Realising the European network of biodosimetry: RENEB-status quo.

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed.

Realising the European Network of Biodosimetry (RENEB).

In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.

Relationship between DNA repair and formation of sister chromatid exchanges and chromatid aberrations under the influence of poly(ADP-ribose) polymerase inhibition by 3-aminobenzamide.

The mechanisms of formation of sister chromatid exchanges (SCEs) and chromosome aberrations following inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide were studied in Chinese hamster ovary cell lines deficient in different repair pathways. The results confirm earlier findings that (a) the 'spontaneous' SCEs are formed due to the incorporated BrdU in the DNA, (b) 'spontaneous' and induced SCEs originate from different mechanisms, and (c) SCEs and chromatid exchanges are formed by different pathways.

DNA repair mechanisms involved in the removal of DBPDE-induced lesions leading to chromosomal alterations in CHO cells.

Polycyclic aromatic hydrocarbons (PAH) such as dibenzo[a,l]pyrene (DBP) are wide-spread environmental pollutants most probably mutagenic and carcinogenic to humans. Detailed data on the cytogenetic effects of anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) in mammalian cells are not available in the literature. The aim of this study is to elucidate the mechanisms involved in the induction of chromosomal aberrations and sister chromatid exchanges (SCEs) by DBPDE in mammalian cells. In order to achieve this a parental (AA8) and different DNA repair-deficient Chinese hamster ovary cell lines such as UV4, UV5, UV61 (nucleotide excision repair, NER), EM9 (base excision repair, BER), irs1SF (homologous recombination repair, HRR) and V3-3 (non-homologous end joining, NHEJ) were used. The most sensitive cell lines for DBPDE-induced chromosome aberrations were EM9 and irs1SF, while EM9 and V3-3 cell lines were the most sensitive in terms of SCEs induction. It can be suggested that the BER pathway plays an important role in the repair of lesions induced by DBPDE, affecting both chromosomal aberrations and SCEs induction. Moreover, the HRR pathway seems to play a role in cellular resistance to DBPDE mainly in terms of chromosomal aberration induction while the NHEJ pathway takes part affecting only the induction of SCEs.

Histone post-translational modifications in DNA damage response.

The fact that eukaryotic DNA is packed into chromatin constitutes a physical barrier to enzymes and regulatory factors to reach the DNA molecule for replication, transcription, recombination and repair. Although most studies in this field have concentrated on how chromatin regulates transcription, there is a recent emphasis on studying the role of chromatin in the response to DNA damage. Two main chromatin-remodeling mechanisms have been identified, namely, ATP-dependent chromatin-remodeling complexes and histone post-translational modifications (PTMs). PTMs constitute reversible covalent modifications in aminoacidic residues, such as serine and threonine phosphorylation, lysine acetylation, lysine and arginine methylation and lysine ubiquitylation, among others. Moreover, nucleosome composition can be modified by the incorporation of histone variants, which are assembled into nucleosomes independently of DNA replication. The phosphorylation of the histone variant H2AX (gammaH2AX) is one of the best examples of histone PTMs in response to DNA damage induction, but many others have recently been revealed. In this review, we focus on and summarize the best-known histone PTMs observed in excision repair (base excision and nucleotide excision) and double-strand break (non-homologous end-joining and homologous recombination) repair pathways. In brief, the interplay between chromatin remodelers and DNA repair factors is discussed in relation to DNA damage response mechanisms.

Asynchronously replicating Eu/heterochromatic regions shape chromosome damage.

In order to shed more light on the influence of DNA replication on the formation and distribution of chromosome aberrations, breakpoints (BP) produced by UV-C and AluI were assigned either to the early replicating short euchromatic arm (Xp(e)) or to the late replicating long heterochromatic arm (Xq(h)) of the Chinese hamster (CHO9) X chromosome. Early (ES) or late (LS) S-phase cells were assessed by pulse incorporating the base analogue 5-bromo-2'-deoxyuridine (BrdU) immediately after UV-C irradiation (30 J/m(2)) or AluI (20 U) poration followed by BrdU immunodetection with FITC-tagged antibodies in metaphase spreads. Short (30 s) UV-C exposures (1 J/m(2)/s) induced BP preferentially in Xq(h) in LS cells and a random distribution of BP along Xp(e) and Xq(h) in ES cells. However, BP induced by long (5 min) UV-C exposures (0.1 J/m(2)/s) clustered according to arm replication time (Xp(e) during ES and Xq(h) along LS). Giemsa-stained metaphases showed elevated frequencies of UV-C induced chromatid-type aberrations and gaps, especially in cells exposed to longer UV-C irradiation. BP induced by AluI clustered in Xp(e) in ES but distributed randomly during LS. In contrast to UV-C, AluI did not produce an increase in the yield of gaps, neither in ES nor in LS cells. Putative mechanisms underlying the observed distributions of chromosome damage in replicating CHO9 cells exposed to UV-C and AluI are discussed.

Distribution of UVC-induced chromosome aberrations along the X chromosome of TCR deficient and proficient Chinese hamster cell lines.

Cells with a transcription coupled repair (TCR) deficiency are characterized by a higher sensitivity to UVC irradiation and by an increase in apoptosis and chromosomal aberration frequencies. It has been claimed that the higher frequency of chromosomal aberrations results from the transcription blockage caused by UVC-lesions located in the transcribed strands of the genome. The distribution of chromosome breakpoints in euchromatic and heterochromatic regions of the X chromosome from TCR deficient and proficient Chinese hamster cell lines was studied. Most UVC-induced breakpoints occurred in euchromatic regions of the X chromosome in both cell lines. No increase of UVC-induced breakpoints in the euchromatic region of the UV61 X chromosome was observed, indicating that TCR failure alone cannot be responsible for the increased frequency of chromosomal aberrations. Differential chromatin remodeling in the TCR defective cell line is proposed as a possible mechanism involved in the distribution of UVC-induced breakpoints along the Chinese hamster X chromosome. A similar explanation for the increase of UVC-induced chromosomal aberrations in TCR defective cells is given.

Apoptosis preferentially eliminates irradiated g0 human lymphocytes bearing dicentric chromosomes.

G(0) human peripheral blood lymphocytes were X-irradiated to determine whether there is a direct relationship between radiation-induced dicentric chromosomes and the triggering of apoptosis. Immediately after X-ray exposure, control and irradiated lymphocytes were analyzed for viability, apoptosis and chromosome damage using the premature chromosome condensation technique. A batch of lymphocytes was kept in liquid holding for 48 h and then loaded on Ficoll-Paque medium to separate apoptotic (high-density) and normal (normal-density) cells. Then the same end points were analyzed in high-density and normal-density fractions of control and irradiated lymphocytes. After 48 h of liquid holding, the majority of apoptotic cells contained dicentric chromosomes. These results demonstrate that in human lymphocytes, the type of chromosome damage influences the induction of programmed cell death and provide direct evidence that cells bearing dicentrics are eliminated by apoptosis. G0 lymphocytes are the most common tissue used in biodosimetry studies, and the amount of chromosomal damage detected depends on the time between exposure and sampling. Since the radiation-induced apoptotic cells show the presence of dicentrics, radiation-induced damage can be underestimated. These results may have relevance in evaluations of the efficacy of radiotherapy based on the frequencies of chromosomal aberrations.

Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX.

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.

Modulation of chromosome damage localization by DNA replication timing.

PURPOSE: Non-random occurrence of induced chromosome breakpoints (BP) has been repeatedly reported. DNA synthesis and chromatin remodeling may influence chromosome BP localization. The CHO9 X chromosome exhibits an early replicating short euchromatic arm (Xpe) and a late replicating long heterochromatic arm (Xqh). We investigated the role played by DNA replication and related chromatin remodeling processes on BP distribution in eu/heterochromatin using the CHO9 X chromosome as a model. MATERIALS AND METHODS: BP induced by etoposide, a topoisomerase II inhibitor, as well as by the S-dependent clastogens ultraviolet-C light (UV-C) and methyl methanesulfonate (MMS) were mapped to CHO9 X chromosome arms. The base analogue 5-bromo-2'-deoxyuridine (BrdUrd) was pulse-added immediately after UV-C irradiation or during etoposide and MMS treatments (40 min) to identify cells in early S-phase (Xpe labeled) or late S-phase (Xqh labeled) after indirect BrdUrd immunodetection in metaphase spreads using primary anti-BrdUrd and secondary fluorochrome-tagged antibodies. RESULTS: During early S-phase, BP induced by etoposide and MMS mapped preferentially to Xpe while BP produced by UV-C localized randomly. BP induced by all agents during late S-phase clustered in Xqh. CONCLUSIONS: Results obtained suggest that replication time of eu/heterochromatin as well as chromatin remodeling may determine BP localization on the CHO9 X chromosome.

Studies on radiation-induced apoptosis in G0 human lymphocytes.

PURPOSE: To determine the relationships between the frequencies of radiation-induced chromosomal alterations and the extent of apoptosis in G0 human lymphocytes. MATERIAL AND METHODS: G0 human peripheral blood lymphocytes (HPBL) were X or gamma-irradiated, in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). Directly after irradiation, a part of the lymphocytes were stimulated to grow while the rest were stimulated 48 h after irradiation. These lymphocyte cultures were analysed for induction of chromosomal aberrations. A subset of lymphocytes was kept in G0 and analysed for cell viability, apoptosis and p53 expression. RESULTS: The fraction of cells bearing dicentrics was reduced in lymphocytes stimulated to grow 48 h post irradiation as compared to lymphocytes stimulated immediately after irradiation. The decrease in the frequency of dicentrics correlated with the increase in the number of apoptotic cells. The operative apoptotic pathway in irradiated Go lymphocytes was dependent on the expression of p53. CONCLUSIONS: The radiation-induced apoptotic response of G0 lymphocytes is p53 dependent and increases with the time they are held in G0. When mitogen was added 48 h after irradiation, cells with dicentrics were either preferentially eliminated or did not enter mitosis. Thus the radiation-induced damage can be underevaluated depending on the time between radiation exposure and the induction of proliferation. These results may have relevance for biodosimetry studies or for evaluations of the efficacy of radiotherapy which are based on the frequencies of chromosomal aberrations.

The novel human gene aprataxin is directly involved in DNA single-strand-break repair.

The cells of an ataxia-oculomotor apraxia type 1 (AOA1) patient, homozygous for a new aprataxin mutation (T739C), were treated with camptothecin, an inhibitor of DNA topoisomerase I which induces DNA single-strand breaks. DNA damage was evaluated by cytogenetic analysis of chromosomal aberrations. The results obtained showed marked and dose-related increases in induced chromosomal aberrations in the patient and her heterozygous mother compared to the intrafamilial wild-type control. The alkaline comet assay confirmed this pattern. Moreover, the AOA1 cells did not show hypersensitivity to ionizing radiation, i.e. X-rays. These findings clearly indicate the direct involvement of aprataxin in the DNA single-strand-break repair machinery.

Mechanisms of formation of chromosomal aberrations: insights from studies with DNA repair-deficient cells.

In order to understand the mechanisms of formation of chromosomal aberrations, studies performed on human syndromes with genomic instability can be fruitful. In this report, the results from studies in our laboratory on the importance of the transcription-coupled repair (TCR) pathway on the induction of chromosomal damage and apoptosis by ultraviolet light (UV) are discussed. UV61 cells (hamster homologue of human Cockayne's syndrome group B) deficient in TCR showed a dramatic increase in the induction of chromosomal aberrations and apoptosis following UV treatment. At relatively low UV doses, the induction of chromosomal aberrations preceded the apoptotic process. Chromosomal aberrations probably lead to apoptosis and most of the cells had gone through an S phase after the UV treatment before entering apoptosis. At higher doses of UV, the cells could go into apoptosis already in the G1 phase of the cell cycle. Abolition of TCR by treatment with alpha-amanitin (an inhibitor of RNA polymerase II) in the parental cell line AA8 also resulted in the induction of elevated chromosomal damage and apoptotic response similar to the one observed in UV61 cells treated with UV alone. This suggests that the lack of TCR is responsible for the increased frequencies of chromosomal aberrations and apoptosis in UV61 cells. Hypersensitivity to the induction of chromosomal damage by inhibitors of antitopoisomerases I and II in Werner's syndrome cells is also discussed in relation to the compromised G2 phase processes involving the Werner protein.

Potassium bromate but not X-rays cause unexpectedly elevated levels of DNA breakage similar to those induced by ultraviolet light in Cockayne syndrome (CS-B) fibroblasts.

It has been previously reported that the elevated accumulation of repair incision intermediates in cells from patients with combined characteristics of xeroderma pigmentosum complementation group D (XP-D) and Cockayne syndrome (CS) XP-D/CS fibroblasts following UV irradiation is caused by an "uncontrolled" incision of undamaged genomic DNA induced by UV-DNA-lesions which apparently are not removed. This could be an explanation for the extreme sensitivity of these cells to UV light. In the present study, we confirm the immediate DNA breakage following UV irradiation also for CS group B (CS-B) fibroblasts by DNA migration in the "comet assay" and extend these findings to other lesions such as 8-oxodeoxyguanosine (8-oxodG), selectively induced by KBrO3 treatment. In contrast, X-ray exposure does not induce differential DNA breakage. This indicates that additional lesions other than the UV-induced photoproducts (cyclobutane pyrimidine dimers, CPD, and 6-pyrimidine-4-pyrimidone products, 6-4 PP), such as 8-oxodG, specifically induced by KBrO3, are likely to trigger "uncontrolled" DNA breakage in the undamaged genomic DNA in the CS-B fibroblasts, thus accounting for some of the clinical features of these patients.

Distribution of breakpoints induced by etoposide and X-rays along the CHO X chromosome.

SORB (selected observed residual breakpoints) induced by ionizing radiation or endonucleases are often non-randomly distributed in mammalian chromosomes. However, the role played by chromatin structure in the localization of chromosome SORB is not well understood. Anti-topoisomerase drugs such as etoposide are potent clastogens and unlike endonucleases or ionizing radiation, induce DNA double-strand breaks (DSB) by an indirect mechanism. Topoisomerase II (Topo II) is a main component of the nuclear matrix and the chromosome scaffold. Since etoposide leads to DSB by influencing the activity of Topo II, this compound may be a useful tool to study the influence of the chromatin organization on the distribution of induced SORB in mammalian chromosomes. In the present work, we compared the distribution of SORB induced during S-phase by etoposide or X-rays in the short euchromatic and long heterochromatic arms of the CHO9 X chromosome. The S-phase stage (early, mid or late) at which CHO9 cells were exposed to etoposide or X-rays was marked by incorporation of BrdU during treatments and later determined by immunolabeling of metaphase chromosomes with an anti-BrdU FITC-coupled antibody. The majority of treated cells were in late S-phase during treatment either with etoposide or X-rays. SORB induced by etoposide mapped preferentially to Xq but random localization was observed for SORB produced by X-rays. Possible explanations for the uneven distribution of etoposide-induced breakpoints along Xq are discussed.

X-irradiated human lymphocytes with unstable aberrations and their preferential elimination by p53/survivin-dependent apoptosis.

PURPOSE: To investigate whether unstable types of chromosomal aberrations are more effective in priming apoptotic cell death in comparison with stable ones. Also, to highlight the phase of the cell cycle at which apoptosis occurs and the mechanism of its execution. MATERIALS AND METHODS: G0 human peripheral blood lymphocytes were X-irradiated in the presence or absence of the repair inhibitor cytosine arabinoside (Ara-C). After irradiation, the lymphocytes were analysed for induction of dicentrics, translocations, apoptosis, p53 and survivin expression at various recovery times. RESULTS: A preferential elimination of cells bearing dicentrics with respect to those with balanced translocations was observed. There was a time-dependent correlation between the decrease in the frequency of dicentrics and the increase in the per cent of apoptotic cells. Most of the apoptotic cells were labelled with bromodeoxyuridine and were mononucleated in cytochalasin B-treated cells cultures (blocked cytokinesis). However, after continuous colcemid treatment, the apoptotic pathway was not induced. Moreover, in the G2/M-phase, an increase in p53 and a decrease in survivin occurred that were X-ray and Ara-C dose dependent. CONCLUSIONS: The apoptotic process is primed when the dicentric-bearing human peripheral blood lymphocytes attempt to exit from metaphase. It is possible that unstable aberrations generate changes in the mitotic spindle causing mechanical tension at the kinetochore, activating the mitotic checkpoint and the execution of p53/survivin-dependent apoptosis.

Towards a standardization of biological dosimetry by cytogenetics.

When individuals are accidentally overexposed to ionising radiations, follow-up investigations may include dose assessment by cytogenetics. Scoring of unstable chromosome aberrations (dicentrics, centric rings and acentrics) in peripheral blood lymphocytes is regarded as the most specific method to estimate the exposure dose. It has acquired, in some countries, a medico-legal recognition. Paradoxically, there is no universally adopted technique and so important variations occur in methods and these may influence the quality of results. The only published documents supplying some standardization background are International Atomic Energy Agency (IAEA) Technical Reports No 260 (1986) and 405 (2001). Even they do not address crucial areas such as the organization of service laboratories and the need for quality assurance programmes. The significant role of biological dosimetry in many countries has proved the need for a standardized technique that is compatible with national radiological protection programmes. Thus, an International Standards Organization working group for the standardization of biological dosimetry by cytogenetics was created. This group comprises 13 scientists from 11 countries plus an IAEA representative. On the basis of a group consensus, a text defining minimal constraints on all the steps of the process was proposed. A working draft was submitted to ISO in 2001 and its structure is presented here.

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro.

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency. The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.

Hypersensitivity to camptothecin in MSH2 deficient cells is correlated with a role for MSH2 protein in recombinational repair.

DNA mismatch repair (MMR) corrects DNA polymerase insertion errors that have escaped proofreading in order to avoid the accumulation of deleterious mutations. While the role of MMR in the correction of replication errors is well established, its involvement in the processing of DNA damage induced by chemical and physical agents is less clear. A role for some of the MMR proteins, such as MSH2, in the repair of double strand break (DSBs) through recombination has also been envisaged. Why MMR- deficient cells are sensitive to agents causing replication fork stalling and thus DSBs remains unclear. To verify a possible role of MSH2 in homologous recombinational repair, we have treated cells from knockout mice for the MSH2 gene and mouse colorectal carcinoma cells also defective for MSH2 with different doses of camptothecin, an agent known to interfere with DNA replication. In the absence of MSH2, we found a reduced survival rate accompanied by higher levels of chromosomal damage and SCE induction. Furthermore, MSH2(-/-) cells displayed an elevated spontaneous RAD51 focus-forming activity and a higher induction of RAD51 foci following camptothecin treatment. Thus, the absence of MSH2 could result in both spontaneous DNA damage and uncontrolled recombination events leading to the observed higher yield of chromosomal damage and the higher induction of RAD51 foci following CPT treatment. Therefore, our results suggest an involvement of MSH2 in the early events leading to correct RAD51 relocalization after the formation of DSBs specifically produced at the blocked replication fork.

Effect of poly(ADP-ribosyl)ation and Mg2+ ions on chromatin structure revealed by scanning force microscopy.

Poly(ADP-ribosyl)ation of nuclear proteins is responsible for major changes in the high-order chromatin structure. The effects of this post-translation modification on nuclear architecture were examined at different Mg2+ concentrations using scanning force microscopy. A quantitative analysis of the internucleosomal distance, the width, and the volume of chromatin fibers imaged in tapping mode reveals that poly(ADP-ribosyl)ation induces a complete relaxation and decondensation of the chromatin structure. Our data, on the center-to-center distance between adjacent nucleosomes and on the fiber width, indicate that the poly(ADP-ribosyl)ated fibers remain significantly decondensed even in the presence of Mg2+. Our results also show that the Mg2+ assumes an important role in the folding of chromatin structure, but Mg2+ is not able to restore the native feature of chromatin, when the fibers are depleted of H1/H5 histones. The combined effect of post-translation modification and cation ions on the chromatin structure shows that poly(ADP-ribosyl)ation could promote accessibility to DNA even in those nuclear processes that require Mg2+.