood, hence much research on the subject has been made possible. Moreover, the stem cells from these sources have the potential to differentiate into many types of cells and being immunologically immature offers them the potential to promote cell survival rather than playing a role in cell replacement, which takes place after cell transplantation. The stem cells derived from the menstrual blood i.e. menstrual blood-derived stem cells (MenSCs) have the additional advantage of being available every month in a woman in her reproductive age; hence, could be collected easily than the human umbilical cord blood cells (huCBs), which could be collected only at the time of birth.

According to research based on neuroscience, it has been found that the transplantation of the stem cells isolated from the umbilical cord blood cells and the menstrual blood cells can help the therapeutics of the various neurological disorders like Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), stroke, etc.

It has been found that the transplantation of huCBs in the animal models of those neurological diseases helped in the reduction of inflammation that is the main component of the neurodegenerative diseases. Although, the transplanted huCBs failed to enter the brain in most of the cases, they often helped in the behavioural improvement. These cells were found to have anti-inflammatory properties as also being pro-angiogenic in nature, that is, capable of encouraging tissue repair and cell growth.

The transplantation of MeSCs in the animal models of stroke resulted in the differentiation of the MeSCs into different neural cell types. It had the potential to protect against the deprivation of oxygen-glucose in the animal models as well as the laboratory cultures of stroke (in vitro and in vivo). The transplanted cells secreted factors that had neuro-protective effect. This may be related to the secretion of vascular endothelial growth factors (VEGF), brain-derived growth factors (BDNF), and neurotrophin-3 (NT-3) by the cells, which play important role in the treatment of stroke.

Both blood vessels as well as neurons are essential for brain repair after stroke. The stem cells have the potential to spur the growth of both the vital components. It has been found that a specific type of stem cell derived from the menstrual blood was more potent than the adult stem cells as it could differentiate into more tissue types i.e. from fat to muscle to nerve.

Research studies have proved the use of MeSCs in preventing amputation of limbs due to peripheral artery disease. Critical limb ischemia is an advanced stage of Peripheral artery disease that caused very low blood flow in the limbs thereby causing withering of limbs leading to its amputation. There is neither medicinal nor surgical treatment for the advanced stage of the disease. Studies in mice models of the advanced stage of the disease were carried out and it was seen that the treatment with the injection of endometrial regenerative cells (ERCs) or the MeSCs were found to help in the revitalisation of the limbs and restored its functionality. The ERCs have regenerative properties and could help in the stimulation of blood vessel growth. Moreover, the ERCS are easily injectable without the use of any complex instruments. The ERCs did not invoke any immunological response, hence could be used in “off the shelf” manner i.e. the injection of ERCs do not require any sort of matching before introduction into the point of care. The translation of the mice studies into human clinical trials are awaited in near future.

Hence, the menstrual blood offers a better alternative for adult stem cells that can circumvent the various logistical and ethical limitations faced by the embryonic stem cells, due to constant debates over the use of embryonic stem cells in medical research, thereby helping in new discoveries related to use of stem cells in medical practice.

From olden days, the intake of proper food has been considered as the first medicine for maintaining good health as it consists of all the nutrients as well as the necessary healing items. However, in the modern times that consist of processed, irradiated, homogenized and genetically modified and engineered food, maintenance of proper diet is itself a huge challenge. Moreover, the addition of preservatives, artificial flavouring, colours, and different harmful additives can further do more harm than good to the public health. Hence, people are switching over to organic foods and foods that were eaten in olden days with natural spices and without any additives to achieve good health.

The use of culinary herbs and spices in the diet was integral to the diet, which has been long forgotten by the present generation, which relies on junk food and readily available ready-to-eat food that are easy on pocket and require minimal or no labour for preparation. Hence, the emergence of various diseases including metabolic disorders, food poisoning, and poor immunity are becoming widely prevalent. The inclusion of different herbs and spices not only adds flavour, variety, aroma, and colour to the foods but also has a big contribution of essential nutrients and bioactive substances leading to health improvement. They also have the added advantage of enhancing the beneficial properties of other components present in the food.

Different herbs and spices are also being used in medicinal therapy, as they are an essential component of most of the drugs given in prescription. Modern tools and techniques of physiology are being used for the elucidation of different underlying mechanisms for the justification of the use of spices as flavours, digestives, secretagogues, antiflatulents, and in constipation and diarrhoea influencing gastrointestinal secretions and motility, secretions of liver and pancreas, various processes involved in absorption, and bacterial microflora.

The phytoestrogens and phytoprogestins that were extracted from some of the herbs and spices were tested for their relative capacity to bind to the receptors of estradiol (ER) and progesterone (PR) in intact breast cancer cells of human competing with estradiol and progesterone acting as their agonists and antagonists in vivo. Some of the them are listed below:

a) Highest ER-binding herbs consumed are turmeric, soy, licorice, thyme, red clover, verbena, hops, etc.
b) Highest PR-binding herbs consumed are turmeric, red clover, and thyme.

Some of the herbs and spices have been shown to have many pharmacological aspects and can be used as an alternative therapy for various fatal diseases like cancer, cardiovascular diseases, diabetes, etc. Below is the list of some of the herbs and spices that contain different pharmacologically important compounds:

a) The phenolic compounds extracted from different edible plants (e.g. Nigerian spices and herbs) have been found to have anti-inflammatory properties as well as act as free radical scavangers thus helping in acting as antioxidants. They were also found to reduce the development of atherosclerosis by affecting the oxidation of the LDL- cholesterol..

b) Garlic contains diallyl sulphides that are associated with the lowering of cholesterol, LDL cholesterol, and triglycerides. It also contains geraniol and other monoterpenes that were found to have antiproliferative properties in human colon cancer cell lines.

c) Clove bud oil has antioxidant and antimicrobial properties and can be effective as antibacterial, insecticidal, and antifungal agent.

d) Presence of eugenol in different essential oils from different herbs and spices has antimicrobial property apart from other beneficial biological actions.

e) Curry leaves have been found to have significant hypoglycaemic action in rat models.

f) The compound piperine in black pepper enhances the absorption of different structurally different drugs thus increasing their bioavailability by altering the dynamics of membrane lipids as well as the conformation of intestinal enzymes. It has also been found to have antioxidant and anti-inflammatory effects apart from anti-peptic ulcer activity.

g) Ginger has been found to have antithrombotic, anticancer, anti-inflammatory, and antimicrobial activities. It is also used as an antihypertensive agent or tranquilizer due to the presence of gingerol and also in treating peptic ulcers.

h) Tamarind is used for the treatment of different liver and bile disorders.

i) Turmeric has been found to be active against different chronic ailments such as cardiovascular diseases, inflammatory disorders, cancer, etc.

j) Onion and its juice have been used for the treatment of blood vessel related diseases, digestive disturbances as well as asthmas and diabetes.

In this way, it can be seen that spices produce a number of diverse compounds, which have multitude of functions in the biological mechanisms within the body. Hence, the inclusion of various spices in the diet is essential for good health.

The whole set of genetic material of a species of plant is known as germplasm of the organism .It is based on the knowledge of germplasm that various breeding techniques of plants are developed. Hence the storage or preservation of germplasm is important. Conventionally seeds were used to store the germplasm. But in case where seeds cannot be used for regeneration of plants or in cases where shoot and root tissue is not stable, it is important to preserve them this can be achieved by mechanisms like:

(i) Cryopreservation:
The freeze preservation of culture of cells or tissues in liquid nitrogen at -196 degree C is known as cryopreservation. The technique involves four steps:

Freezing:
The procedure of freezing may be conducted slowly, rapidly or initial freezing by dropping temperature slowly and followed by a rapid decrease in temperature. In order that the plants are not affected by the sudden decrease in temperature, treatment of cells with plant vitrification solution helps cells and tissue to overcome the harsh temperature. The medium was added with cryoprotectant like DMSO, glycerol, and proline to the culture medium to protect cells from injury. The addition of cryoprotectant protects the cell by prevention of large crystals inside cell, protect from water loss from cell. The frozen cells are stored in a refrigerator containing liquid nitrogen. The temperature of such refrigerator is maintained at or below -130 degree C. Organised tissues like shoot tips, somatic and zygotic embryos are usually chosen for storage. Alternatively cells can be immobilised in sodium alginate and then cryopreserved.

Thawing:
Thawing of cultures is done in a rapid process. The freeze preserved culture is dipped in a water bath containing water at about 37-40 degree C for about 90 seconds. This process in done rapidly so that no ice crystals are formed. The thawed culture is washed several times to remove cryoprotectant. In the recent times, the cryprotectant is removed by diluting. This is done by fixing the culture along with a cryoprotectant onto a disk and is kept on a suitable medium. This disk is frequently transferred into a fresh medium. This frequent transfer dilutes out the cryoprotectant.

Reculture:
The culture which is freeze preserved need to be thawed and cultured to bring it back to normal life. The optimum conditions of freeze preserved plants have to be determined for developing a successful reculture. After cryopreservation, some plants tend to show special requirement for growth which was not necessary under normal propagation of the corresponding plants. For eg: tomato shoot tips when cryopreserved, thawed and recultured, the culture required some levels of abscisic acid in their medium in order to initiate and develop shoot tip from callus formed.

It is found that mostly meristematic cells survive cryopreservation than other cells. In plants where the germplasm cannot be stored in seeds or other parts the cryopreservtaion provides a good option of storage and future usage.

(ii)Slow growth cultures:
Slow growth of cultures involves limiting the conditions of growth so that the culture does not grow and propagate in ordinary pace. This can be achieved by limiting the factors affecting the growth. This provides an attractive alternative to cryopreservation as the procedure is cost effective and simple comparatively. The various factors affecting the growth of cultures are:

Temperature:
The lowering of temperature beyond optimum level was found to affect the cultures by lowering the growth pace.
Nutrient restriction: The limiting of certain nutrient which is vital for growth and differentiation helped in achieving the slow growth culture.
Growth regulators: In some case where temperature and nutrient control was not seen to be effective the culture is added with some growth regulators which regulate the growth of culture. The various growth regulators added include tri-idobenzoic acid (TIBA), chlormequat(CCC), abscisic acid(ABA).

Osmotic concentration: the level of osmotic concentration is another important method by which the slow growth of cultures can be achieved. The high levels of sucrose, mannitol or sorbitol were shown to reduce the growth of cultures.

Other factors: certain other factors such as oxygen concentration, culture vessel used for culturing, restricting the illumination received by cultures all affect the growth of cultures.

DNA clones:
The germplasm can also be conserved in DNA segments cloned into appropriate vectors but the process demands high expertise and is costly.

Artificial seeds:
Another mechanism of germplasm conservation is by desiccating embryos and storing it as artificial seeds. This has proved to be an effective mechanism, but was possible only with somatic embryos and in certain cases by shoot tips. The process of germplasm conservations offers several advantages like cost effective, availability of germplasm of specific plants to propagate, small storage space, and longer terms of storage. It also includes risks such as cell damage by cryopreservation, high technology involved etc.

Drug delivery is process of administering pharmaceutically active compound with a goal to produce therapeutic effect. Technology applied in drug delivery system development is important because it affects drug release and its absorption, distribution, metabolism and elimination from the body. Drug delivery needs to ensure safe application of the precise amount of drug and ensure that used method is convenient for the patient. Drug can be released by diffusion, degradation, and swelling or by affinity-based mechanisms. Most popular drug administration routes are oral (through the mouth), transdermal (through the skin), transmucosal (through nasal, sublingual, vaginal, rectal or through some other mucosa) or via inhalation.

After drug is ingested, it undergoes metabolic changes and its concentration in the body is dropping down. Conventional therapeutics contains larger doses of pharmaceutically active compound to ensure desired medical effect after metabolic degradation of the drug. High level of drug could impair a lot of other biochemical processes in the body and induce different kind of side effects. Some types of drugs (peptide and protein based drugs and different antibodies…) undergo fast enzymatic degradation when applied using conventional methods. Some other types of drugs are not suitable for the systemic circulation due to large molecular size. Targeted or smart drug delivery is relatively new field of medicine that is focused on systems that could deliver drug to a targeted tissue in amount that is medically essential. Using these systems, medicine can be delivered to specific location (certain tissue or group of cells) without damaging healthy (surrounding) tissue. Also, treatment duration can be regulated (prolonged) by specifically designed drug releasing methods.

To optimize drug delivery, team of scientists including chemists, biologists, doctors and engineers is needed. Drug vehicle needs to be non-toxic, non-mutagenic, biodegradable, biocompatible and non-immunogenic. Different types of drug vehicles are used today. Most famous are polymeric micelles, liposomes, lipoprotein-based drug carriers and nano-particle drug carriers.

Liposomes are most commonly used drug vehicles because they are biocompatible and biodegradable and don’t produce toxic or immunogenic response. They could be specifically designed to skip renal clearance and chemical or enzymatic inactivation. Main problem with liposomes is their high instability when applied in vitro and immediate reuptake and clearance by reticuloendothelial system when applied in vivo. Addition of PEG to the surface of the liposome prolongs their circulation time significantly.

Polymeric micelles are created using hydrophilic and hydrophobic monomer units. They are mostly used as vehicles for poorly soluble drugs.

Biodegradable particles are used in controlled release therapy. These particles are bearing ligands to P-selectin, E-selectin and ICAM-1 and successfully target inflamed endothelium.

Artificial DNA nanostructures are manufactured using DNA as structural and chemical material (not as a carrier of hereditary characteristics). Idea is to use DNA that will respond (release drug) when certain stimuli, such as specific mRNA, are applied.

Newly invented drug-delivery system is microorganism-powered thermo-pneumatic pump. All “equipment” is placed in a single patch. Drug is placed in the reservoir made of stacked layers of polydimethylsiloxane and a silicon substrate while baker's yeast and sugar are placed in a small working chamber. Before applying a patch to the skin, water needs to be added. Body heat will then trigger yeast fermentation resulting in small amount of carbon dioxide. Gas production is directly dependent upon time and temperature; created carbon dioxide pushes the membrane (thanks to ~5.86 kPa generated pressure) allowing drug pump to work continually for more than two hours. Autoimmune disorders and cancer treatment has been associated with large molecules that can’t penetrate the skin using traditional drug delivery patch. Invention of micro-needle transdermal patch partially solved the problem. Researchers believe that numerous drugs could be delivered transdermally using thermo-pneumatic patch able to generate necessary force to pump the drug and micro-needles that could penetrate cutaneous barrier. Besides being able to solve a problem associated with large molecules and inefficient transdermal application of some drugs, robustness of yeast would provide long shelf life for transdermal patches with thermo-pneumatic pump.

Targeted drug delivery has a lot of applications, mainly in cardiovascular and cancer therapy. Advantages of this kind of treatment are numerous. Future experiments in this field will probably increase the number of drug delivery systems and expand their application options.

It increases your self-confidence and people get attracted towards your pearly smile,
Well-maintained teeth also prevent progress of oral health related problems,
Evidence also exists that bacteria infested teeth and gums lead to a swelling inside the body.....

Chronic infection by Hepatitis B Virus (HBV) is one of the most prevalent diseases worldwide. It usually leads to Hepatocellular carcinoma (HCC), chronic liver injury, liver cirrhosis, end-stage liver disease (ESLD) and ultimate death in many cases. New therapeutic agents are being developed for the possible treatment of HBV infection, as almost all the current treatments require the lifelong administration of the drug making the optimization of the therapy essential.

The in-depth knowledge regarding the life cycle of the virus and different genetic mechanisms involving the virus within the body is essential for the development of various therapeutic approaches for the HBV, although it is only partially understood. The HBV is an enveloped virus with its viral genome in the form of partially double-stranded DNA and its entry into the hepatocytes takes place by endocytosis with the help of unknown receptors. It is uncoated after entry into the cytoplasm with the DNA being transported into the nucleus, whereby the relaxed circular partially double-stranded DNA is converted to covalently closed circular DNA (cccDNA), a stable episomal form, that serves as a template for the transcription of 4 viral mRNAs. The pregenomic RNA (pgRNA), which is the largest mRNA is translated in the cytoplasm to form core protein and viral polymerase. The pgRNA is then reverse transcribed to form DNA by the newly formed HBV polymerase, which is the main site of action of most of the oral anti-HBV therapeutics. The DNA is, then incorporated by the core proteins to form capsid within which the viral DNA replication takes place. This DNA then either moves to the nucleus to form more copies or encapsulated for secretion by the three viral surface proteins, which are produced by the translation of the subgenomic RNA. The secreted forms are the progeny virions.

The onset of the HBV infection triggers the host immune response triggering the virus-specific cytotoxic T lymphocytes (CTLs) to produce antiviral cytokines such as interferon and tumor necrosis factors (TNFs) and also in the killing of infected liver cells. Hence, the current therapeutics for the infection focus mainly on

a)the immune modulators including the conventional and the pegylated interferon-α that helps in the enhancement of the host immune defense against the infection. However, the low response rate about 20%-30% of this treatment in the chronic HBV patients as well as the possible development of serious side effects limits its tolerability.

b)The nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) constitute the other method of treatment. It consists of nucleoside or nucleotide analogues that bind to the polymerase and inhibit the reverse transcription of the pgRNA to HBV DNA. However, a major drawback of this treatment is the development of mutations in the polymerase gene of HBV, which have been confirmed by different studies, giving rise to drug resistance.

Introduction of different innovations can help in development of effective therapeutics for the infection. Since, the presence of viral mutations has been confirmed, the genotyping of these mutations before treatment can help in better choice of NRTIs. Moreover, a combination of different NRTIs and introduction of new NRTIs can help in reducing the chance of drug resistance development, thereby decreasing the viral load. Further, the combined therapy of pegylated interferon α with NRTIs can be more effective than the monotherapy on any one by increasing the interferon tolerability and efficacy of the treatment.

The development of different drugs targeting specific points in the life cycle of the HBV has also become possible. The entry inhibitors have been found to reduce the chance of HBV infection especially in case of acute HBV infection or in new liver cells i.e. before liver transplantation when used in combination with NRTIs. The targeting of cccDNA is difficult due to its high stability, however in studies using duck models of HBV, some zinc finger proteins have been found successful in binding to the enhancer region of the duck HBV (DHBV) DNA model and reduced the viral replication. The encapsulation of the viral pregenome and the capsid formation are also found to be potential targets for the development of therapeutics and have helped in the discovery of successful antiviral agents targeting them.

Several studies have been conducted for the development of vaccines against the HBV infection. It has been found that CpG oligodesoxynucleotides (CpG ODN), which are synthetic agonists of Toll like receptor 9, in combination with NRTI Lamivudine (LMV) shows to be a promising combination for the suppression of HBV infection by helping in the innate response. Moreover, a therapeutic vaccine consisting of the immune complexes composed of YIC i.e. yeast derived HBsAg and antibodies has been demonstrated to be successful and safe in phase I and phase IIa trial, whose evaluation is ongoing in phase III trials. Further, a DNA vaccination expressing envelope proteins has been found to be successful in restoring and activation of T cell responses in chronic HBV (CHB) patients by Mancini-Bourgine et al.

Thus, the development of various novel therapeutic agents for HBV infection is ongoing and the clinical outcomes and survival of the CHB patients is possible only with the discovery of potent antiviral agents that can cross the anti-resistance barrier.