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Circulating DNA in the bloodstream has been studied since the 1940s, leading to its identification as a possible early marker for the presence of a primary tumor. Recently, it has been more successfully employed in liquid biopsies to determine the early presence of a metastatic tumor arising after chemotherapy, radiotherapy, and surgery. The appearance of such circulating tumor DNA permits the identification of the metastatic tumor before it is detected by either palpation or radiological analysis. Nevertheless, the liquid biopsy may possibly be affected by the removal of circulating tumor DNA via the kidneys and spleen as it is released. Furthermore, the liver removal of cell-free DNA has not yet been considered to be involved in this process. Here, we review the literature on the removal of free single- and double-stranded DNA and nucleosomal, vesicular, and exosomal DNA via the liver and examine its possible impact on circulating DNA levels. The removal of all forms of DNA by the liver, together with that removed by the kidneys and spleen, may delay the timing of positive results from liquid biopsies.

(1) Although long noncoding RNAs (lncRNAs) are known to be precursors of microRNAs (miRNAs), they frequently act as competing endogoneous RNAs (ceRNAs), yet still their interplay with miRNA is not well known. However, their interaction with miRNAs may result in the modulation of miRNA action. (2) To determine the contribution of these RNA molecules in tumor resistance to chemotherapeutic drugs, it is essential to consider not only the oncogenic and tumor suppressive function of miRNAs but also the impact of lncRNAs on miRNAs. Therefore, we performed an extensive search in different databases including PubMed. (3) The present study concerns the interplay between lncRNAs and miRNAs in the regulatory post-transcriptional network and their impact on drugs used in the treatment of breast cancer. (4) Consideration of this interplay may improve the search for new drugs to circumvent chemoresistance.

Exosomes, small extracellular vesicles mediate intercellular communication by transferring their cargo including DNA, RNA, proteins and lipids from cell to cell. Notably, in the immune system, they have protective functions. However in cancer, exosomes acquire new, immunosuppressive properties that cause the dysregulation of immune cells and immune escape of tumor cells supporting cancer progression and metastasis. Therefore, current investigations focus on the regulation of exosome levels for immunotherapeutic interventions. In this review, we discuss the role of exosomes in immunomodulation of lymphoid and myeloid cells, and their use as immune stimulatory agents to elicit specific cytotoxic responses against the tumor.

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging “DNA” with “RNA” in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with “NAs” to indicate nucleic acids.

Exosomes are small extracellular vesicles engaged in intercellular communication in both healthy and tumor cells. When released by the primary tumor, they transfer their cargo including nucleic acids, proteins, and lipids to target cells, thus modulating the character and fate of the recipient cells. By propagating their oncogenic content, exosomes are able to promote tumor progression, angiogenesis, metastases, and drug resistance. Their functions as delivery vehicles of biological material make exosomes promising biomarkers for the early prediction of disease progression and drug resistance in breast cancer, as well as for therapeutic targeting of molecules to treat this deadly disease. In the present review, we accentuate the relevance of exosomes as vehicles of prognostic and predictive markers and target molecules, and describe their potential therapeutic applications as drug cargo suppliers. We made an extensive literature research to clarify the association of their cargo, including exosomal DNA and RNA molecules, with the propagation of drug resistance.

Ovarian cancer has a poor outcome because it is usually detected at advanced tumor stages, and the majority of the patients develop disease relapse as a result of chemotherapy resistance. This most lethal gynecological malignancy metastasizes within the peritoneal fluid or ascites to pelvic and distal organs. In ovarian cancer progression and metastasis, small non-coding RNAs (ncRNAs), including long noncoding RNAs and microRNAs have been recognized as important regulators. Their dysregulation modulates gene expression and cellular signal pathways and can be detected in liquid biopsies. In this review, we provide an overview on circulating plasma and serum ncRNAs participating in tumor cell migration and invasion, and contributing to recurrence and metastasis of ovarian cancer. We will also discuss the development of potential, novel therapies using ncRNAs as target molecules or tumor markers for ovarian cancer.

The identification of exosomes, their link to multivesicular bodies and their potential role as a messenger vehicle between cancer and healthy cells opens up a new approach to the study of intercellular signaling. Furthermore, the fact that their main cargo is likely to be microRNAs (miRNAs) provides the possibility of the transfer of such molecules to control activities in the recipient cells. This review concerns a brief overview of the biogenesis of both exosomes and miRNAs together with the movement of such structures between cells. The possible roles of miRNAs in the development and progression of breast, ovarian and prostate cancers are discussed.

Initially, most ovarian tumors respond to the treatment with platinum components, but frequently recurrence occurs within the following two years in advanced ovarian cancer patients. In this regard, previous studies have shown changes in the epigenetic patterns in ovarian cancer that are linked with resistance to cis- and carboplatin therapy. Thus, epigenetic changes mediated by a treatment with cis- or carboplatin could identify such patients who do or do not respond to this therapy. Therefore, an understanding of the impact of platinum on epigenetics in ovarian cancer is important in overcoming platinum resistance. In this review, we delineate epigenetic abnormalities in cis- and carboplatin-resistant ovarian tumors, such as changes in DNA methylation, histone modifications and deregulation of microRNAs, and discuss the potential of epigenetic therapies in combination with platinum.

The monitoring of therapy during the treatment of cancer patients is currently assessed by the levels of circulating tumor cells or by PET/CT scans. Neither approach has the sensitivity or specificity to be very sure of the efficacy of the treatment. Moreover, PET/CT scans can be both comparatively expensive and produce low levels of radiation for the patient. The advent of the possibility of using circulating DNA released from the tumor permits (1) a possible early marker of the presence of the cancer, (2) an indication of the success of the primary treatment, (3) an indication of the early presence of possible metastasis, (4) a marker of the success of secondary subsequent treatment, (5) determining which patients can benefit from a particular treatment, and (6) offering a prognosis. These aspects will be discussed concerning the application of circulating tumor DNA analysis to the monitoring of cancer patients undergoing therapy.

The monitoring of therapy during the treatment of cancer patients is currently assessed by the levels of circulating tumor cells or by PET/CT scans. Neither approach has the sensitivity or specificity to be very sure of the efficacy of the treatment. Moreover, PET/CT scans can be both comparatively expensive and produce low levels of radiation for the patient. The advent of the possibility of using circulating DNA released from the tumor permits (1) a possible early marker of the presence of the cancer, (2) an indication of the success of the primary treatment, (3) an indication of the early presence of possible metastasis, (4) a marker of the success of secondary subsequent treatment, (5) determining which patients can benefit from a particular treatment, and (6) offering a prognosis. These aspects will be discussed concerning the application of circulating tumor DNA analysis to the monitoring of cancer patients undergoing therapy.

The presence of circulating nucleic acids in man, animals and plants is well documented. It is clear that such nucleic acids can not only circulate freely within an organism, but can also enter cells when their biology may be changed either epigenetically or genetically. Evidence is presented concerning a possible influence of these nucleic acid fragments on the genetics of the F1 generation of man, animals and plants. The data presented also offer a mechanism by which the incorporation of horizontally transferred genes between organisms may be achieved. The role that circulating nucleic acids might play in modifying the F1 generation and possibly the evolutionary process is considered. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110, 931–948.

Biotechnology uses substances, materials or extracts derived from living cells, employing 22 million Europeans in a (sic)1.5Tn 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.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

Studies on a range of prokaryote and eukaryote cells and tissues have shown that a newly synthesized DNA/RNA-lipoprotein complex is released in a regulated manner. This complex, termed a virtosome, is a novel cytosolic component of eukaryote cells. The released virtosomes can readily enter other cells where they can modify the biology of the recipient cells. Such modifications include immunological changes and transformation from normal to cancer cells. The virtosomes form a normal component of the circulating nucleic acids in plasma and serum currently used for clinical diagnostic purposes. Given the transformative powers of virtosomes released from tumour cells, the presence of such a complex in human plasma could readily offer the basis of an alternative mechanism for the initiation of metastases.

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, and urine of both healthy individuals and patients. The ability to isolate, quantify, and analyze these molecules has led to the identification of specific nucleic acid fragments related to particular disorders such as diabetes, cancer, myocardial infarction, and stroke, threby permitting their early diagnosis. Currently, a number of methods for isolating the nucleic acids are employed and although a start has been made to compare the efficiencies of these methods, there is still a way to go before there are precise protocols for nucleic acid extraction. The older chemical methods of extraction still outperform some of the available kits. Some progress is being made to determine the origin of the circulating nucleic acids, although there are still many questions to be answered, including whether the source is through the spontaneous release of newly synthesized nucleic acid or whether it just derived from necrotic and apoptotic cells. In addition, it can be demonstrated that the nucleic acids can enter cells and exhibit a biological activity in the recipient cells. Hence, the question remains: Are the circulating nucleic acids freely entering tissues and cells from the blood and inducing changes in those tissues and cells? Further work is needed to elucidate these areas, and the various protocols must be standardized if the new methodology is to be widely and accurately applied in the diagnosis of disease and the monitoring of therapy. This chapter summarizes the work reported in this volume.

A DNA fraction is spontaneously released from living, but not dead or dying, human, other mammalian, avian, amphibian, plant, and prokaryote cells. The spontaneously released DNA fraction has been shown to be (a) present in both actively dividing and nondividing, differentiated cell populations; (b) labile; (c) associated with DNA-dependent RNA or DNA polymerase; (d) associated with an RNA fraction; and to have (e) a lower molecular weight than the typical genetic DNA fraction; and (f) Alu repeat sequences in increased proportions compared to a unique gene in plasma/serum. On the other hand, early autoradiographic and biochemical and quantitative cytochemical and cytophysical studies on DNA permitted the identification of a DNA fraction which was (1) present in both actively dividing and nondividing, differentiated cell populations; (2) labile; and (3) had a lower molecular weight than the typical genetic DNA fraction. This DNA fraction was termed metabolic DNA (m-DNA) and was proposed as possibly forming extra gene copies for the rapid production of m-RNA, to be destroyed subsequently. Therefore, we suggest that the metabolic DNA fraction might represent the precursor to the formation of the spontaneously released DNA fraction.

In vitro studies of partially purified virtosomes from rat liver showed inhibition of cell multiplication in four normal and two tumour cell lines. In vivo, the liver virtosomes slowed tumour growth and limited metastases in rats bearing DHD/K12-PROb cell initiated tumours.

The book will present the progress made since the last meeting in fall 2013 concerning the analysis of circulating extra-cellular nucleic acids. There are a modest number of laboratories involved in this field, nevertheless the number of papers published by researchers is extensive. The articles which will be published in this meeting report will be a valuable contribution for researchers and research students alike and will help them to stay on top of the developments in different research areas and to cross borders between them

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.

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 addition to plasma and serum as sources of nucleic acids circulating in the whole body, amniotic fluid, saliva, urine, pleural effusion, bronchial lavage, bronchial aspirates, breast milk, colostrums, tears, seminal fluid, peritoneal fluid, pleural effusion and stools are all available for minimally invasive analysis of nucleic acids. This chapter introduces the possibilities of using nucleic acids from amniotic fluid, saliva, urine, cerebrospinal fluid and bronchial lavage/aspirates in attempts to produce reliable early markers for diagnosis, prognosis and treatment monitoring using minimally invasive methodology. Moreover, the data from amniotic fluid can be used also to further the understanding of normal and abnormal fetal development in utero. In addition, the data from saliva can be employed for monitoring the progress of premature born infants.

1948 marked the identification of DNA in peripheral blood by Mendel and Metais and the beginning of modern cell biology after the 2nd world war. During the period that followed, little was heard of the peripheral blood DNA, but there was research activity concerning the movement of DNA about plants that led to parallel studies on animal systems. This resulted in the establishment of the capability of DNA to leave and enter cells as well as to circulate about plants and animals. Subsequently, together with improved technology and analytical genetics, both DNA and RNA markers have been identified that may act as early indicators of a pathological state and also have a use in the monitoring of treatment. This chapter offers some historical background to CNAPS and indicates both the current state of the art as well as the directions in which the research is developing. Attention is drawn to the ethical and legal problems arising through the development of the genetic information and the testing systems. The topics discussed are expanded in the subsequent chapters.

Although nucleic acids have been known to circulate in the blood since 1948 their biology has been studied only since the 1960s. This chapter contains discussion of (a) the presence of DNA and RNA circulating in human plasma and serum from both healthy individuals and patients, (b) the amounts of DNA/RNA present together with the variables affecting these amounts, (c) possible sources of the DNA/RNA in blood and (d) the ability of the circulating nucleic acids to enter other cells and to modify the biology of the recipient cells. The relationship of the DNA from cancer patients is considered with respect to the formation of metastases.

A cytochemical and biochemical study of Agrobacterium rhizogenes-transformed (hairy) roots of Valerianella discoidea has demonstrated: (a) the presence of a soluble valepotriate fraction in root sections in addition to the stored valepotriates in lipid droplets; (b) that roots need to be at least 3.5 cm long before they will synthesise/store valepotriates; (c) that there is a loss of valepotriates from the pericycle cells about to form lateral roots; and (d) an accumulation of seven times more monoene and diene valepotriates compared with non-transformed, control roots. Copyright © 1999 John Wiley & Sons, Ltd.

The earliest biochemical marker of ¯oral evocation in the shoot apex of S. oleracea is the doubling of the rate of glucose-6-phosphate dehydrogenase (G6PD) activity 12±15 h after transfer of 4-week-old plants from short days to continuous light i.e. 1±2 h after the leaves are raised to the ¯oral state. Quantitative cytochemical analysis of G6PD activity in the vegetative apices showed that addition of 10y7 M Ca2‡ to the cytochemical enzyme reaction medium for G6PD activity raises the rate of enzyme activity to that seen in the induced apices. Higher concentrations of Ca2‡ result in G6PD inhibition in the vegetative apices and any added Ca2‡ at concentrations of 10y7 M or higher inhibit the G6PD activity seen in both the induced apices and leaf primordia of both types of apex. The addition of EGTA abolishes the cytochemical reaction. The ability of the Ca2‡ to activate the G6PD activity in addition to the incubation medium occurs during the periods of 8±11 h of continuous light, but is already lost by 12 h when no change is achieved by Ca2‡ treatment. This can be interpreted as indicating a point in time close to the moment of ¯oral evocation. A model is proposed in which Ca2‡ is able to activate the inactivated-G6PD molecules in the vegetative apex through increased Ca2‡ ¯ux possibly through the action of plasmalemmal Ca2‡-ATPase activity as part of the ¯oral evocation process.

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.

A quantitative cytochemical study of intact root apices and wounded roots of Pisum sativum, and of cotyledons from Solanum aviculare induced to form vessels from mesophyll cells, has demonstrated a sharp increase in glucose-6-phosphate dehydrogenase activity at a very early stage in the differentiation of the vascular tissues. However, UDP-D-glucose dehydrogenase activity appears to increase only at a time corresponding to the initiation of secondary cell wall events. Data from the callus cultures from Malus M27 and Cox endosperm indicated that glucose-6-phosphate dehydrogenase activity changes may be a possible marker for determining between the regenerability and recalcitrance of calluses. © 1997 John Wiley & Sons, Ltd.

Evidence is presented that, as in animal and human cells, plant cells can release a newly-synthesized DNA which can freely circulate in the plants. This DNA enters cells and their nuclei where it may be integrated and be expressed so acting, apparently, as a messenger-DNA.

The presence of DNA and RNA circulating in human plasma and serum is described. The possible sources of the DNA/RNA in blood, their ability to enter other cells and to express in the recipient cells are discussed and the relationship with metastases considered. The possible role(s) of the DNA/RNA in clinical diagnosis, in monitoring treatment and in prognosis are considered for diabetes and oncology.

The National Health System (NHS) in the UK has evolved to become one of the largest healthcare systems in the world. In 2010 the UK government in its 2010 White Paper “Equity and Excellence: Liberating the NHS” has announced a strategy on how it will “create a more responsive, patient-centred NHS which achieves outcomes that are among the best in the world”. Here we present an overview of the UK healthcare system as it stands at the end of the first decade of the twenty-first century, with emphasis on established and emerging Predictive, Preventive and Personalised Medicine (PPPM) elements.

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.

Although cinnamyl alcohol dehydrogenase is an important enzyme in the biosynthesis of lignins, its localization at the tissue and cellular levels is not well known. A quantitative cytochemical method has been devised to determine the localization and level of activity of this enzyme in plant tissues. Unexpectedly, the enzyme activity was present not only in the developing xylem elements and phloem fibres, but also in the epidermal and subepidermal layers of both roots and shoots. This pattern of localization implies that several distinct sites of cinnamyl alcohol dehydrogenase exist and that the role of this enzyme could be different according to the cell types in which it is located.

The presence of DNA and RNA circulating in human plasma and serum is described. The known sources of the DNA/RNA in blood, the ability of these nucleic acids to enter other cells and to express in the recipient cells are considered along with their relationship to metastases. The possible role(s) of the DNA/RNA in personalized clinical diagnosis, monitoring of treatment and prognosis in oncology are discussed.

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, urine, saliva and spinal fluid from both healthy individuals and patients. The ability to isolate, quantify, and analyze these molecules has led to the identification of specific nucleic acid fragments related to a variety of clinical disorders thereby permitting their early diagnosis and prognosis. This chapter summarizes the work reported in this volume.

Immunocytochemistry, one of the two basic approaches to the in situ study of enzymes, allows the enzyme molecules to be localized, but indicates nothing about their activity. Quantitative cytochemistry, on the other hand, indicates the sites of localization of enzyme activity and, in many cases, the rates of these activities. This latter approach, which is the subject of this review, permits the assessment of activity in individual cells or groups of cells in a given tissue. Details of tissue preparation, enzyme procedures, methods of quantification and some case histories illustrate the specificity and flexibility of the method.

DNA and RNA fractions have been isolated from the whole blood, serum, plasma, the surface of blood cells, and urine of both healthy individuals and patients. The ability to isolate, quantify, and analyze these molecules has led to the identification of specific nucleic acid fragments related to particular disorders such as diabetes, cancer, myocardial infarction, and stroke, thereby permitting their early diagnosis. Currently, a number of methods for isolating the nucleic acids are employed and although a start has been made to compare the efficiencies of these methods, there is still a way to go before there are precise protocols for nucleic acid extraction. The older chemical methods of extraction still outperform some of the available kits. Some progress is being made to determine the origin of the circulating nucleic acids, although there are still many questions to be answered, including whether the source is through the spontaneous release of newly synthesized nucleic acid or whether it just derived from necrotic and apoptotic cells. In addition, it can be demonstrated that the nucleic acids can enter cells and exhibit a biological activity in the recipient cells. Hence, the question remains: Are the circulating nucleic acids freely entering tissues and cells from the blood and inducing changes in those tissues and cells? Further work is needed to elucidate these areas, and the various protocols must be standardized if the new methodology is to be widely and accurately applied in the diagnosis of disease and the monitoring of therapy. This chapter summarizes the work reported in this volume.

The presence of DNA and RNA circulating in human plasma and serum is described. The possible sources of the DNA/RNA in blood, their ability to enter other cells and to express in the recipient cells are discussed and the relationship with metastases considered. The possible role(s) of the DNA/RNA in clinical diagnosis, in monitoring treatment and in prognosis are considered for diabetes and oncology.

A cytochemical and electrophoretic analysis of alpha-naphthyl and naphthol AS-D acetate esterases from the radicles of pea seeds germinated for 24, 36 and 48 h permitted the detection of the loss of activity from the procortex with time after germination, but a stability of this activity in the prostele. Inhibitor studies showed the procambial enzyme as a single band of carboxylesterase activity the remainder being acetylesterase activity. The procambial carboxylesterase which is present in the more mature root apex corresponds to that of the 48-h embryo (separating at Rf 0·74).