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

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    Expensive, complex, and very rarely performed procedure of cutting the DNA plays a role in replacing damaged genes to cure genetic diseases and AIDS. But scientists have made to the development of simple, accurate and inexpensive method of cutting the DNA, which could lead to a significant shift in genetic medicine.

    The new technique was first discovered last year, but when cutting the DNA of bacteria. This discovery, published as a research paper in June 2012, is already declared as an outstanding creative achievement in the review of scientific contributions in 2012 of the journal Nature Biotechnology.

    After that, the inventors of new method, Jennifer Doudna from the Howard Hughes Medical Institute, Jinek Martin from the University of Berkeley and Emmanuelle Charpentier from the Laboratory for Molecular Infection in Sweden, published two new papers in the journal Science Express, which have proven successful innovative methods for cutting DNA of human cells.

    New Technology To Cross The Barriers

    Major obstacle in the research and development of gene therapy in humans are modifications of gene technology, which are also the main precondition for the overall understanding of biology and human health. However, by using the new technology of DNA cutting, these barriers should not represent a problem any more, so almost everyone will be able to use this genetic reprogramming in mammals, and quite probably in other eukaryotic organisms, said Doudna, a professor of molecular and cell biology and chemistry and a researcher at the Howard Hughes Medical Institute and Lawrence Berkeley National Laboratory.

    The scientists explain how, on the basis of feedback received, can be concluded that this method will lead to a revolution in genetic engineering of plants and animals. Not only makes programming easier, but it is probably just as strong as the polymerase chain reaction (PCR). George Church, professor of genetics at Harvard Medical School and lead author of an article in Science Express, adds that the ease of use and compactness of these techniques will allow the reproduction of small segments of DNA into millions of copies that will cause major changes in biological and medical research genetics.

    The Principles of the New Method of DNA Cutting

    Previous techniques that use cutting DNA synthesized zinc-ring nuclease (ZFN) and protein TALEN(Transcription Activator-Like Effector nucleases) Science magazine named as one of the top 10 scientific discoveries in year 2012. They named them "cruise missiles" as they allow researchers to "land" in a certain part of the genome and precisely cut the DNA double helix at this specific place. Using two of these cuts it is possible to remove the part of DNA, and to replace it with a new part. In this way, the damaged or mutated genes are replaced by healthy ones. Clinical studies conducted by bio-pharmaceutical company Sangamo Biosciences, have shown that the replacement of a specific gene in individuals infected with HIV, may make them resistant to AIDS.

    By comparing the new technique, which uses an enzyme Cas9 with TALEN method, Church proved that the technique is five times more efficient in inserting genes into mammalian cells. This difference is attributed to the size of Cas9 that is smaller than TALEN, proteins and is therefore possible to use it to cut a small pieces of DNA. Since the smaller part of the DNA consists of a short series of nucleotides, that means the transfer of the nucleotide sequence will require RNA with less nucleotids which significantly facilitates the modification and synthesis of new DNA. Not only is it easier, but it is also possible to use Cas9 to reprogram hundreds of DNA sequences simultaneously. It is also shown that this technique is less toxic to cells than other methods. Church says it is too early to declare complete victory over the ZFN proteins and TALEN, but promising new technique works.

    Discovery of Cas9 Enzyme

    Doudna came to the discovery of the enzyme Cas9 while studying the immune system of bacteria that have developed an enzyme to cut the DNA as a way to defend form viruses. Using this enzyme, bacteria cut the viral DNA, then insert DNA parts into its own DNA, and uses it to produce RNAs which bind to the virus and deactivate it.

    For this unusual antiviral immune system Doudna was intrigued by Jill Banfield, a professor of Earth and planetary systems at the University of Berkeley. But, in her research she focused on the study of the ways in which cells use RNA that are essentially copies of genes from DNA and are essential for its synthesis and replication.

    Doudna, together with her associates Jinek East and Alexander and Aaron Cheng and Ma Enbom from the Department of Molecular and Cellular Biology at the University of Berkeley, continued to investigate how the DNA is being cut using an enzyme-RNA complex. First, the protein Cas9 binds to both the ends of the RNA, and then this complex binds to one particular piece of DNA which is determined by the RNA sequence. Researchers have simplified this whole system to use only one part of the RNA, rather than the whole RNA chain, to cut and target specific parts of the bacterial DNA, as presented in the previous article journal Science.

    The main advantage of this system is the use of only one enzyme for genetic modification. There is no need to change the enzyme for every part of DNA we want to cut, because it just reprograms itself using different RNA transcripts. Comparing to the method used in the past few decades, this method is much easier to design and implement, says Doudna. Scientists claim this is the best solution so far that would allow to interfere with human genome.

    Last three articles pay attention to the effectiveness of the studied bacterial DNA cutting system in human cells. Out of partly unknown bacterial immune system, the new technology is being developed, that completely changes the way of studying the cells of different animals and plants, and the way we manipulate with them. This opens the way for major scientific discoveries that would have a significant impact on human health, says Doudna.

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    Current discoveries suggest that the cure for aging is not that far from us. There was a new study recently conducted with a substance normally present in red wine called resveratrol, which proved some fascinating effects of this substance on aging and longevity in preclinical studies on laboratory animals. Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a compound of grapes, peanuts and berries. At the moment, the clinical studies of substances with similar mechanism of action are being conducted, and scientists hope to develop useful drugs to treat and prevent common diseases in elderly.

    The Role of Sirtuins

    There is a big interest among scientists dealing with this problematic for the proteins called sirtuins. Sirtuins are proteins involved in regulation of biological processes in the cells, and, as have been recently discovered, they are very important factors to prevent cell damage due to aging process which keeps the integrity of the tissues, and therefore prevents the development of aging related diseases. The mechanism of action, although not fully described, is focused on mitochondria, the cell organelles responsible for energetic metabolism of the cell. Those organelles seem to age faster than other cell parts, so their preservation and reparation is of the greatest importance when it comes to the aging process.

    The Discovery of Resveratrol

    It is recently shown that resveratrol has the ability to increase the activity of certain sirtuin called SIRT1. That discovery encouraged the scientists to start the investigation of this substance effects. The experiments have first been conducted on mice, and have shown prolonged lifespan, lower incidence of obesity, an aging related disorders.

    "In the history of pharmaceuticals, there has never been a drug that binds to a protein to make it run faster in the way that resveratrol activates SIRT1," said David Sinclair, professor of genetics at Harvard Medical School and senior author on the paper. "Almost all drugs either slow or block them."

    Debates About Relevancy of The Results

    David Sinclair’s study results were confirmed in many other studies, and all of them proved the relationship between resveratrol and SIRT1. Anyway, there was one thing that bothered skeptic scientists. They were suspicious because SIRT1 protein was studied in vitro, using one chemical group as a fluorescing indicator for increased concentration of SIRT1. Actually, if SIRT1 activity increases, the fluorescing also increases, and that was the way for the detection of resveratol induced increase in SIRT1 activity. This chemical group was artificial, synthetic, and did not exist anywhere in the nature, which guided some scientists to characterize this study as inaccurate or even pure coincidence and experimental artifact.

    These happenings were followed by serious debates on the matter, with the central question: Are the effects of resveratrol directly or indirectly affecting the activity of SIRT1? After six years of debate, and serious research, scientists can now tell that the results were definitely not artifacts. "Still, we needed to figure out precisely how resveratrol works. The answer was extremely elegant.", said Sinclair.

    Proving The Hypothesis

    Sinclair first teamed with other researchers from gonvermental and other institutions to examine the relevancy of his previous results by conducting new research. Their goal was to discover why the cell needs the presence of the chemical group to increase the activity of SIRT1as a response to resveratrol. They surmised that the chemical group was mimicking some other substance already present in the human cell. Only three protein components, amino acids, had a structural similarity with this chemical. The most important of them was tryptophan. So they managed to replace the synthetic chemical group with the residue of tryptophan for the purpose of the new experiment, and as a result, resveratrol again affected the activity of SIRT1 in the sense of increase.

    "We discovered a signature for activation that is in fact found in the cell and doesn't require these other synthetic groups," said Hubbard, first author of the study. He also said that this was a critical result, which allowed them to bridge the gap between biochemical and physiological findings.

    Further experiments are intended to describe the full process of resveratrol induced activation of SIRT1 on molecular level. For that purpose, scientists have already tested the effects of resveratrol on various mutated forms of SIRT1in order to detect which mutation causes the loss of the effect. Some of them were successful in blocking resveratrol effects. The experiment was confirmed using some newly synthesized chemicals with the same mechanism of action as resveratrol, but much more potent.

    So far, there are some assumptions on how resveratrol works, of which the most important is the change of structural organization of SIRT1 induced by resveratrol molecule, followed by increased activation of SIRT1.

    First Experiment on a Living Cell

    As soon as they succeeded to create mutant genes for SIRT1, scientists were able to conduct the tests on living cells. They have actually replaced the normal gens with mutated, and then compaired the effects of resveratrol on mutated and normal cells. The results were, as expected, very good. Normal cells reacted on resveratrol much stronger than mutated cells by much stronger activation of SIRT1.

    "This was the killer experiment," said Sinclair. "There is no rational alternative explanation other than resveratrol directly activates SIRT1 in cells. Now that we know the exact location on SIRT1 where and how resveratrol works, we can engineer even better molecules that more precisely and effectively trigger the effects of resveratrol."

    Next Goals

    Resveratrol efficiency and safety were already investigated in animal studies, mostly on rats and mice, where its anticancer and antiinflammatory effects were reported. Positive effects on cardiovascular system and decrease in blood sugar concentration were also noted.

    Further investigations are most probably headed to the goal of developing drugs and pharmaceuticals with same mechanism of action as resveratrol. Of course, before these drugs become available for use in human medicine, there are a lot of clinical studies to be done in order to investigate their efficiency and safety.

    This could not possibly be the drugs that would make us younger, but they most certainly could bring us longer and healthier life, and significant improvement in quality of life.

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    Introduction:
    Bio-nanotechnology is used for developing biosynthesis and environmental-friendly technology for synthesis of nanomaterials and nanoparticles. Nano particles are considered as a powerful tool to develop new approaches in the field of designing new antimicrobial drugs. Nowadays fungal infections resulted from opportunistic fungi have been common especially in people talented in being affected by special conditions like immune weakness , pregnancy and diseases like HIV and the candidates of affection to these infections. As like the bacterial stains, fungal stains also developed their resistence to antifungal drug. Now days several types of nanomaterials like copper, zinc, titanium, magnesium, gold, alginate and silver are used. Silver or silver ions have long been known to have strong inhibitory and bactericidal effect as well as a broad spectrum of antimicrobial activities. The current investigation supports the theory that the use of silver ions or metallic silver as well as Nano-Ag can be exploited in medicine for burn treatment, dental materials, and possesses low toxicity to human cells, high thermal stability and low volatility. Silver nanoparticles (NPs), shows a very strong bactericidal activity against both gram-positive and gram-negative bacteria including multi resistant strains. The antifungal effect of silver NPs has received only marginal attention and just a few studies on this topic have been published. Recent studies revealed the effects of silver NPs as antifungal agents. Recent studies also revealed that silver nanoparticles are comparatively better than other antibiotics which are currently used as antifungal agent.

    Synthesis of nanoparticles

    The silver nano particles can be synthesized either by biological or non-biological method. The biological method includes silver nanoparticle synthesis from bacteria (ex: Bacillus licheniformis), marine microalgae (ex: Isochrysis galbana), yeast (ex: MKY3), fungi (ex: Aspergillus flavus), plants (ex: Aloe Vera). At the stationary phase of the marine microalgal culture the 0.01 normality of the silver nitrate solution is added, the silver is reduced to silver nanoparticles. Exposure of the fungal biomass to aqueous Ag+ ions resulted in the intracellular reduction of the metal ions and formation of silver nanoparticles. Silver nanoparticles were synthesized extracellular by a silver-tolerant yeast strain MKY3, with 1 mmol L–1 soluble silver in the log phase of growth. In plants, the leaf extracts were treated with silver to synthesize the silver nanoparticles. The non-biological methods for the synthesis of silver nanoparticles are physical method and chemical method. The physical method includes physical vapor condensation, arc discharge method. The chemical methods include chemical reduction, photochemical method, electrochemical method (electrolysis), and pyrolysis.


    Antifungal activity of silver nanoparticles

    Fungi are usually saprophytic. Once they enter the host cells it becomes parasite. Fungi often develop morphogenetic virulence mechanisms, e.g., formation of yeasts, hyphae, and spherules that facilitate their multiplication within the host. The transition of the fungi yeast to the mycelial growth in the host cell is said to be the dimorphic transition. This transition is responsible for pathogenicity in the host cells. In order to find the antifungal activity of the silver nano particles the dimorphic transition of the fungi was investigated. A mycelial form can be induced by temperature, pH, and serum. Silver nanoparticles attach to cell membrane and penetrate in the fungi then produce a site witch little molecular weight in center of fungi, and then Silver nanoparticles attach to respiratory sequence and finally cell division stop lead to cell death, Silver nanoparticles release silver ion in fungal cell which increase antifungal function as result. The serum-induced mycelia were significantly inhibited from extending and forming in the presence of Silver nanoparticles but the mycelia formed was normal in the absence of Nano-Ag.

    Antifungal effect of silver nanoparticles

    The antifungal effect of the silver nanoparticles is identified by various methods depends on the fungal species. One of the most commonly used method in the broth dilution method. The broth dilution method is used for identification of the minimum inhibitory concentration (MIC). The 80% inhibitory concentration (IC80) was defined as the lowest concentration that inhibited 80% of the growth as determined by a comparison with the growth in the control. MIC was found for silver nanoparticles and also for the antifungal drugs such as fluconazole, amphotericin B, geriscofalvin. Anti-fungal drug fluconazole is more effective than the amphotericin B. the inhibitory concentrations of silver nanoparticle and marketly available antibiotics were found at 50, 80, 90% of concentrations. On an average of these inhibitory concentrations of Amphotericin B, Fluconazole, geriscofalvin, silver nanoparticles were 16mg/ml, 64mg/ml, 8mg/ml, and 4 mg/ml respectively. This it shows that silver nanoparticles is more effective than the marketly available chemical antifungal drugs. The changes in the cell structure of the fungi due to the silver nanoparticles are viewed and identified by scanning electron microscopy or transmission electron microscopy. This shows “pits” in the cell membrane of the fungus due to action of silver nanoparticles which leads to the cell death. This method is double attenuated method.

    Along with the anti-fungal activity, the hemolytic activities of the silver nanoparticles against the human erythrocytes are also determined. Many antimicrobial agents are limited regarding clinical applications, as they can induce cytolysis of human cells. The hemolytic activity of Nano-Ag was investigated as an indicator of its cytotoxicity to mammalian cells. The hemolytic activity is measured by the release of hemoglobin from the 4% suspension of the human red blood cells. Hemolytic effect of silver nanoparticles is compare with the chemical antifungal drugs such as amphotericin B. it is found that silver nanoparticle caused 6% lists of erythrocyte whereas amphotericin B induced 10% lysis. This proves that Silver nanoparticle could be applied to therapeutic agents regarding human fungal diseases with low cytotoxicity. This can act as broad spectrum antifungal agent. Silver nanoparticles exhibited potent antifungal effects on fungi tested, probably through destruction of membrane integrity; therefore, it was concluded that silver nanoparticles has considerable antifungal activity, deserving further investigation for clinical applications.

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  • 03/12/13--02:30: Effects of transgenic plants
  • What is difference between control weeds by transgenic plants, chemical herbicide and bio-herbicide? Chemical herbicide control weed through spray chemicals but how transgenic plants control weeds? I need our suggestion and share your idea.

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    Are you an omnivore? Or are you a vegetarian strictly on animal welfare grounds? Would you consider eating cultured meat (meat grown in the lab)? Is an animal flesh product that has never been part of a complete, living animal more ethical? My friend, Michael Brogan, is doing a survey on the subject for his thesis in Culinary Arts, it takes less than 5 mins if you'd like to ponder the question. -Rob Dunlop


    Please consider taking part in this survey, your opinion is important and your participation would be greatly appreciated

    https://www.surveymonkey.com/s/Attitudes...tured_Meat

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    Metagenomics : The transformation of modern microbiology.

    Since the invention of microscope to the revolutionize knowledge of microbiology and microbial world, the emerging field of metagenomics can bring transformation in the modern microbiology. This has been again confirmed by the new report from the National research council. The Metagenomics is the emerging field where in the DNA of entire strains and communities of microorganisms are being studied in detail. This is obviously the greatest of opportunities to learn and apply microbiology for the well being of mankind. Metagenomics is nothing different than Human Genome project in this, genetic mapping of entire microbial community is being done. This further calls for a initiative to drive advances in this field of microbial mapping, globally. The mapping of genetic code of microorganism will not only give us details of the genetic materials but also this knowledge can be applied in various fields like Medical, Pharmaceutical, Agriculture and even in developments of innovative products.

    Microorganisms are important in maintaining the chemical balance in the nature and atmosphere, also microorganisms has helped a lot to transform key elements of earth into energy, providing nutrients to plant and animals and other necessary nutrients for survival of plant and animals. Today Microbes are used in production of biofuels, antibiotics, remediating oil spills, and agriculture and plant enhancement.

    Microorganims are studied by using microscope and it has been understood that not only individual organism perform essential activities, but it is the combination of complex enzymatic reactions carried out by group of organism. Microorganims are grown in various selective media and their cultural characteristics are identified. They are thus differentiated and the useful are isolated. The knowledge of Genetic Mapping of such organism will help to understand their relationship during complex reactions. This can further help in aligning the systems and production of required bio-molecules.
    With Metagenomics we can see into the previously invisible world of microorganism, opening new dimensions of unimaginable science which was not understood until recently. This Shedding light of new microbial activities and genetics will help in developing new concepts as well as can be applied practically. The best example of such genomic study is of Nif Gene, these genes are present in microbes found in leguminous plant. These bacteria with Nif gene transform atmospheric nitrogen into nitrate. This knowledge came into light only after the discovery of Nif Gene. This knowledge further was applied in biotechnology and tissue culture. With this, many other plants without these types of bacteria were made capable of Transforming atmospheric nitrogen into nitrate. This helped to meet the vetegation requirements of earth and thus further maintained various natural cycles. If metagenomics can identify such important genes in microbial world, this can further be applied for benefits of all life forms on the earth including, plants and animals. This symbiosis of bacteria and humans, plant and animals always remained from ancient time but metagenomics can bring a revolution in such associations.

    The study of Genetic material of microorganism starts with extraction of DNA from microbes and this is the basics of metgenomic study. One such sample could contain even millions of fragmenst of DNA. All these are cloned into a form. Such forms can be easily maintained in the laboratory and can be used as and when required. Such bacteria are used as a library which includes genomes of all the microbes which are found in same habitat. This is the most common or natural environment of the organism. Such metagenomic jigsaw puzzles are solved with the help of modern DNA sequencing technology and super computers. These are compared with gene sequences of previously unknown microorganism and thus help genetic engineer to screen bacteria that express protein and are capable of production of vitamins and other useful antibiotics.

    During this project, care is taken to characterize in detail chosen microbial community those are present in various habitats worldwide. Such project unit scientist from all over the world and the mass knowledge gives a fine way to success. The great thing about such projects is that the completion of this project is not success, but it is a beginning of success and has multiple opportunities ahead with this base. During this project, brain storming can help to work out on soil and seawater issue, environmental issue like sludge processing apart from other developing fields of science.

    This project work would serve as database and as a incubators for the development of technique and new concepts in various field of science. The availability of such data to public will enhance researcher which can be used in their research. This database will not only include gene sequences but also information on Sampling, DNA extraction techniques and all other details of project.

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    It seems that Microorganisms are both friends as well as foe of human beings. But it is certainly true that we cannot live without their association in our metabolism. They play a central role in the lives of not only humans but also animals and plants. Most of the organisms are harmless, most are beneficial for our life and biological metabolism, at the same time many causes diseases in humans, plants as well as animals. This type of mixed behavior is observed when we think of microorganisms and our biological association with them. The question is obvious microorganism are our friends or enemies?
    This question can be answered by comparing both the attitudes of microbes. The one who is more is the answer. Now let us look at the causes of microorganism. We fight with bacteria when they infect us and our immune system stimulates. We use enzymes of many bacteria when we digest our food. We use many micro-nutrients and vitamins as synthesized by bacteria which are present in our colon. The deterioration of food by microbes leads to unsafe food our consumption. The quality of such food is very bad and we cannot consume them. At some level animals can consume them but it is always a risk due to presence of toxins in such food. It means when bacteria eat their own food and accumulates by products like toxins these are harmful. But when we are the habitat of microbes and the association is symbiotic, we both are beneficial. It means they had their own way to live and we had our own biological ways to respond to certain foreign matters like bacteria. Apart from this extreme, the centre is association of both in which both are beneficial, and they are not termed as foreign matter by our immune system.
    Many bacteria are aggressively infecting our system and this leads to fighting between the two. They had their weapons like endo toxins and other enzymes and try to survive with our system. We react to such actions using our immune system. The one which is stronger survives. Therefore in this case it cannot be the question of friends or foe, it is natural that all happens and is genetically coded. This is nothing with friendship or enemy, this is simply the rule of evolution and a part of Darwins Concept of survival of the fittest, and this is natural selection. If so, then what are symbiotic associations or friendship with microbes!

    The activities of bacteria which we call as friendly behavior is nothing but a give and take system in which both are jointly running in the race of natural selection or for their own survivals alike aggressive infections which is one sided race. When to look further deep, we will find one more surprising thing and that is we as Humans, Animals and plants are more dependent on microbes than they are. It can be possible for microorganism to survive in saprophytic conditions, it can be possible for microbes to live without any association with Humans but this is not true for us. We may fail to survive in absence of many bacteria which helps us in our metabolic activities with their enzymatic reactions. This means they are friends but not completely. They are our friends when we are strong and are associated with them in a way where we also return them things like we can be their host and then can help us with enzymes. “They are friends when we are strong” has a sense, this is observed with many bacteria turning violent and infecting us even when they are in symbiotic associations. Such Bacteria are called as “Opportunistic pathogens”. The best example is E.coli. E.coli is the bacteria which lives in our gut and provides useful vitamins like B2 and B12 to us and in return utilizes our system and a place to live. We act as their host. But this scenario is in existence only when we are strong immunologically. The moment we are infected with other pathogens, they take a u- turn and infect us too like an enemy never before.
    Many bacteria like LAB- Lactic acid bacteria are important group of food processing and production. They ferment food for us which is easily metabolized then. Examples are yoghurt, Sausages, they also have probiotic properties. The primary metabolism by LAB is conversion of carbohydrates into acids. This inhibits growth of other food deteriorating bacteria. This way they are enemy of their own group “Bacteria”. Therefore this is true that no one is enemy and no one if friend when it is comes to natural selection and struggle for existence. This is what is called as natural, where everyone has to act for his own propagation and growth.
    Things happen (within) so naturally that we get time to think of it, as “if they are friends or enemy”. The totality of all is nature which many times act in a way which can be irrespective to what we assume and what we think. But not always!

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    cloning is one of the best way to to develop a new individual of the same kind. then y people do not use the technique of cloning and make organs like kidney,eyes etc for the tranplantation process. if the cell of a human who is need of an organ is taken and cultured and then transplanted then we can save many lives..

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    A group of scientists have recently conducted two studies that will possibly make a contribution to discovery of cure for three hematological diseases: beta thalassemia, hemochromatosis and polycythemia vera.

    In this extensive research project, 24 researchers from 6 scientific institutions across North America and Europe were involved and thanks to their great effort, iron metabolism and life cycle characteristics of red blood cells now seem to be much more clear.

    "When you tease apart the mechanisms leading to these serious disorders, you find elegant ways to manipulate the system," says Dr. Stefano Rivella, associate professor of genetic medicine in pediatrics at Weill Cornell Medical College. He pointed out that although the genetic causes of hemochtomatosis and beta thalassemia are different, the consequences of that mutations are the same – iron overload. Therefore, the treatment which would eliminate the excess of iron from the body would be effective for both disorders.

    The scientists then decided to take a closer look at the possibilities of controlling red blood cell production, as it seemed like a perfect way to treat thalassemia and polycytemia vera.

    Erythropoesis and Iron Levels

    The first study was published on March 17, 2013, and it focused on red blood cells production process called erythropoesis. That was the key for revealing pathological processes of two diseases – beta thalassemia and polycytemia vera.

    Beta thalassemia is hematological disease which develops because of the mutation of the gene which carries information for synthesis of proteins that build hemoglobin. In thalassemia minor, only one of two beta hemoglobin chains is missing, so the symptoms are mild – slight mycrocyte anemia. In thalassemia major, on the other hand, two beta chains are missing leading to severe anemia. Distruction of blood cells leads to excess iron which accumulates in various organs and makes damage.

    Hemoglobin chains in polycitaemia vera are normal, but there is a mutation of regulatory gene that should be limiting the production of red blood cells. As it doesn’t work properly, the main finding in this disease is increased number of circulating erythrocytes.

    New Crucial Erythropoesis Regulator

    Until now, it was well known that two crucial factors contribute to regulation of erythropoesis – hormone erythropoietin and iron level. In this research it was found that macrophages play important role in this process. Functions of macrophages are recognized long time ago; they digest extracellular pathogens, gather dead cells and tissues, and they are in one word “cleaners” of the body. Dr. Rivella discovered that macrophages are also crucial for the initiation of stress erythropoesis. He pointed out the importance of physical contact between macrophage and erythroblasts in order for erythropoesis to begin. "No one knew macrophages were a part of emergency red blood cell production. We now know they provide fuel to push red blood cell factories to work faster," says the study's lead author Dr. Pedro Ramos, a former postdoctoral researcher at Weill Cornell.

    The Treatment Strategy

    This discovery implies that by disabling macrophage activity, the excessive erythropoesis as pathological process in diseases mentioned above could be slowed down. That is why researchers conducted an experiment on mice with polycytemia vera by inactivating their macrophage cells. As a result, a normal production of red blood cells was established.

    The benefits of this discovery for patients with beta thalassemia are also very predictable, bearing in mind the fact that their body is desperately trying to compensate genetic defect by accelerating erythropoesis, which is not effective.

    And that is not the end, it seems that the process of inactivation of macrophages also slows down their other functions such as transporting the iron to erythroblasts. That way, the erythrocytes decrease not only in number but also in iron content and they become very much like normal erythrocyte.

    "I estimate that up 30 to 40 percent of the beta-thalassemia population could benefit from this treatment strategy," Dr. Rivella says.
    Another parallel made by Dr. Rivellaa emphasizes the similarities between the ways macrophages affect tumor growth and red blood cells proliferation in polycytemia. He says that polycytemia could be considered as a type of red blood cell tumor. His estimations are that 30-40 percent of people with thalassemia could benefit from this discovery.

    Regulation of Iron Absorption Revealed

    The second study, conducted by researchers from Weill Cornell and from Isis Pharmaceuticals of Carlsbad, Calif and published on March 25, 2013, focused on investigating iron metabolism. It is well known that decreased iron level causes anemia, and on the other hand, excessive amount of iron is toxic for almost all organs.

    Hemochromatosis is a genetic disorder of iron metabolism with iron levels up to 7 times greater than in healthy persons. The common consequences of long-term increased iron level are liver cirrhosis, kidney failure, diabetes mellitus etc.

    In this study, several molecules that regulate iron metabolism are detected and explained. Ferroportin is a transport protein located in the mucosa of the small intestine, and it is considered as an “iron gate”. The other protein has the regulatory function and it is called hepcidin or Hamp. If the food contains much iron, the level of Hamp increases leading to closing the iron gate, thus blocking the iron absorption. In patients with hemochromatosis and beta thalassemia, levels of Hamp are too low, leading to increased absorption of iron and consequently to high iron blood levels.

    The Treatment Strategy

    To solve this problem, scientists used an opposite disorder which occurs in childhood and is based on genetic mutation of Tmprss6 gene which causes increase of Hamp level, thus disabling iron absorption. Therefore, the research team leaders developed Tmprss6 blocker drug in order to increase Hamp expression in patients with beta thalassemia and hemochromatosis. The drug is designed to target mRNA transcribed from Tmprss6 gene, and change its configuration thus disabling translation process, process of protein synthesis. "When you destroy that RNA, you destroy the ability of the Tmprss6 to make any protein," Dr. Monia says. Dr. Rivella says that this technology could be implemented and used in clinical studies very soon.

    As these disorders didn’t have a proper treatment so far, this discoveries are great hope for the patients in order to improve their quality of life.

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    Hi,

    I am know doing my A levels but this year I am finishing my second course. I like biotechnology and I think that this is the degree which I am going to choose.

    Have you studied this degree? Do you recommend it?

    I am undecided because I don´t know how could it be its future and if I will be able to pass my exams in the university, I have heard that it's quite difficult.

    I would like to read your personal experiences.

    Thanks

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    Mushrooms, with the exception of shitake and some species, are mainly associated with moldy bread and moist smell of mold. But they deserve more respect. Mushrooms have fantastic possibilities and, under certain conditions, they can be grown in almost all forms, and are fully biodegradable. And if all that is not enough, fungi have the potential to replace plastics one day.

    The Secret Is in The Mycelium

    Mycelium is the vegetative part of a fungus, consisting of a mass of branching, thread-like hyphae. The mass of hyphae is sometimes called shiro, especially within the fairy ring fungi. Fungal colonies composed of mycelia are found in soil and on or within many other substrates. A typical single spore germinates into a homokaryotic mycelium, which cannot reproduce sexually; when two compatible homokaryotic mycelia join and form a dikaryotic mycelium, that mycelium may form fruiting bodies such as mushrooms.

    One of the primary roles of fungi in an ecosystem is to decompose organic compounds.

    Professor of Biology from the University Union, Steve Horton, explains this, mostly underground placed part of fungus (what appears on the surface are the reproductive parts) as tiny biological chain of tubular cells.

    "This chain of stations can communicate with the outside world, in the sense that it gets information what are the conditions in terms of food, light and moisture," he said. "The mycelium takes nutrients from the available organic materials such as wood and used them as food, and as a result of that we get the growth of fungus."
    "When you think about mushrooms and their mycelia, you realize that their function is in ecological sence very important in the breakdown of materials," says Horton. "Without fungi and bacteria we would be buried in God knows how many meters of waste which would consist of plant and animal tissue."

    Observing something extremely subtle as dental floss, we see that the mycelium grows into, through and around any organic substrate. Whether it leaves or mulch, mycelium digests these natural materials and it can combine them all together into a cohesive cover.

    The company Ecovative Design of Green Island, New York, exploits this mycotic power, with the help of Professor Horton and another researcher from the University Union, Ronald Bucinella, associate professor of mechanical engineering.

    The Products From Fungi

    Evocative use of several types of fungi to produce environmentally friendly products. The process starts by raising products, such as cotton trash, seed husks of rice, buckwheat and oat, hemp, and other plant materials. All they are sterilized, mixed with nutrients and then put on cooling. Then, mycelium spores are added that are extremely well reproduced, so that soon every cubic inch of material contains millions of tiny fibers of mushrooms.

    This complex network then grows in the mold of whatever shape we want. In Ecovative, everything is about creation. When you get the desired texture, strength and other characteristics of the product, it is removed from the mold, and heated and dried to kill the mycelium and stop its growth.

    Advantages of Using Fungi Products

    These products are completely natural, they are created in less than five days, they do not cause allergic reactions and are absolutely non-toxic. Even more impressive is the fact that they are resistant to fire up to a certain point and as resistant to water as styrofoam, but they will not just lie around and needlessly take up space in landfills. They are also more UV stable than foam as they are not based on petrochemicals and they will not emit volatile organic compounds. When exposed to the real microbes, they will be decomposed within 180 days on any landfill or in any yard.

    Mycelium is also cheap because it can be grown on farms using the trash that can not be used to feed the animals or from which we can not get fuel. Besides that, fungi can be multiplied without sunlight and excessive human surveillance in simple containers at room temperature - no need for vast greenhouses with expensive systems to control temperature. It also means a lower level of carbon dioxide and Evocative hope that the moment will come when these products are going to replace all the plastic and foam on the market.

    The professors and researchers from the Union, Steve Horton and Ronald Bucinell are helping with that too. In Horton’s laboratory he and his students are working with the types of fungus that Evocative uses in production.

    "We manipulate single strain in different ways, to see if we can create other types of fungi that would suit the specific demands of the company," says Horton. "For example, it may be useful for Evocative to have some strains which could grow faster."

    Associate professor of mechanical engineering Bucinell Ronald and his students also contribute to the research and development of Evocative. Bucinell’s specialty is experimental mechanics and mechanics of reinforced materials and his task is to examine the samples of solid materials by various parameters. This includes testing whether the mycelium binds better to one herbal material or to another, and whether the way of producing it makes it stronger or weaker.

    The important method used in this production is Polymerase Chain Reaction (PCR). Evocative company founders are grateful to their premium partners.

    "Steve is truly unique because his research over the past 28 years has focused on the effects of genetic pathways of physiology of fungi, which is an important factor in what we can do with the mycelia," said one of the founders of Evocativea - Gavin McIntyre. "Ron is one of the foremost experts in the field of composite design. We are very pleased and we appreciate the opportunity to work closely with the two remarkable scientists. "

    "This is a complete novelty in the field of materials and collaboration allows us to learn quickly," McIntyre continued. "It's really important when you are trying to replace the plastic."
    The project is financed through the funds of the National Science Foundation (NSF) and the Agency for Energy Research and Development of the Federal State of New York (NYSERDA).

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    Our nervous system is composed of two types of cells - neurons and glial cells (glial cells, neuroglia). A new discovery shows that glial cells are not just "glue" of the nervous system, as previously thought, but that they are much more important in the unique complexity of our brains. Scientists came to this conclusion after seeing that transplantation of glial cells of the human nervous system in the mouse nervous system can affect connections within the mouse brain, thus enabling it to learn a lot faster.

    The Importance of Astrocytes

    The study, published in the journal "Cell Stem Cell", suggests that the evolution of astrocytes (a type of glial cell) - which are much larger and more complex in humans compared to other species - may be one of the key events that have caused the higher cognitive functions that make us so different from other species.

    "This study shows that glial cells are not necessary only for neural data transfer, but suggests that the evolution of human cognition is driven by evolution of glial cells, which are by form and function specific to humans," explains neurologist dr Steven Goldman, MD from University of Rochester Medical Center – URMC and co-author of the research. "We believe this is the first evidence that human glial cells have unique functional benefits. This discovery gives us important new research model for a number of diseases in which these cells may play a significant role. "
    Over the past few years, scientists have begun to understand and appreciate the role of glial cells (especially astrocytes) on our brains.

    URMC researchers are the pioneers in unlocking the secrets of astrocytes and proving that they not only serve as a support to neurons in the brain, but that they also communicate with neurons and with each other.
    "The role of astrocytes is to form a perfect environment for neural transmission," continues Prof. Maiken Nedergaard, coauthor of the research and one of the managers at Center for Translational Neuromedicine at URMC's. "However, we noticed that when this station evolves and becomes more complex, larger and more diverse - as in humans - the brain's functions are becoming increasingly complex."
    Astrocytes are far more numerous, bigger and more diverse in the human brain comparing to other species. In humans, one astrocyte ejects the fiber sufficient to simultaneously connect a large number of neurons, and particularly with their synapses, transfer points where two adjacent neurons are touching. Therefore, one astrocyte in humans has the potential to coordinate thousands of synapses, which is much more than mouse astrocyte can.

    The Astrocytes as Key Constituent of Human Inteligence

    This observation hinted that human astrocytes may play a significant role in integrating and coordinating highly complex signaling activity in the human brain, thereby assisting in the regulation of our higher cognitive functions. Also, this observation is what gave the idea to transplant human glial cells into mice, assuming that our glial cells would affect the underlying patterns of brain activity in mice.

    "In essence, we are very different from lower species," says Goldman. "Our advanced cognitive processing abilities are not only due to the size and complexity of neural networks, but also because of increased functional capacity and coordination that the glial cells enable."

    "I always thought that the concept that the human brain is more capable because we have a little more complex neural networks is too simple, because if you bring together the entire neural network and all of its functions together - we would get super computer," says Nedergaard. "But human cognition is far more than mere data processing, it is also the coordinating of emotion and memory, which shapes our ability to learn."

    The Experimental Design

    The researchers decided to find out if the human glial cells are the cause of unique abilities of the human brain, by testing what would happen if human glial cells coexisted with ordinary nerve cells in mice. In order to succeed, the researchers first isolated glial progenitors- cells in the central nervous system, which allow the growth of astrocytes - from brain tissue. Then these cells were transplanted into the brains of newborn mice. As the mice grew, the human glial cells exceeded hosts glial cells, but at the same time they have left the existing neural network of mice intact.

    "In essence, the human glial cells spread to the point where almost all glial progenitor and a large amount of astrocytes in mice were of human origin, and have developed and behaved as if they werewould in the human brain," says Goldman.

    The team of scientists then decided to investigate the functional impact that these cells have on animal brains, with special attention to the speed and retention of signals between cells in the brain and its plasticity - the brain's ability to form new memories and to adopt new tasks. They found that two important indicators of brain function have improved dramatically in mice with transplanted human glial cells. First, the measurement of the phenomenon called "calcium wave" (as astrocytes connect with the help of calcium) - speed and distance at which the signal is spread within and between adjacent astrocytes in the brain - the researchers noted that the speed of the wave transmission in mice with transplanted human glial cells is faster than in normal mice, similar to the movements that take place in the tissues of the human brain.

    Second, the researchers checked long-term potentiation , a process that measures how long the neurons in the brain are affected during a brief electrical stimulation. Long-term potentiation is considered one of the most important points of the molecular mechanisms responsible for learning and memory. In this test the researchers found that the mice with transplanted human glial cells developed faster and more sustained long-term potentiation, which means that learning ability of these mice increased.

    Based on these findings, series of behavioral tasks were designed to test memory and learning ability. The results were as follows; mice with transplanted human glial cells learned more quickly, they gained new associations faster, and they performed a number of different tasks significantly faster than mice without the transplanted human glial cells.
    "The point is that the mice with transplanted human glial cells exhibited increased plasticity and increased learning capacity within its neural networks, which significantly changed their functional abilities," said Goldman. "This tells us that human glial cells have a specific role in human body in the sense of intellectual capacity and cognitive processing. Though we have for some time suspected that this could be the case, this is really the first evidence of this hypothesis. "

    Possible Uses of The Experimental Model

    Researchers believe that this animal model, which now has the name of human glial chimera mouse, provides the medical community a new tool for understanding and treating neurological disorders that occur because of abnormalities in the glial cells. This could be especially important for those of neurological and neuropsychiatric disorders that are more likely to occur in humans than in other species. In these diseases, astrocytes’ features specific to the human species would be of particular importance for the process of development of these diseases. Goldman, Nedergaard and colleagues have used these mice to study human neuropsychiatric and neurodegenerative diseases, in which the pathology of glial cells could significantly contribute to the research results.

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    Clinical trials are set of test in drug development and medical research. These tests generate safety and efficacy data as required for developing confidence for safety of patients. These tests are done well before the product or new drug is brought in market. These trials give information about adverse drug reactions and other effects for health interventions. According to the results, evaluation of drugs, diagnostics, devices and therapy protocols are done. The approval of drug for bringing them in market is given by regulatory authorities of Government , example USFDA..

    Initially pilot studies are done and depending upon the stage of its development, an investigator enrolls volunteers and other animals. After this, large group of patients and volunteers are covered to evaluate the drug under trial. Many a times, it is country specific but certain potential drugs are tested in various countries at a time. During trials, the investigator recruits patients with characteristics which are predetermined. After administration of treatments, the data is collected and analysis is done. Depending upon the results, conclusions are drawn. This conclusions and results are verified in detail by various regulatory authorities including Governmental departments.

    The drug is effective in treating the disease but has side effects, in such cases; the drug is rejected and fails to pass clinical trials. This is because even though the patients get relief from disease, the later side effects are worse than it. Therefore for safety of patients, it is mandatory requirement to have lesser side effects. For example, if a drug is having clinical trials for treatment against cancer and is found to be effective in treating the deform, then it can just not be approved immediately. The patients under test are re-tested for side effects for a larger period of time for estimation or investigation of any later side effects. And if the latter side effects shows that with such drug which is under test, re-occurrences of cancer is observed, such drugs are rejected completed because it is at last of no use. This is the way of clinical trials to ensure patients safety first.

    Also there are many cases , were in the side effects are not observed in first trial and even missed to identify it during later effect stage, but after approval of drugs if anything goes wrong with respect to safety of patients, after many years also, such drugs are banned by FDA or respective regulatory authorities of that country.

    Below are some examples of best designs of what a clinical trial may need.
    • Assess the safety and effectiveness of a new medication on patients who have been diagnosed with Alzheimer’s disease.
    • Assess the safety and effectiveness of a different dose of a medication than eg. Instead of 5mg will be tested at 10mg.
    • Assess the safety and effectiveness of an already marketed medication or device for a new finding, i.e. a disease for which the drug is earlier not approved.
    • Assess and evaluate new device or drug with effective standard drugs. This is also known as Gold standard or the standard therapy.
    • Compare the effectiveness in patients by comparing the already approved drugs and interventions for the diseases. For example if drug A is curing disease A then can new drug along with A drug can cure Large number of diseases or can treat broad spectrum of Gram Negative Organisms.
    • Compare two or more number of medications at time. Comparing it with devices against each others.

    Clinical trials are of different types , it depends upon its purpose. The US National Institutes of Health that is NIH organizes trials into five types.

    The first one is known as Prevention trials, in this, the objective is to look for preventing diseases which were never in certain groups of people or patients. This includes minerals, vitamins, medicines and even changes in life styles. The second type is Screening trial this is the only way to detect certain disease or health conditions. The third type is Diagnostic trials this is for finding better test or methods for diagnosing a particular condition or disease. The fourth type is Treatment trial in this, new combination of drugs is tested and also new approaches to therapies are identified. The Quality of life trials are test which explores way to improve comfort or life quality for patients with chronic illness. The fifth is Compassionate use trials or expanded access trial. This is used to test unapproved therapeutics to a small number of patients and also to test completely the drugs which are earlier test partially.

    The clinical trials are truly "safety shield for patients" against unknown new drugs!

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    Now, when scientists discovered that mammals, birds and many other animal species have consciousness, it is the time for the society to take action in this direction.

    Do animals have consciousness? This question has a long and respectful history. It was submitted by Charles Darwin when he was thinking about the evolution of consciousness. His ideas about continuity of evolution- that the differences between species are actually differences in level of development and not in kind - lead to the firm conclusion that if we have "something", then "they" (other animals) also have it.

    The Problems in Investigation Process

    Of course, something like that could be only supposed, but not yet conclusively proven. Consciousness in animals is a very tricky thing to prove since it is certainly not on the high enough level to be expressed clearly and to be available for observation. One of the problems lies in the fact that certainly not all animals have consciousness, and that raise the question at what level of development should a living organism be considered as consciousness being.

    Last year, a group of scientists gathered at the first Annual Francis Crick Memorial Conference at the University of Cambridge, in detail discussed the issue. Crick, one of the discoverers of DNA, spent the second half of his career studying consciousness, and in 1994 he published a book about it, "Astonishing Hypothesis: The Scientific Search for the Soul".

    The Cambridge Declaration on Consciousness

    The outcome of the meeting was The Cambridge Declaration on Consciousness, which was publicly declared by three recognized neuroscientists, David Edelman from the Institute of Neuroscience in La Jolla, Philip Low, Stanford University and Christof Koch Institute of Technology in California.

    Declaration of consciousness concludes that: "The non-human animals have neuroanatomical, neurochemical and neurophysiological basis of conscious state, combined with the ability to show intentional activity. Thus, the evidence shows that humans are not unique in terms of having neurological substrate that is the cause of consciousness. Non-human animals, including octopuses, also possess the neural substrate. "
    The first reaction on the Declaration was disbelief. Did we really need an official confirmation of obvious? Many mainstream scientists came to this conclusion long ago.

    However, the Declaration contains some glitches. The Declaration was signed by the scientists involved in laboratory studies (all but one), but the declaration would have more credibility if they took into account the perspectives of researchers who have conducted long-term research and observation of wild animals, nonhuman primates, carnivores, whales, rodents and birds . It is also disappointing that the Declaration did not include fish, because the evidence supporting the existence of consciousness in this group of vertebrates is very convincing.
    However, we welcome the publication of this Declaration which is intended not only for scientists, because as the author before publishing Low said: "We agreed that it is perhaps now time to make public declaration. Although perhaps everyone in this room is clearly aware that animals have consciousness, the rest of the world may not be clear. "
    There are a few important questions: Will this declaration change something? What will scientists and others do now when they agreed that awareness is widespread in the animal kingdom?

    Application of The Agreement

    I hope that the Declaration will be used to protect animals from violent and inhumane acts. Too often, the scientific knowledge about animal cognition, emotion, and consciousness is not recognized in law for the welfare of animals. We know, for example, mice, rats and chickens show empathy, but this knowledge has not been implemented in the U.S. Federal Animal Welfare Act. Approximately 25 million of the animals, and fish, are included in an aggressive research each year. Scientific community is continually amazed by how those who decide on the rules on the use of animal neglect that data.

    But on the other hand, all the legislation does not ignore science. Lisbon Act of the European Union, which entered into force on 1 December 2009, recognizes that animals are sentient beings and calls on member states to "take into consideration the welfare of animals very seriously,” during the agricultural policy, fisheries, transport and space policy, and the politics of research and development.

    Also, there are still scientific skeptics about animal consciousness. In his book, Crick wrote: "idealizing animals is sign of sentimentality," and continued that for many of the animals living in captivity is better, longer and less cruel than life in the wild.

    Similar views are still prevalent in some scientists. In his latest book, "Why are animals important: Awareness of animals, animal welfare and the welfare of man”, Marian Stamp Dawkins, from Oxford University argues that there is still really not known whether the other animals have consciousness and will remain skeptical and agnostic, belligerently agnostic if necessary. Dawkins ignores the data that scientists have used to form the Declaration and goes so far as to argue that it is actually harmful to animals if we base decisions about their well-being on the basis of their awareness.

    Those who want to harm the animals can easily take Dawkins' opinion in order to justify their actions.

    The Cambridge Declaration of awareness is here, and now we have to work on how to provide animal protection they deserve. Let's hope that the Declaration is not just a gesture to show, but it's something that has teeth, something that leads to action. We owe it to the animals to use our knowledge and to show some mercy and compassion in our treatment of them.

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    There are many instruments in laboratories which are important for performing various operations and testing. But when we talk of Microbiology laboratory, the most important and critical instrument is Autoclave. One cannot consider any microbiology laboratory without Autoclave. Autoclave is used for sterilization of Microbiological media, for garments as well as for destruction of Biological waste. Biological wastes are generated during microbiological operations in which life is involved. For example any microbiology media when used, has many bacteria in it. These bacteria cannot be exposed to environment directly as they can be potential sources of diseases. Therefore, such media with bacteria after use is known as Biological Waste and is categorized as “Biohazard”. Autoclave is used to inactivate such biohazard. In Autoclave, such biological waste are steam sterilized at specific time that is generally 50-65 minutes and the temperature is maintained at not less than 121 degree Celsius during this period. Autoclave is also used for sterilization of various microbiological media this helps in avoiding cross contamination during microbiological activities. Such sterilized media are used to get error free results as they can be used for specific purpose as required. Therefore autoclave is very important in any microbiology laboratory. The validation of autoclave is done periodically to ensure that the desired results are meeting consistently. Autoclave works on principal of steam pressure. As the steam pressure increases, temperature of autoclave also increases. Pressure of steam is directly related to temperature. Therefore for sterilization of any media, garment or biological waste, time and temperature relationship is very important. Autoclave sterilization is done at not less than 121 degree Celsius for times between 10 to 65 minutes depending upon the material in for sterilization. The temperature of 121 degree Celsius is set for the reason that this is the temperature where the most resistant organism (against temperature) starts dyeing. And after certain time at this temperature, they are killed completely. So this concept is taken into consideration that if most resistant organism is killed at 121 degree Celsius then obviously the sterilization will he high in SAL (Sterility assurance level).

    The next in list of most important instrument in any microbiology laboratory is obviously the “Microscope”. There are different types of microscopes. Some are simple microscope, while others are compound microscopes. The type of it is used depending up on the requirement and sample under test. Now days, the most sophisticated microscope is Electron Microscope. It is able to show very micro sized viruses also. Microscope are complex optical set aligned in such a way that the object is magnified 100 to 1000 times that it is and thus are visible to eyes. The classification of bacteria can easily be done using microscope wherein, the stain obtained by bacteria, differentiate them as either Gram Positive or Gram Negative in nature. This is most used method of classifying bacteria. Electron Microscope is widely used for study of viruses and other microorganism at molecular level.

    Laminar flow bench is another most important instrument in any microbiology laboratory. Laminar flow is used to maintain the clean environment. Laminar flow is generally class 100, level of classified area. LF is used to perform various testing in which contamination is not acceptable. The places or environments were testing of pathogenic organism or their handling is involved, Biological safety cabinets are used. These are laminar flow bench designed in such a way that the air does not come on to the microbiologist or operator and he is safe in handling pathogens in biological safety cabinet.

    Hot air oven is also important instrument and is used for sterilization of glassware especially petri plates. This instrument is slightly different than autoclave in which temperature is the factor for killing bacteria but the temperature is not generated due to steam but it is due to dry heat. The moisture level is low and therefore higher temperature than 121 is required for dry heat to kill most resistant organism for dry heat. The hot air oven is run for generally 2-4 hours at 180 degree Celsius for sterilization of glassware.

    Other instruments are incubators, deep freezers, rotary shakers, BOD incubators, Temperature-RH incubators etc. Incubators are used to incubate various media for growth of bacteria. This is done by providing specific temperature to bacteria in such incubators. The temperature is set and maintained accordingly in it. Refrigerators are used to preserve bacteria and other required microorganism.

    Therefore with these basic instruments, all microbiological testing are completed and each instrument has its own role in completing microbiological testing and related activities.

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    Hi all,

    I try to express human protein in E.coli, but some of them is not expressed at all or in a very small amount. I realize that that can be many parameters that affect protein expression at cellular level (like codon usage for example) but I am more intressed of parameters in the physiology of cultivation like media and the cell lysis. Today we are using Tryptic soy broth + yeast extract and for cell lysis, lysis buffer. Can a change this two parameters for better protein expression and where can i find resourses for this?

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    INTRODUCTION:

    After years of research on liposome it has been possible to create a new class of drug delivery vehicles called COCHLEATES. They are stable, protects drug from harsh environmental condition and also prevent drying of drug. Cochleate are calcium-phospholipids structures that engulf the drug to be introduced to body within itself.

    DISCOVERY:
    Cochleate were discovered by Dr. D. Papahadjoupoulos and co-workers, in 1975 and have been used in late 80s and early 90s for delivery of antigens. Nanocochleates were introduced in 1999 to develop smaller particles for encapsulation of hydrophobic drugs of small size. They are cigar like micro structure composed of multiple series of lipid bi layer. They are elongated structures unlike liposome which are circular in shape.

    COMPOSITION:
    Nanocochleates consists of a purified soy based phospholipids that contains at least about 75% by weight of lipid which can be phosphotidyl serine (PS), dioleoylphosphatidylserine (DOPS), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidyl glycerol (PG) and /or a mixture of one or more of these lipids with other lipids. A multivalent cation, which can be Zn+2 or Ca+2 or Mg+2 or Ba+2 and a drug.
    Drug can be protein, peptide, polynucleotide, antiviral agent, anesthetic, anticancer agent, immunosuppressant, steroidal anti inflammatory agent, non steroidal anti inflammatory agents, tranquilizer, nutritional supplement, herbal product, vitamin and/or vasodilator agent .

    PREPRATION:
    Cochleates are generally prepared by four major methods:
    a) Hydrogel method:
    b) Trapping method
    c) Dialysis method
    d) Direct calcium dialysis method

    STABILITY OF COCHLEATES:
    Even though the outer layer of the cochleate is exposed to harsh conditions or enzyme the component within the multilayer remains intact. The interior of cochleates is essentially free of water and resistant to penetration of foreign components. Even after drying cochleates to powder and storing at freezing temperature they can be reconstructed and the process of drying have no adverse effect on the working of them.

    ADVANTAGES:
    • They are easily observed under microscope and are produced with ease and safety.
    • Due to limited oxygen permeability, drugs are prevented from oxidation.
    • Provide ease of shipping and storage of drugs before administration.
    • Hydrophobic molecules and tissues impermeable drugs can be transferred into body via cochleates.
    • Specificity of target part of the body can be maintained using them.
    • They are made unique by their structure that enables them to be a great delivery vehicle.
    • With administration of live vaccine they are many life threatening risk associated like allergies , inversion of vaccine effect to wild infection etc. these can be neglected by use of Nanocochleates
    • Multiple administrations of high doses of cochleate formulations to the same animal show no toxicity and do not result in either the development of an immune response to the cochleat.

    APPLICATION:
    • Production of ApoA1 (i.e. Apo lipoprotein) in treatment of Atherosclerosis and other heart diseases.
    ApoA1 is a High Density Lipoprotein (HDL) which carries out esterification of cholesterol and transfers it to lever thus protecting it from arthrosclerosis. As ApoA1 is a protein it degrades gastrointestinal enzymes hence are not used as in tact molecule. So Nanocochlaetes provide a good platform for ApoA1 delivery.
    • Used in delivering Amphotericin B, an anti fungal agent orally with least harm, good safety and also reduced cost of treatment.
    • They can also deliver omega 3 in cakes, soups and cookies without harming products taste appearance or odor
    • They are also used to deliver protein, DNA for vaccine and gene therapy.

    CONCLUSION:
    Nanocochleates could be a major breakthrough in scientifically made drugs suitable for large level of drug delivery with up most care and accuracy of treatment.

    REFERENCES:
    1. V. Ravi Shankar, Y. Dastagiri Reddy ; NANOCOCHLEATE –A NEW APPROCH INLIPID DRUG DELIVERY
    2. Dr. David Deimarre, Dr. Susan Gould Fogerite, Dr. Rapheal J. Mannino ; FORMULATION OF HYDROPHOBIC DRUGS INTO COCHLEATES DELIVERY VEHICLE

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    Syringes and needles have been used for immunization since 1853. This way of application is very old and very good, but it has a lot of disadvantages like high expenses in preservation of vaccines, complications with needle application, discomfort at 10% of the patients and phobia caused by needles etc. A revolution in human vaccines is currently under development. This revolution is called Nanopatch vaccines. This Nanopatch approach is made of an array of thousands of vaccine- coated microprojections which perforate skin but only its outer layers. This is maybe one of the greatest achievements, because this way of application is absolutely painless. This way of application is made not to reach the deeper layers of the skin where nerve endings are located. Major doubt about this way of application was the question- will there be enough material which will activate immune system, because there is used only a tiny fraction of the dose. The answer is- YES. Yes, there is sufficient amount of immune material because there is a huge difference between conservative way and the Nanopatch way of presentation.

    Conservative way with syringes and needles is oriented on muscle cells, but the Nanopatch technology is oriented on skin cells. It has been proven in past 30 years that skin, unlike muscle, is rammed full of immune cells. Those immune cells are dendritic cells (DC) which are potent antigen presenting cells. Whereas the biological characteristics and immunological functions of epidermal DC known as Langernahs cells (LC) have been the focus of intense research in the past, less is known regarding their dermal counterparts named dermal dendritic cells (DDC). Because of the lack in immune cells in muscle cell, skin is preferable place for vaccines.

    Benefits of Nanopatch vaccines

    The outer layer of skin is different from person to person. It varies in thickness, humidity, age, gender and other factors. This is one of the potential problems for Nanopatch vaccine application, but Nanopatch application achieves delivery of vaccine by choosing the coated Nanopatch arrays with an adequate applicator. This is the way of skipping possible problems related to dose.

    Another important advantage of Nanopatch vaccines is the way of preserving. This vaccines are not like conventional vaccines because they don’t need cold chain for transport. Professor Mark Kendall from University of Queensland's Institute for Biotechnology and Nanotechnology and his team have tested these vaccines in extreme conditions. They traveled to Papua New Guinea, which is one of the leading countries in infection with HPV virus. They were in extreme conditions with nearly 100% humidity and they have not noticed loss in vaccines activity. This was a huge achievement for this team, because they cut out expenses, and there was no danger that vaccines will be ineffective or possibly harmful.

    Next benefit of these Nanopatch vaccines is expenses cost effectiveness. This is so far the main benefit for most of the countries in development. These vaccines could be made for fifty times less cost than many current vaccines. This price could cure the people from the countries which cannot afford many current vaccines. Also cost effectiveness can be increased with cost savings due to using less vaccine to reach an effective immunization, reduction of cold chains and, for sure, reduction in the costs which are connected with needle injuries. There are some opinions that these vaccines could be sent by mail in remote parts of the countries or even remote parts of the world! Those mailed vaccines could be applied by people themselves even by people who have fobia of needles, because it is absolutely not painful and there are no complications which we have in conventional way of application.

    Needles and syringes have also risk of blood-transmitted diseases like Hepatitis B, Hepatitis C, Human Immunodeficiency Virus and other viruses. With this way of vaccine application there is no risk of infection with similar viruses.

    This benefits from above are strong and unique in world of vaccines. Therefore this way of application may be suitable for a vast majority of vaccines.

    History of Development

    This way of application is still under development. Professor Mark Kendall from University of Queensland's Institute for Biotechnology and Nanotechnology and his team have only tested this type of immunization on mice using various inoculations. In animals treated with influenza vaccines, Nanopatch induced immune response with a tiny dose which was ten times better than any other way of application.

    Beside this influenza vaccine there were good results with Human Papiloma Virus vaccine, Human Simplex Virus, Chikungunya Virus and West Nile Virus. Although this type of vaccination has shown great results, it has a long, long way to go to bring what is still an experimental device to market, but Professor Mark Kendall from University of Queensland's Institute for Biotechnology and Nanotechnology and his team are sure that this way of immunization will be the future of vaccines.

    There were attempts of creation similar with this Nanopatch system but all of them were failures. The Austrian biotech firm Intercell tried to create experimental VEP (vaccine enhancement patch) system against pandemic flu. This project was a complete failure. However this system had completely different way of action which was applied after a needle vaccination and it was designed to boost vaccines effect. But this Nanopatch vaccine applies directly, in outer layers of the skin. Than it is presented to immune skin cells without adjuvant.

    This way of application is definitely utopia of all vaccine applications as it is, according to recent studies, more effective than any other way of application and costs about fifty times less than then many current vaccines. If it shows good results in human trials, it will definitely bring bright future to human vaccination.

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    The most common cause of unsuccessful implanting embryo in the uterus and abortions in the first trimester of pregnancy are genetic anomalies. Genetic information is contained in the chromosomes.

    Genomic DNA analysis includes preimplantation genetic diagnosis - PGD and preimplantation genetic screening - PGS. Basis of PGD and PGS involves genetic analysis of embryos before implantation and transferring certain "normal" embryos. The first successful application of PGD for hereditary diseases is published in 1990. For the application of PGD / PGS patients must use IVF to create embryos for in vitro analysis. For diagnosis are described three distinct developmental stages: polar corpuscles removed from the oocyte / zygote (polar body biopsy); blastomers 6-8-cell embryos, day three (cleavage stage biopsy); or biopsy blastocysts cell, 5 or 6 days develops (blastocyst biopsy).

    Biopsy samples are analyzed by PCR (polymerase chain reaction) or FISH (fluorescence in situ hybridization) of relevant techniques. PCR techniques are applied for the diagnosis of specific genetic diseases, whereas the FISH technique to analyze the number of chromosomes in patients with chromosomal anomalies or for sex selection of embryos for diseases transmitted on the X-chromosome.

    PGD

    PGD is an important scientific advance for patients who are carriers of genetic diseases, and who are at risk for transmission of inherited diseases (haemophilia, cystic fibrosis, Fragile X, Duchenne muscular dystrophy, etc.).

    Statistic shows that PGD procedures are increasing each year as a result of new and successful methods of detection of abnormal gene on one level blastomers and completing the human genome sequence. One of the techniques that have been developed recently is amplification genome. This method allows the collection of DNA from one microgram blastomers, analysis and application of whole-genome microarrays.
    It is very important for the detection of chromosomal abnormalities, mutations, and aneuploidies. The indications for PGD biopsy is done on the third day of embryonic development, in one or two blastomers, and transferred to a "normal" embryo of the fifth or sixth day.

    PGS

    PGS, unlike PGD was developed to improve the success of IVF, especially in elderly women. Indications for PGS are: elderly patients, multiple miscarriages, repeated IVF failure and male infertility.

    Concerns with PGD/PGS

    The embryos could be traumatized by the biopsy procedure - particularly during cleavage stage biopsy. There is some evidence that carefully performed trophoectoderm biopsies blastocysts might not weaken the embryo at all.

    As with any new technique and technology, there is a "learning curve". There could be large differences between centers performing these techniques, and even between technicians within the same IVF center.
    Mosaicism can complicate matters. An embryo is a mosaic if there are 2 (or more) different chromosomal patterns in cells of that embryo. There is evidence that mosaic embryos sometimes "self-repair", or possibly designate abnormal cells preferentially to the placenta instead of the fetus. More research on mosaicism is needed.

    Many people believe that because life begins at conception and that the destruction of an embryo is the destruction of a person. In some cases, a genetically defective fertilized egg will mature without the presence of disorder or disease.


    Sampling of Embryonic DNA Without Biopsy

    On the fifth day of developmental processes in the embryo begins to absorb liquid (endometrial gland secretions), which initially accumulates in the form of vacuoles in blastomers and soon extracellularly. In this way, it creates a cavity within the embryo - blastocists cavity and structure of the embryo at this stage is called a blastocyst developmental processes.

    In a recent study published in Reproductive Biomedicine Online, a group of researchers sought to achieve diagnose of genetic disease in embryonic DNA without the use of a biopsy. They found that extracting fluid from human embryos at the blastocyst stage contains DNA from embryo. Blastocysts are embryos, which are 5 or 6 days old, and they are the last free-living stage that can be studied in the laboratory prior to transfer into the uterus. They contain between 50 and 300 cells that surround a fluid-filled cavity called the blastocoels. The scientists got the idea to the blastocyst stage liquid removed, leaving the cells intact. The analysis of this fluid have proven that it contained cell-free DNA in a sufficiently good state, enough to determine several known genes of the sex chromosomes by polymerase chain reaction (PCR).

    This method removes some ethical issues that arise using PGD methods. The study above is a good start. However, it is important to consider that regardless of the outcome of these studies, we need a large number of studies.

    These methods cannot identify all the genetic and chromosomal disorders. These methods are not determined at the level of gene disorders or other developmental malformations and congenital anomalies. Performing these methods in any case does not reimburse prenatal screening for congenital anomalies of development during pregnancy.

    Conclusion

    These procedures are used for determining the presence of a particular genetic condition in the embryo prior to implantation. In this way it is possible to determine whether it is in the process of artificial insemination to damage genetic material, a disturbance in the number of chromosomes, etc. These procedures are used to mother spared spontaneous abortions, reduce the chances of multiple birth, as well as to select out an embryo that is in the best condition, which gives a greater chance of success interventions. In addition, it is possible to test for the presence of gene alleles inherited disorders, as well as a wide range of other information.

    These methods are used to determine the different genetic abnormalities, and predisposition to a variety of serious diseases, and the number of genes associated with pathological conditions that can be examined is increasing. Same as with any new technology, here is also the main problem - the problem of borders. Like everything else, these technologies should be used within the limits of the moral.

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