Meeting EBTNA 9 ottobre 2015

 

Lo scorso  9 Ottobre 2015 si è svolto presso la Facoltà di Agraria dell'Università degli Studi di Perugia un Meeting Internazionale sulle Biotecnologie.

 

Munis Dundar Erciyes University, Turkey

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Epigenetics

Epigenetics refers to the study of changes in the regulation of gene activity and expression that are not dependent on gene DNA sequence. While epigenetics often refers to the study of single genes or sets of genes, epigenomics refers to more global analyses of epigenetic changes across the entire genome. C.H. Waddington coined the term epigenetics to mean above or in addition to genetics to explain differentiation.How do different adult stem cells know their fate?Myoblasts can only form muscle cells, Keratinocytes only form skin cells Hematopoetic cells only become blood cells, but all have identical DNA sequences!Modern definition is non-sequence dependent inheritance.How can identical twins have different natural hair colors?How can a single individual have two different eye colors? How can identical twin liter mates show different coat colors?How can just paternal or maternal traits be expressed in offspring? (This is called genetic imprinting), How can females express only one X chromosome per cell?How can acquired traits be passed on to offspring? Some changes in gene expression that are, in fact,  heritable. Many more …. DNA Methylation & the Epigenetic Code, Histone Modifications, Different Methylation pattern and stem cell totipotency and unipotency, X-chromosome inactivation, RNAi, epigenetics and cancer, epigenetics misregulation and related diseases and above mentioned questions will be summaries in the lecture.

 

 

Kevan M.A. Gartland  Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, SCOTLAND

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Biotechnology for Food and Environmental Security

Biotechnology is an invaluable tool for meeting the current and future needs of society affected by climate change and global population growth. Climate change is having increasing impacts on food security globally. With 925 million citizens expected to be undernourished by 2020, the contribution of green biotechnology to meeting world food and environmental security needs is discussed.  Applications of biotechnology including marker aided selection, genomics, genetic modifications and emergent novel technologies of relevance to drought resistance and salt tolerance to meet the needs of an increasing population under stressed environmental conditions are described.  Environmental applications of biotechnology are considered from a sustainable development perspective.  The regional and global nature of acceptance or rejection of green biotechnology is considered.

 

Oscar Vicente Institute of Plant Molecular and Cellular Biology (IBMCP, UPV-CSIC),

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Oscar Vicente¹* and Monica Boscaiu²

¹Institute of Plant Molecular and Cellular Biology (IBMCP, UPV-CSIC),

²Mediterranean Agroforestal Institute (IAM, UPV). Universitat Politècnica de Valencia

'Green/red’ biotech: GM plants as biofactories of pharmaceutical proteins

Since the production of human insulin in E. coli – the first commercial therapeutic protein provided by recombinant DNA technology – several GMO systems have been used as "bio-factories" for the synthesis of a wide range of proteins with pharmacological activities, by expression of the corresponding cloned genes: hormones, growth factors, blood clotting factors, enzymes, vaccines, antibodies... in general, any protein with application in the diagnosis, prevention or treatment of human (or animal) diseases. Bacterial cultures have several advantages for the production of recombinant proteins, being reliable and relatively cheap systems, easy to establish and maintain, and providing high protein production levels. Nevertheless they have also serious drawbacks, especially for the expression of complex and multimeric proteins, which in many cases are not produced in an active form in the cells, since they do not fold or are not assembled properly. However, their most important limitation is that the bacteria do not possess the machinery responsible for post-translational modification of proteins; most human proteins are modified by phosphorylation, acetylation, glycosylation, etc., and the presence of these groups (especially the correct glycosylation) is essential for their biological activity. Mammalian cells cultures are also very robust and reliable systems, there is a long experience in their industrial use and approval by the competent regulatory authorities, and a whole body of ‘good manufacturing practices’ (GMP) have been developed over the years. Unlike bacteria, however, they ensure (in general) the synthesis of pharmacologically active products, since post-translational modifications of the recombinant protein are the same than that of the native human protein in vivo. That is why animal cell cultures – for example, of Chinese Hamster Ovary (CHO) cells, the 'golden standard' in the industry – are currently the system of choice for the production of biopharmaceuticals, despite having several drawbacks and limitations: high costs of up-front investment, development and maintenance, slow growth of the cells and limited productivity, the difficulty (technical and economic) to scale up or down production, or the risk of contamination by human pathogens (e.g., viruses or prions). Production of recombinant proteins in the so-called “3^rd generation” of transgenic plants provides an alternative, or rather a complement, to systems based on GM-microorganisms or animal cells in in vitro cultures. This is what is known as 'molecular farming' (... or 'molecular pharming' if we refer specifically to pharmacological proteins), and has – at least theoretically – a number of advantages over other commercial production platforms:

i) the methods of plant genetic transformation and regeneration of transgenic plants are relatively cheap and simple, as compared for example to the generation of transgenic animals

ii) the production systems can be established with low up-front investment and maintained cheaply, since they are based on common techniques used for centuries in agriculture (low-tech, low-cost)

iii)the production can be scaled (up or down) easily and cheaply, to adapt to market demands (in principle, simply by increasing or decreasing the cultivation area)

iv) in general, proteins are synthesized in a pharmacologically active form, since the systems of post-translational modification (e.g. glycosylation) in plants are very similar to those of mammalian cells

v) the synthesis of the recombinant protein can be directed to specific organs (and organelles) by using tissue-specific promoters and proper subcellular localization signals. Thus, the protein can be 'encapsulated' in natural plant structures, for example in the endosperm of seeds, facilitating in this way the storage of the protein in an active form, without requiring special conditions such as refrigeration

vi) there is the possibility of developing simple and efficient purification methods

vii) there is no risk of contamination with human pathogens

Another specific application of “pharma-crops” is the production of edible vaccines: ingesting plant material containing a suitable recombinant antigen will activate the immune system at the level of the intestinal mucosa. This approach would eliminate many of the problems associated with the production and application of traditional vaccines, such as high cost, transport and distribution issues (mostly in developing countries): no need to maintain the 'cold chain', avoiding risk of transmitting infections by the use of non-sterile syringes, etc.

Besides stable genetic transformation, protocols have been developed for transient expression of recombinant proteins in plant tissues (tobacco or alfalfa leaves are the commonest), using Agrobacterium tumefaciens, plant viruses or hybrid constructs as vectors. These systems allow the rapid production of substantial quantities of biopharmaceuticals, which would be needed to treat large numbers of individuals in a short span of time in case, for example, of epidemics or bioterrorist attacks. For over 20 years a large number of recombinant proteins, many with possible clinical applications, have been produced in transgenic plants, proving the aforementioned advantages of these platforms; in most cases this work has been limited to academic or 'proof-of-concept' studies. Several recombinant proteins are currently produced in plants, marketed as reagents for research or used in various industries: cosmetics, detergents, food, etc. However, commercial production of biopharmaceuticals in GM plants is lagging far behind with regard to the system of cultured mammalian cells. This is due more to regulatory and technical issues, rather than to purely scientific advances. There is no clear specific rules applicable to the production of pharmaceuticals in plants, and it is very difficult to adapt those existing at present, established for such different biological systems as cells in in vitro cultures (similar regulatory problems exist in the case of the production of therapeutic proteins in transgenic animals). In addition, worldwide, there are only a few facilities authorised for the production of recombinant proteins in plants according to 'good manufacturing practice' (GMP). In recent years, nevertheless, there has been a substantial boost in commercial development and potential applications of molecular pharming, and several specific products are currently undergoing clinical trials, at different phases. Some of the specific milestones that mark this development are: i) the approval by the FDA in May 2012, of 'Elelyso' (recombinant glucocerebrosidase), enzyme used to treat Gaucher's disease (a lysosomal storage disorder), produced by Protalix Biotherapeuticals (Israel) in a carrot cell culture; ii) production by transient expression in Nicotiana benthamiana of the experimental drug ZMapp, which has proven effective against Ebola virus in primates, and contain a combination of three humanized monoclonal antibodies, which recognize a surface glycoprotein of the virus; iii) production in transgenic tobacco of a neutralizing anti-HIV monoclonal antibody, which is used as a topical prophylactic to prevent virus infection (by vaginal application prior to sexual intercourse); the relevant authorities approved a phase I clinical trial, which demonstrated the safety of its use. Although ‘molecular pharming’ will probably never replace the systems of animal cell cultures, much more developed (and where the industry has made major investments), the advantages of using transgenic plants as bio-factories for the production of biopharmaceuticals provide a very interesting market niche for specific products. For example, as mentioned before, when large amounts of protein are required in a short time, which cannot be produced in vitro culture systems. Moreover, technical simplicity, the low initial investment and the also limited running costs, make ‘molecular pharming’ (or, more generally 'molecular farming') an affordable technology to less developed countries. Further developments on scientific, technical and regulatory issues regarding these plant-based platforms for production of recombinant proteins are, therefore, expected in the coming years.

Further reading:

The following recent reviews (and the references therein) provide a complete and up-to-date overview of the issues discussed above:

  -Eva Stoger, Rainer Fischer, Maurice Moloney and Julian K.-C. Ma (2014) Plant Molecular Pharming for the Treatment of Chronic and Infectious Diseases Annu. Rev. Plant Biol. 65: 743-768.

-Shah Fahad et al. (2015). Recent developments in therapeutic protein expression technologies in plants Biotechnol. Lett.  37:265–279

 

 

 

 

Fabio Veronesi Università degli Studi Di Perugia

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 Is coexistence among genetically engineered, conventional and organic crops possible?Coexistence is not a problem of public health; it is set up on the farmer's capability to make a choice among organic, conventional and biotechnological agriculture with reference to the legal commitments concerning labelling and purity standards defined by low. If this possibility is missing, problems of interference among the three agricultural approaches are rising. Based on the actual scientific knowledge and of the genetically modified crops that at the moment are of interest for Italy (mainly maize and soy), the coexistence is more a political-economical problem than an environmental one. The possibility of gene flow from transgenic crops such as mais and soy to the environment is extremely limited, because these species do not present sexually compatible wild relatives in the Italian flora. This could be different considering others crops such as beets or canola. For the above reported reasons, coexistence problems should be considered one by one and not overall.

 

 

 

Peter B. Gahan King’s College, London, UK .

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Components of the cytosolic and released virtosomes from stimulated and non-stimulated human human lymphocytes.

 

A number of early investigators demonstrated that both stimulated and non-stimulated lymphocytes released DNA (1-5). Subsequently, Stroun and Anker showed the released DNA to be newly synthesized with ³H-thymidine labeling studies (6-8). Furthermore, the DNA was associated with RNA (9). Since both nucleic acids were resistant to nuclease activity, it was considered that they were protected by lipoprotein. The presence of protein was identified when RNAse activity affected RNA only after a prior treatment with either pronase or proteinase k (9,10-13) while that of lipids was identified from the complex’s low density during upward sucrose density gradient centrifugation (10), freezing and thawing (10, 11) and the incorporation of radioactive phospholipid precursors (10,13,). Subsequent studies using radioactive precursors permitted the demonstration that the RNA, protein and associated phospholipids were (a) newly synthesized and (b) synthesized at about the same time (9-12). This DNA/RNA-lipoprotein complex, has an estimated size of ~5 x 10⁵ daltons (13) although the complex released from stimulated rat lymphocytes had a higher density than that released fom non-stimulated rat lymphocytes (10). The complex, termed a virtosome (14) is released in an apparently energy-dependent step (10), only from living cells (10,13,15-17) and in a controlled manner (18-21). Experiments employing radioactive precursors have shown that the DNA, RNA, phospholipid and proteins appear in the cytoplasm at about 3 h after commencing labeling and that the complex is released from cells 3 – 6 h later, depending on which cells were studied i.e. human, other mammalian, avian, amphibian and plant cells (1,6,8,10,15,16,20-22).

 

The complex does not appear to have a limiting membrane as shown by studies on the uptake and release of virtosomes between chick embryo fibroblasts (15) and on release from J774 cells and their uptake by non-stimulated lymphocytes (23).

 

Importantly, virtosomes released from one cell type can enter a different cell type resulting in a biological modification of the recipient cells e.g. transformation of NIH 3T3 cells on uptake of released mutant k-ras from SW480 cells (24), an allogenic T – B lymphocyte co-operation involving lymphocyte subsets from human donors with different allotypes (25,26) and DNA synthesis initiation in non-stimulated lymphocytes on uptake of virtosomes released by J774 and P497 tumour cells (23). Thus, the virtosome appears to be a novel cytoplasmic component that may act as an inter-cellular messenger.

 

However, the full structure of the complex has not been ascertained. In the present study, experiments were designed (a) to identify the lipids and proteins associated with both the cytosolic and released complexes, (b) the comparative amounts of proteins, lipids, DNA and RNA in cytosolic and released virtosomes and (c) the nature of the proteins present in the released virtosomes as opposed to those absent from the cytosolic virtosomes. However, as a first step to ensure that the virtosomes released from stimulated and non-stimulated lymphocytes were biologically active, the cytosolic and released virtosomes were isolated and tested for their biological activity, as previously described (23-26).

 

In addition to obtaining the overall content of DNA, RNA and phospholipids, the analysis of the individual phospholipids gave further confirmation for the absence of a classical membrane limiting the virtosome.