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Biotechnology Forums - https://www.biotechnologyforums.com

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  • 11/03/12--22:51: RNAi against HIV infection
  • 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.

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    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.

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  • 11/04/12--18:41: What is Functional Genomics?
  • 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.

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

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    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.

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    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.

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    Recent talk at the Open Science Summit in silicon valley.







    youtube.com/watch?v=zCQmDUURe3I

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    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.

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    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.

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    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.

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    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.

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    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.

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    The unique environment of the human and animal body like presence of warmth, moisture and nutrients makes them a perfect habitat for various bacteria and viruses. In order to compete all the pathogens and survive and execute its function properly body has to protect itself by developing a defensive mechanism against all the invading pathogens. So developed defensive system of the body is called as Immune System and the collective study of immune system – its components, functions, response developed against an infection, protection from a disease all falls under one roof called as ‘Immunology’.

    The two factors governing the immune system are detection and reaction. Detection is the recognition and identification of the molecule foreign to the body and once detected, a reaction is developed (Immune response) to eliminate the foreign substance. The immune response otherwise known as effector response is the reaction developed by the immune system towards a pathogen entering the system for the first time. The pathogen is either destroyed or neutralized by the effector response of the immune system and the response is stored in the intelligence of the immune system. Recurrence of the exposure of the body to the same pathogen initiates the memory response of the immune system which enhances the immune response.

    The article by Thucydides on Plague in Athens around 430 B C could be the first citation on immunology. During that time, the practice of people recovered from the plague infection serving the affected people was observed. But it was correlated to the phenomenon of immunity in later years. It took 2000 years from then to apply this phenomenon in medical field. In China around 15th century in an attempt to develop immunity against the deadly disease small pox, people were either made to inhale the dried crust of the pustules from small pox affected people or it was injected into the body through wounds and this method was coined as Variolation. The beneficial effect of variolation was addressed by Lady Mary Wortley Montagu in 1718. The fame goes to the English physician Edward Jenner for his establishment of the Variolation technique in 1798. He observed milkmaids acquired cow pox disease showing resistance to small pox and validated his observation by experimenting on an eight year old boy by injecting him with cowpox causative and tested for his defense against smallpox.

    The further development of immunology has a foot step of the scientist Louis Pasteur whose discovery of immunity to cholera is an interesting story. He cultured the organism potential to cause fowl cholera in his laboratory and introduced this organism deliberately into healthy chicks and observed infection in the birds. After a while return to his laboratory from a short vacation, he used the old culture to infect the chicks and to his wonder the chicks did not develop any signs of infection. He again allowed the growth of the old culture under laboratory condition and injected them into previously infected chickens and found the chickens ability of protection from the disease. This act of Pasteur made him understand that aging of the culture has reduced the virulence of the pathogen and hence it can be used as a protective agent against disease. Pasteur conducted his first vaccination programme in 1881 using Bacillus anthracis a causative agent of anthrax, on sheeps which initiated the further development of immunology. Also in the year 1885, Pasteur’s attempt to vaccinate a boy against Rabies was successful. Inspite of the discovery of vaccination by Louis Pasteur, the insight into the phenomenon behind immunity was well established by a scientist named Emil ven in 1890.

    The discovery of immune cells the WBC and Serum component antibody lead to the debate by researchers on cell mediated immunity and immunity produced by antibody (Humoral immunity) until the inter related roles of the immune cells and the antibody in developing immunity were understood. The various researches and discoveries like, the discovery of serum antitoxin, understanding the cellular immunity in Tuberclosis, role of phagocytosis, Type I anaphelaxis hypersensitivity reaction, complement mediated bacteriolysis, discovery of human blood groups, vaccine for yellow fever, antihistamines, research on acquired immunological tolerance, study on the chemical nature of antibodies, immunological techniques like radio immuno assay, immunogenetics (histocompatibility antigens), production of monoclonal antibodies and study on gene rearrangement in antibody production, immune response in transplantation, specific cell mediated immunity and vaccine for human Papilloma virus cites the drastic progress of immunology in the 20th century and its applications.

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    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.

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  • 11/07/12--21:08: Dental care
  • 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.....

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    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.

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    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.

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    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.

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    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.

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    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.

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