Origins, structures, and functions of circulating DNA in oncology.

While various clinical applications especially in oncology are now in progress such as diagnosis, prognosis, therapy monitoring, or patient follow-up, the determination of structural characteristics of cell-free circulating DNA (cirDNA) are still being researched. Nevertheless, some specific structures have been identified and cirDNA has been shown to be composed of many "kinds." This structural description goes hand-in-hand with the mechanisms of its origins such as apoptosis, necrosis, active release, phagocytosis, and exocytose. There are multiple structural forms of cirDNA depending upon the mechanism of release: particulate structures (exosomes, microparticles, apoptotic bodies) or macromolecular structures (nucleosomes, virtosomes/proteolipidonucleic acid complexes, DNA traps, links with serum proteins or to the cell-free membrane parts). In addition, cirDNA concerns both nuclear and/or mitochondrial DNA with both species exhibiting different structural characteristics that potentially reveal different forms of biological stability or diagnostic significance. This review focuses on the origins, structures and functional aspects that are paradoxically less well described in the literature while numerous reviews are directed to the clinical application of cirDNA. Differentiation of the various structures and better knowledge of the fate of cirDNA would considerably expand the diagnostic power of cirDNA analysis especially with regard to the patient follow-up enlarging the scope of personalized medicine. A better understanding of the subsequent fate of cirDNA would also help in deciphering its functional aspects such as their capacity for either genometastasis or their pro-inflammatory and immunological effects.

Progress towards the 'Golden Age' of biotechnology.

Biotechnology uses substances, materials or extracts derived from living cells, employing 22 million Europeans in a € 1.5 Tn endeavour, being the premier global economic growth opportunity this century. Significant advances have been made in red biotechnology using pharmaceutically and medically relevant applications, green biotechnology developing agricultural and environmental tools and white biotechnology serving industrial scale uses, frequently as process feedstocks. Red biotechnology has delivered dramatic improvements in controlling human disease, from antibiotics to overcome bacterial infections to anti-HIV/AIDS pharmaceuticals such as azidothymidine (AZT), anti-malarial compounds and novel vaccines saving millions of lives. Green biotechnology has dramatically increased food production through Agrobacterium and biolistic genetic modifications for the development of 'Golden Rice', pathogen resistant crops expressing crystal toxin genes, drought resistance and cold tolerance to extend growth range. The burgeoning area of white biotechnology has delivered bio-plastics, low temperature enzyme detergents and a host of feedstock materials for industrial processes such as modified starches, without which our everyday lives would be much more complex. Biotechnological applications can bridge these categories, by modifying energy crops properties, or analysing circulating nucleic acid elements, bringing benefits for all, through increased food production, supporting climate change adaptation and the low carbon economy, or novel diagnostics impacting on personalized medicine and genetic disease. Cross-cutting technologies such as PCR, novel sequencing tools, bioinformatics, transcriptomics and epigenetics are in the vanguard of biotechnological progress leading to an ever-increasing breadth of applications. Biotechnology will deliver solutions to unimagined problems, providing food security, health and well-being to mankind for centuries to come.

Biotechnology worldwide and the 'European Biotechnology Thematic Network' Association (EBTNA).

The European Biotechnology Congress 2011 held under the auspices of the European Biotechnology Thematic Network Association (EBTNA) in conjunction with the Turkish Medical Genetics Association brings together a broad spectrum of biotechnologists from around the world. The subsequent abstracts indicate the manner in which biotechnology has permeated all aspects of research from the basic sciences through to small and medium enterprises and major industries. The brief statements before the presentation of the abstracts aim to introduce not only Biotechnology in general and its importance around the world, but also the European Biotechnology Thematic Network Association and its aims especially within the framework of education and ethics in biotechnology.

Circulating DNA: intracellular and intraorgan messenger?

The circulation of both foreign and endogenous DNA within plants and its ability to be expressed in the host plants and FI generation is described. These data, together with those from animal systems are used to support the concept that a DNA fraction can act as a messenger between cells and tissues.

Evidence that direct DNA uptake through cut shoots leads to genetic transformation of Solanum aviculare Forst.

The reporter genes GUS, NPTII and BAR, either separately or in combination, have been exploited to determine if DNA which can directly enter plants, circulate within the plant and enter nuclei, can also integrate into the genome in a manner which will permit gene expression. Feeding of either seed-derived or adventitious cut shoots of Solanum aviculare with the GUS gene followed by rooting of the shoots and growing on, resulted in all tissues of the plant showing GUS activity as detected cytochemically. Southern blot analysis of plants derived from the adventitious shoots confirmed the presence of the reporter gene in roots. Reporter gene expression was observed also in the F1 generation. If GUS and NPTII or GUS, NPTII and BAR were fed together, then in each case it was possible to have both expression and Southern blot confirmation of each of the genes. There was a relatively high rate of transformation of approximately 5% of the fed stems across all experiments conducted during the present study.

In vitro stimulation by tumour cell media of [3H]-thymidine incorporation by mouse spleen lymphocytes.

Mouse spleen lymphocyte (SL) cells show a three to four-fold increase in [3H]-thymidine incorporation when incubated in tumour cell media, or in media containing tumour cell cytosol. Agarose gel chromatography of both [3H]-thymidine-labelled tumour cell media and cytosol shows a sharp peak of DNA-associated material eluting at about 60 kDa. This DNA-associated material is imported rapidly and efficiently by SL cells and is recoverable from their cytosol. The stimulating effect on SL cell thymidine incorporation resides primarily, if not exclusively, in this extruded/cytosolic 60 kDa DNA material. Tumour cells incubated in media containing normal or liver, but not tumour, cytosol show a reduced rate of [3H]-thymidine incorporation, indicating competition between normal and tumour associated DNA complexes. The results indicate that such cell-extruded DNA complexes may transmit 'genetic messages' to other cells, and are discussed in terms of interactions in the tumour-bearing host.

Mechanisms involved in living systems organisation, especially the programming necessary to enable the construction of individuals in three dimensions.

The complexity of the organization of living systems escalates by orders of magnitude in the development, from single precursor cells, not only of the three-dimensional structures characterizing each species but also in the variations necessary to accommodate the 1 million or more separate and identifiable species on this planet. Although the genetic information controlling such information is currently considered to reside in cellular DNA, it is also held that such information is restricted to a linear form encoding specifically for protein. However, this not only fails to explain the co-ordination of the vast number of processes occurring in simple, single cell, organisms, but also the integration of cellular activities to serve the interest of the total system. In particular how can a homeobox containing only genes encoding specifically for proteins organize and implement the mechanisms necessary for three-dimensional development? This, and the organization and implementation of the massive amount of information necessary to execute the construction of such a wide range of species, each in its own unique and exquisite detail, calls for internal programming of a highly complex and sophisticated nature. It is proposed here that such a central computer-analog program does exist, housed in the molecular electronic structure of cellular nucleic acid, primarily in DNA: its possible nature is discussed. Since precursor cells contain only of the order of 10(-10) g of DNA, this proposal involves an increase in information storage efficiency comparable with that already achieved by silicon microprocessors over mechanical calculators.

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes.

The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.

Chromatin phospholipid changes during rat liver development.

The chromatin extracted from rat hepatocytes of different ages has been shown to contain a phospholipid fraction representing 0.47-0.59 per cent of total chromatin in newborn animals and 0.22 per cent in 45-day-old animals. No such age-related differences are observed in the nuclei. The phospholipid composition of the nuclei at different ages shows a higher level of sphingomyelin and a lower level of phosphatidylserine in newborn than in adult animals. Chromatin phospholipids have a completely different composition from that of nuclei with respect to age, particularly in newborn rats, where there is a decrease in phosphatidylcholine and an increase in phosphatidylserine.

Glycolytic activity in embryos of Pisum sativum and of non-dormant or dormant seeds of Avena sativa L. expressed through activities of PFK and PPi-PFK.

A quantitative cytochemical assay for PPi-PFK activity in the presence of Fru-2,6-P2 is described along with its application to determine levels of activity in embryos of Pisum sativum and Avena sativa. The activity of ATP-PFK has also been studied in parallel as have PFK activities during the switch from dormant to non-dormant embryos in Avena sativa. PPi-PFK activity has been demonstrated in all tissues of Pisum sativum embryos and of Avena sativa embryos including the scutellum and the aleurone layers. The PPi-PFK activity was greater than that of ATP-PFK in both dormant and non-dormant seeds though with only marginally more activity in the dormant as opposed to the non-dormant state.

Immunocytochemical localization of neurosecretory amines and peptides in the free-living nematode, Goodeyus ulmi.

Mammalian antibodies to the neuroamines, serotonin and gamma-amino-butyric acid (GABA) and to the neuropeptides, adrenocorticotrophic hormone (ACTH) and FMRF-amide evoked a response to Goodeyus ulmi, a free-living nematode. Serotonin-like immunoreactivity was found in cell bodies in the nerve ring and in the ventral nerve cord in all developmental stages. Neurons in the vulva, implicated in egg-laying, were immunoreactive to anti-serotonin in G. ulmi females, while in males serotonergic nerve fibres was found in the spicular region. Immunoreactivity to ACTH was also seen to differ depending on the developmental stage of G. ulmi, being present only in the ventral cord from the late L3 stage. Anti-GABA immunoreactivity was localized in two cell bodies near the amphids in all life stages and FMRF-amide immunoreactivity was seen in the nerve ring in all developmental stages. No reactivity was found with antibodies to vasointestinal peptide and somatostatin-14.

The relationship between the activities of the pentose phosphate pathway and glycolysis during early stages of floral induction in spinach.

A quantitative cytochemical study was made of fructokinase, glucokinase, and fructokinase (both PFK-ATP and PFK-PP + F-2:6-P) activities in shoot apices of 4-week old Spinacia oleracea. The rates of activity of these enzymes in the central zone of the shoot apex of plants kept on a short day regime were compared with those from plants transferred from a range of timing up to 24 h to a continuous light regime when floral induction occurred. A mechanism is suggested explaining how no measurable change in activities of the enzymes assayed could still account for the availability of adequate levels G-6-P as substrate for pentose pathway activity which is almost doubled early on in cells of the central zone of shoot apices induced to flower.

Synthesis of chromatin phospholipids.

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.

Cytochemical and autoradiographic studies of the localization and turnover of chromatin and nucleolar associated phospholipids in plant and animal tissues.

The localization of chromatin-associated phospholipids has been demonstrated on chromosomes and on chromatin of interphase nuclei by cytochemical methods either in plant and in animal tissues. Three methods of fixation are suggested which can be combined which two cytochemical methods for phospholipids detection. An additional method is represented by autoradiographic technique after incorporation of a radioactive precursor such as [3H] ethanolamine. This method has been used with good results in nuclei of lateral root apices of Vicia faba on 1 micron thick section, thus avoiding any possible contamination by nuclear membrane. In these nuclei the phospholipids appear associated with the chromatin and more intensely with the nucleolar regions. The positivity of the cytochemical reaction disappears after extraction of fixed tissue with acidified methanol chloroform and after treatment of unfixed sections with phospholipase D. The use of phospholipid precursor has allowed the study of chromatin-phospholipids synthesis in root apices of Vicia faba with respect to timings of the cell cycle. The results show that there is a strong case for a pattern of chromatin phospholipid synthesis which operates during S phase. Concerning the role of phospholipids it is suggested that they may be linked to acidic protein and may have a structural function, particularly on the nucleoli.

Phospholipids in chromatin: incorporation of [32P]O4(2-) in different subcellular fractions of hepatocytes.

Nuclear, chromatin and microsomal fractions were isolated from hepatocytes prepared from rats injected with [32P]O4(2-) and killed subsequently at times between 1 and 48 h. Specific activities of the total phospholipids (PL) were determined for each subcellular fraction. The major points noted were the initial specific activity of the chromatin PL was half that of both nuclear and microsomal PL at 1 h; the first peak of labelling occurred at 6 h in both nuclear and microsomal PL, but was 3 h later (9h) in the chromatin PL; and a second peak of labelling occurred in the chromatin and microsomal PL, but not in those of the nuclei. On fractionation of the PL, the major and most metabolically active components were phosphatidylcholine + phosphatidylethanolamine, whilst sphingomyelin accounted for only about 8 per cent of the total PL. The chromatin and microsomal fractions were somewhat similar in their labelling patterns though with a delayed peaking of activity in the chromatin. This is indicative of a synthesis and transport of PL from the microsomes to the chromatin.

A quantitative cytochemical study of UDP-D-glucose: NAD-oxidoreductase (E.C. 1.1.1.22) activity during stelar differentiation in Pisum sativum L. cv Meteor.

A quantitative cytochemical assay for UDP-D-glucose dehydrogenase (UDPGD) activity employing scanning and integrating microdensitometry has been revised and applied to a study of this enzyme during the initiation of secondary cell wall biosynthesis during the formation of primary vascular tissues in roots of Pisum sativum L. cv Meteor. The reaction involves the use of NBT as final electron acceptor and is inhibited 10-fold by either 10 mM UDP-D-xylose or 25 mM UDP-D-glucuronic acid, two molecules involved in feed-back inhibition of UDPGD activity in vivo. UDPGD is a far-from equilibrium enzyme representing a flux-generating step in the biosynthesis of precursors for hemicelluloses involved in secondary cell wall construction, and can be demonstrated to increase sharply in activity in cells of the developing primary vascular elements. This changed activity occurs 18-20 cells back from the root cap junction and coincides with the first cells containing the activated programme for secondary cell wall synthesis.

Chromatin phospholipids and DNA synthesis in hepatic cells.

The synthesis of phospholipids found in microsomes, in the nuclei and in chromatin has been studied in rat liver after partial hepatectomy. [32P]O4(2-)incorporation in phospholipids has been compared with that of (3H) thymidine over a period of 48 h after operation. The presence of two peaks of DNA synthesis has been observed at 18 and 36 h; nuclear phospholipids show a continuous synthesis starting from 12 h, whereas the microsomes show two peaks at 12 and 24-30 h. The specific activity of the chromatin phospholipid fraction increases at 12h, doubles its initial value at 18 h, shows a peak at 30 h and comes back to the initial value at 48 h. It is concluded that chromatin phospholipids increase their synthesis in relation to the S phase of the cell cycle, whereas those of the nuclear membranes do not change the rate of synthesis throughout the cell cycle. The possibility is suggested that chromatin phospholipids are synthesized in the microsomes and transferred to the nucleus.

Phospholipids in plant and animal chromatin.

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.

Microcomputer control applied to the Vickers M85/86 microdensitometer.

An electronic interface for a Research Machines 380Z microcomputer and a Vickers M85/6 integrating microdensitometer is described, together with outlines of the control software. A typical applications programme, which may be written in several high-level languages, is outlined for the determination of a time course plot for enzyme activity in a tissue section. This system facilitates rapid accurate measurement and analysis of experimental data.

Euploidization of human hepatocytes from donors of different ages and both sexes compared with those from cases of Werner's syndrome and progeria.

Quantitative cytochemical studies of ploidy values for mono- and bi-nucleate hepatocytes from human livers of individuals from 15 weeks to 80 years of age, confirm earlier reports that the main ploidy shifts occur at 6-7 years, puberty and 21 years of age. In addition, the pattern of ploidy increase is similar to that described for other mammals. Ploidy values obtained for hepatocytes from two patients with Werner's Syndrome are in excess of those from livers of normal individuals of similar age. In contrast, ploidy values from the liver of a single case of Hutchinson-Gilford Syndrome were similar to those from children of a similar age group.