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8 p.-3 fig.-3 tab., Background: PIWI-interacting RNAs (piRNAs) are an abundant single-stranded type of small non-coding RNAs (sncRNAs), which initially were discovered in gonadal cells, with a role as defenders of genomic integrity in the germline, acting against the transposable elements. With a regular size range of 21-35 nt, piRNAs are associated with a PIWI-clade of Argonaute family proteins. The most widely accepted mechanisms of biogenesis for piRNAs involve the transcription of longer precursors of RNAs to be processed, by complexes of proteins, to functional size, preferentially accommodating uridine residues at the 5’ end and 3’ methylation to increase the stability of these molecules. piRNAs have also been detected in somatic cells, with diverse potential functions, indicating their high plasticity and pleiotropic activity. Discovered about two decades ago, piRNAs are a large and versatile type of sncRNA and that remain insufficiently identified and analyzed, through next-generation sequencing (NGS), to evaluate their processing, functions, and biogenesis in different cell types and during development. piRNAs’ distinction from other sncRNAs has led to controversial results and interpretation difficulties when using existing databases because of the heterogeneity of the criteria used in making the distinction., Description: We present “piRNA-IPdb”, a database based uniquely on datasets obtaining after the defining characteristic of piRNAs: those small RNAs bound to PIWI proteins. We selected and analyzed sequences from piRBase that exclusively cover the binding to PIWI. We pooled a total of 18,821,815 sequences from RNA-seq after immunoprecipitation that included the binding to any of the mouse PIWI proteins (MILI, MIWI, or MIWI2)., Conclusions: In summary, we present the characteristics and potential use of piRNA-IPdb database for the analysis of bona fide piRNAs., This study was supported by the Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades (BFU2017-87095-R), Spain. Miguel A. Brieño-Enriquez was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (R00HD090289) and the Magee Auxiliary Research Scholar (MARS) endowment.

14 p.-4 fig.-1 tab., Although mitochondria are widely studied organelles, the recent interest in the role of mitochondrial small noncoding RNAs (sncRNAs), miRNAs, and more recently, piRNAs, is providing new functional perspectives in germ cell development and differentiation. piRNAs (PIWI-interacting RNAs) are single-stranded sncRNAs of mostly about 20–35 nucleotides, generated from the processing of pre-piRNAs. We leverage next-generation sequencing data obtained from mouse primordial germ cells and somatic cells purified from early-differentiating embryonic ovaries and testis from 11.5 to 13.5 days postcoitum. Using bioinformatic tools, we elucidate (i) the origins of piRNAs as transcribed from mitochondrial DNA fragments inserted in the nucleus or from the mitochondrial genome; (ii) their levels of expression; and (iii) their potential roles, as well as their association with genomic regions encoding other sncRNAs (such as tRNAs and rRNAs) and the mitochondrial regulatory region (D-loop). Finally, our results suggest how nucleo-mitochondrial communication, both anterograde and retrograde signaling, may be mediated by mitochondria-associated piRNAs., This study was supported by the Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades (BFU2017-87095-R), Spain. Miguel A. Brieño-Enriquez was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (R00HD090289) and the Magee Auxiliary Research Scholar (MARS) endowment.

57 p.-12 fig.-6 tab., EFSA was asked by the European Commission to provide information on the levels of domoic acid (DA) in whole scallops that would ensure that levels in edible parts are below the regulatory limit after shucking. This should include five species of scallops. In addition, EFSA was asked to recommend the number of scallops to be used in an analytical sample. To address these questions, EFSA received suitable data on DA for only one scallop species, Pecten maximus, i.e. data on pooled samples of edible and non-edible parts. A large part of the concentration levels was above the limit of quantification (LOQ) and only these data were used for the assessment. Shucking in most cases resulted in a strong decrease in the toxin levels. Statistical analysis of the data showed that levels in whole scallops should not exceed 24 mg DA/kg, 59 mg DA/kg and 127 mg DA/kg to ensure that levels in, respectively, gonads, muscle and muscle plus gonads are below the regulatory limit of 20 mg DA/kg with 99% certainty. Such an analysis was not possible for the other scallop species. In the absence of data from member states, published data of variations between scallops were used to calculate the sample size to ensure a 95% correct prediction on whether the level in scallops in an area or lot is correctly predicted to be compliant/non-compliant. It was shown that 10 scallops per sample would be sufficient to predict with 95% certainty if DA levels in the area/lot were twofold below or above the regulatory limit for the highest reported coefficient of variance (CV) of 1.06. To predict with 95% certainty for levels between 15 and 27 mg DA/kg, a pooled sample of more than 30 scallops would have to be tested.

12 p.-2 fig.-2 tab., Following a request from the European Commission, the EFSA Panel on Contaminants in the Food Chain (CONTAM) provided a scientific opinion on an application for a detoxification process of groundnut press cake for aflatoxins by ammoniation. Specifically, it is required that the feed decontamination process is compliant with the acceptability criteria specified in the Commission Regulation (EU) 2015/786 of 19 May 2015. The CONTAM Panel assessed the data provided by the feed business operator with respect to the efficacy of the process to remove the contaminant from groundnut press cake batches and on information demonstrating that the process does not adversely affect the characteristics and the nature of the product. Although according to the literature the process may be able to reduce aflatoxin levels below the legal limits, the Panel concluded that the proposed decontamination process, on the basis of the experimental data submitted by the feed business operator, cannot be confirmed for compliance with the acceptability criteria provided for in Commission Regulation (EU) 2015/786 of 19 May 2015. The Panel recommended sufficient sample testing before and after the process, under the selected conditions, to ensure that the process is reproducible and reliable and to demonstrate that the detoxification is not reversible. In addition, genotoxicity testing of extracts of the treated feedingstuff and of the identified degradation products would be necessary. Finally, information on the transfer rate of AFB1 to AFM1 excretion in milk for animals fed the ammoniated product, in comparison to the starting material and on the ammoniation process changes of the nutritional values of the feed material should be provided., The Panel wishes to thank Federico Cruciani and Carina Wenger for the support provided to this scientific output, and the hearing expert Professor Dr Wayne L Bryden, for the overview on aflatoxin inactivation by ammoniation.

130 p.-4 fig.-23 tab.-Appendix A-D (118-129), The European Commission asked EFSA to update its 2011 risk assessment on hexabromocyclododecanes (HBCDDs) in food. HBCDDs, predominantly mixtures of the stereoisomers a-, b- and c-HBCDD, were widely used additive flame retardants. Concern has been raised because of the occurrence of HBCDDs in the environment, food and in humans. Main targets for toxicity are neurodevelopment, the liver, thyroid hormone homeostasis and the reproductive and immune systems. The CONTAM Panel concluded that the neurodevelopmental effects on behaviour in mice can be considered the critical effects. Based on effects on spontaneous behaviour in mice, the Panel identified a lowest observed adverse effect level (LOAEL) of 0.9 mg/kg body weight (bw) as the Reference Point, corresponding to a body burden of 0.75 mg/kg bw. The chronic intake that would lead to the same body burden in humans was calculated to be 2.35 lg/kg bw per day. The derivation of a health-based guidance value (HBGV) was not considered appropriate. Instead, the margin of exposure (MOE) approach was applied to assess possible health concerns. Over 6,000 analytical results for HBCDDs in food were used to estimate the exposure across dietary surveys and age groups of the European population. The most important contributors to the chronic dietary LB exposure to HBCDDs were fish meat, eggs, livestock meat and poultry. The CONTAM Panel concluded that the resulting MOE values support the conclusion that current dietary exposure to HBCDDs across European countries does not raise a health concern.An exception is breastfed infants with high milk consumption, for which the lowest MOE values may raise a health concern.

66 p.-22 fig.-18 tab-Appendix A-B (45-65), EFSA was asked by the European Commission to provide information on levels of lipophilic shellfish toxins in whole scallops that would ensure levels in edible parts below the regulatory limits after shucking, i.e. removal of non‐edible parts. This should include the okadaic acid (OA), the azaspiracid (AZA) and the yessotoxin (YTX) groups, and five species of scallops. In addition, EFSA was asked to recommend the number of scallops in an analytical sample. To address these questions, EFSA received suitable data on the three toxin groups in two scallop species, Aequipecten opercularis and Pecten maximus, i.e. data on individual and pooled samples of edible and non‐edible parts from contamination incidents. The majority of the concentration levels were below limit of quantification (LOQ)/limit of detection (LOD), especially in adductor muscle but also in gonads. Shucking in most cases resulted in a strong decrease in the toxin levels. For Pecten maximus, statistical analysis showed that levels in whole scallops should not exceed 256 μg OA eq/kg or 217 μg AZA1 eq/kg to ensure that levels in gonads are below the regulatory limits of 160 μg OA or AZA1 eq/kg with 99% certainty. Such an analysis was not possible for yessotoxins or any toxin in Aequipecten opercularis and an assessment could only be based on upper bound levels. To ensure a 95% correct prediction on whether the level in scallops in an area or lot is correctly predicted to be compliant/non‐compliant, it was shown that 10 scallops per sample would be sufficient to predict with 95% certainty if levels of OA‐group toxins in the area/lot were 25% below or above the regulatory limit. However, to predict with a 95% certainty for levels between 140 and 180 μg OA eq/kg, a pooled sample of more than 30 scallops would have to be tested.

101 p.-5 fig.-16 tab., The European Commission asked EFSA to update its previous Opinion on nickel in food and drinking water, taking into account new occurrence data, the updated benchmark dose (BMD) Guidance and newly available scientific information. More than 47,000 analytical results on the occurrence of nickel were used for calculating chronic and acute dietary exposure. An increased incidence of post‐implantation loss in rats was identified as the critical effect for the risk characterisation of chronic oral exposure and a BMDL10 of 1.3 mg Ni/kg body weight (bw) per day was selected as the reference point for the establishment of a tolerable daily intake (TDI) of 13 μg/kg bw. Eczematous flare‐up reactions in the skin elicited in nickel‐sensitised humans, a condition known as systemic contact dermatitis, was identified as the critical effect for the risk characterisation of acute oral exposure. A BMDL could not be derived, and therefore, the lowest‐observed‐adverse‐effect‐level of 4.3 μg Ni/kg bw was selected as the reference point. The margin of exposure (MOE) approach was applied and an MOE of 30 or higher was considered as being indicative of a low health concern. The mean lower bound (LB)/upper bound (UB) chronic dietary exposure was below or at the level of the TDI. The 95th percentile LB/UB chronic dietary exposure was below the TDI in adolescents and in all adult age groups, but generally exceeded the TDI in toddlers and in other children, as well as in infants in some surveys. This may raise a health concern in these young age groups. The MOE values for the mean UB acute dietary exposure and for the 95th percentile UB raises a health concern for nickel‐sensitised individuals. The MOE values for an acute scenario regarding consumption of a glass of water on an empty stomach do not raise a health concern., The Panel wishes to thank the following for the support provided to this scientific output: Elena Rovesti. The Panel wishes to acknowledge all European competent institutions,Member State bodies and other organisations that provided data for this scientific output.

13 p.-5 fig., Vinclozolin is a pesticide with antiandrogenic activity as an endocrine disruptor compound. Its effects upon the progression of primordial follicles were assessed in cultures of mouse fetal ovaries from the onset of meiotic differentiation of germ cells (13.5days post coitum) and from both in vivo exposed mice and in vitro exposed ovaries. Exposure of ovaries to vinclozolin-at in vitro dosages ranging from 10 to 200muM and in 3D ex vivo culture following in vivo exposure to 50mg/kg bw/day-showed delays in meiocyte differentiation and in follicle growth, even at the lowest in vitro dose exposure. Immunofluorescent analysis showed the presence of the proteins MSY2 and NOBOX in the primary follicles but no difference in the level of protein signals or in the number of follicles in relation to treatment. However, assessing the cytological differentiation of germ cells by detecting the synaptonemal complex protein SYCP3, the exposure to vinclozolin delayed meiotic differentiation from both in vitro- and in vivo-exposed ovaries. These effects were concomitant with changes in the energy metabolism, detected as a relative increase of glycolytic metabolism in live-cell metabolic assays in exposed ovaries., This study was supported by the Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades(BFU2017-87095-R), Spain. Miguel A. Brieño-Enriquez was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (R00HD090289) and the Magee Auxiliary Research Scholar (MARS) endowment.

44 p.-8 fig.-3 tab.-4 fig. supl.-1 tab. supl., piRNAs are small non-coding RNAs known to play a main role in defence against transposable elements in germ cells. However, other potential functions, such as biogenesis and differences in somatic and germline expression of these regulatory elements, are not yet fully unravelled. Here, we analysed a variety of piRNA sequences detected in mouse male and female primordial germ cells (PGCs) and gonadal somatic cells at crucial stages during embryonic differentiation of germ cells (11.5–13.5 days post-coitum). NGS of sncRNA and bioinformatic characterization of piRNAs from PGCs and somatic cells, in addition to piRNAs associated with TEs, indicated functional diversification in both cell types. Differences in the proportion of the diverse types of piRNAs are detected between somatic and germline during development. However, the global diversified patterns of piRNA expression are mainly shared between germ and somatic cells, we identified piRNAs related with molecules involved in ribosome components and translation pathway, including piRNAs derived from rRNA (34%), tRNA (10%) and snoRNA (8%). piRNAs from both tRNA and snoRNA are mainly derived from 3ʹ and 5ʹ end regions. These connections between piRNAs and rRNAs, tRNAs or snoRNAs suggest important functions of specialized piRNAs in translation regulation during this window of gonadal development., This study was supported by the Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades (BFU2017-87095-R), Spain. ; ; M.A.B-E was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development [4R00HD090289-03] and the Magee Auxiliary Research Scholar (MARS).

190 p.-8 fig.-47 tab.-7 appendix tab., The European Commission asked EFSA for a scientific opinion on the risks for animal and human health related to the presence of glycoalkaloids (GAs) in feed and food. This risk assessment covers edible parts of potato plants and other food plants containing GAs, in particular, tomato and aubergine. In humans, acute toxic effects of potato GAs (a-solanine and a-chaconine) include gastrointestinal symptoms such as nausea, vomiting and diarrhoea. For these effects, the CONTAM Panel identified a lowest-observed-adverse-effect level of 1 mg total potato GAs/kg body weight (bw) per day as a reference point for the risk characterisation following acute exposure. In humans, no evidence of health problems associated with repeated or long-term intake of GAs via potatoes has been identified. No reference point for chronic exposure could be identified from the experimental animal studies. Occurrence data were available only for a-solanine and a-chaconine, mostly for potatoes. The acute dietary exposure to potato GAs was estimated using a probabilistic approach and applying processing factors for food. Due to the limited data available, a margin of exposure (MOE) approach was applied. The MOEs for the younger age groups indicate a health concern for the food consumption surveys with the highest mean exposure, as well as for the P95 exposure in all surveys.For adult age groups, the MOEs indicate a health concern only for the food consumption surveys with the highest P95 exposures. For tomato and aubergine GAs, the risk to human health could not be characterised due to the lack of occurrence data and the limited toxicity data. For horses, farm and companion animals, no risk characterisation for potato GAs could be performed due to insufficient data on occurrence in feed and on potential adverse effects of GAs in these species.

220 p.-4 fig.-37 tab.-15 fig. compl.-16 tab. compl.-2 tab. append., The European Commission asked EFSA for a scientific opinion on the risks for animal and human health related to the presence of chlorinated paraffins in feed and food. The data for experimental animals were reviewed and the CONTAM Panel identified the liver, kidney and thyroid as the target organs for the SCCP and MCCP mixtures tested in repeated dose toxicity studies. Decreased pup survival and subcutaneous haematoma/haemorrhage were also identified as critical effects for an MCCP mixture. For the LCCP mixtures tested, the liver was identified as the target organ. The Panel selected as reference points a BMDL10 of 2.3 mg/kg bw per day for increased incidence of nephritis in male rats, and of 36 mg/kg bw per day for increased relative kidney weights in male and female rats for SCCPs and MCCPs, respectively. For LCCPs, a reference point relevant for humans could not be identified. Due to the limitations in the toxicokinetic and toxicological database, the Panel concluded that derivation of a health-based guidance value was not appropriate. Only limited data on the occurrence of SCCPs and MCCPs in some fish species were submitted to EFSA. No data were submitted for LCCPs. Thus, a robust exposure assessment and consequently a complete risk characterisation could not be performed. A preliminary risk characterisation based only on the consumption of fish was performed, and the calculated margins of exposure suggested no health concern for this limited scenario. The Panel noted that dietary exposure will be higher due to the contribution of CPs from other foods. The Panel was not able to identify reference points for farm animals, horses and companion animals. No occurrence data for feed were submitted to EFSA.Therefore, no risk characterisation could be performed for any of these animal species., The Panel wishes to thank the hearing expert: Kerstin Krätschmer and EFSA staff member: Kelly Niermans for the support provided to this scientific output. The CONTAM Panel acknowledges all European competent institutions and other stakeholders that provided occurrence data in food and human milk and data on the toxicity of CPs, and supported the data collection for the Comprehensive European Food Consumption Database.

150 p.-15 fig.-37 tab., The European Commission asked EFSA to update their 2006 opinion on ochratoxin A (OTA) in food. OTA is produced by fungi of the genus Aspergillus and Penicillium and found as a contaminant in various foods. OTA causes kidney toxicity in different animal species and kidney tumours in rodents. OTA is genotoxic both in vitro and in vivo; however, the mechanisms of genotoxicity are unclear. Direct and indirect genotoxic and non-genotoxic modes of action might each contribute to tumour formation. Since recent studies have raised uncertainty regarding the mode of action for kidney carcinogenicity, it is inappropriate to establish a health-based guidance value (HBGV) and a margin of exposure (MOE) approach was applied. For the characterisation of non-neoplastic effects, a BMDL10 of 4.73 lg/kg body weight (bw) per day was calculated from kidney lesions observed in pigs. For characterisation of neoplastic effects, a BMDL10 of 14.5 lg/kg bw per day was calculated from kidney tumours seen in rats. The estimation of chronic dietary exposure resulted in mean and 95th percentile levels ranging from 0.6 to 17.8 and from 2.4 to 51.7 ng/kg bw per day, respectively. Median OTA exposures in breastfed infants ranged from 1.7 to 2.6 ng/kg bw per day, 95th percentile exposures from 5.6 to 8.5 ng/kg bw per day in average/high breast milk consuming infants, respectively. Comparison of exposures with the BMDL10 based on the non-neoplastic endpoint resulted in MOEs of more than 200 in most consumer groups, indicating a low health concern with the exception of MOEs for high consumers in the younger age groups, indicating a possible health concern. When compared with the BMDL10 based on the neoplastic endpoint, MOEs were lower than 10,000 for almost all exposure scenarios, including breastfed infants. This would indicate a possible health concern if genotoxicity is direct. Uncertainty in this assessment is high and risk may be overestimated.

3 p.-2 fig., Jesús del Mazo Martínez recibe fondos de la Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades (BFU2017-87095-R).

50 p.-6 fig.-1 tab., The increase in male idiopathic infertility has been associated with daily exposure to endocrine disruptors chemicals (EDCs). Nevertheless, the mechanisms of action in relation to dysregulating proteins and regulatory microRNAs are unknown.We combined proteomic and miRNome analyses of mouse testis chronically exposed to low doses of a define mixture of EDCs [phthalates: bis (2-ethylhexyl), dibutyl and benzyl-butyl; 4-nonylphenol and 4-tert-octylphenol], administered in the drinking water from conception until adulthood (post-natal day 60/75) and compared them with no-exposed control mice.We analysed fertility parameters and global changes in the patterns of mice testis proteome by 2D-electrophoresis/mass spectrometry, along with bioinformatic analyses of dysregulated microRNAs, and their association with published data in human infertile patients.We detected a decrease in the potential fertility of exposed mice associated with changes in the expression of 18 proteins (10 up-regulated, 8 down-regulated). Functional analysis showed that 89% were involved in cell death. Furthermore, we found a group of 23 microRNAs/isomiRs (down-regulated) correlated with six of the up-regulated target proteins (DIABLO, PGAM1, RTRAF, EIF4E, IVD and CNDP2). Regarding this, PGAM1 up-regulation was validated by Western blot and mainly detected in Sertoli cells. Some of these microRNA/protein dysregulations were reported in human testis with spermatogenic failure.Overall, a chronic exposure to EDCs mixture in human males could potentially lead to spermatogenic failure through changes in microRNA expression, which could post-transcriptionally dysregulate mRNA targets that encode proteins participating in cell death in testicular cells. Finally, these microRNA/protein dysregulations need to be validated with other EDCs mixtures and concentrations., This work was supported by grants from FONDECYT 1150352 to R.D.M., FONDECYT 3160273 to P.U-M and CONICYT 21120505 to J.B., Chile, and MINECO BFU2013-42164-R and BFU2017-87095-R to J. d. M., Spain

113 p.-8 fig.-32 tab., The European Commission asked EFSA for a scientific opinion on the risks for animal and human health related to the presence of quinolizidine alkaloids (QAs) in feed and food. This risk assessment is limited to QAs occurring in Lupinus species/varieties relevant for animal and human consumption in Europe (i.e. Lupinus albus L., Lupinus angustifolius L., Lupinus luteus L. and Lupinus mutabilis Sweet). Information on the toxicity of QAs in animals and humans is limited. Following acute exposure to sparteine (reference compound), anticholinergic effects and changes in cardiac electric conductivity are considered to be critical for human hazard characterisation. The CONTAM Panel used a margin of exposure (MOE) approach identifying a lowest single oral effective dose of 0.16 mg sparteine/kg body weight as reference point to characterise the risk following acute exposure. No reference point could be identified to characterise the risk of chronic exposure. Because of similar modes of action for QAs, the CONTAM Panel used a group approach assuming dose additivity. For food, the highest mean concentration of Total QAs (TotQAs) (i.e. the 6 most abundant QAs) was found in lupin seed samples classified as ‘Lupins (dry) and similar-’. Due to the limited data on occurrence and consumption, dietary exposure was calculated for some specific scenarios and no full human health risk characterisation was possible. The calculated margin of exposures (MOEs) may indicate a risk for some consumers. For example, when lupin seeds are consumed without a debittering step, or as debittered lupin seeds high in QA content and when ‘lupin-based meat imitates’ are consumed. For horses, companion and farm animals, other than salmonids, the available database on adverse effects was too limited to identify no-observed-adverse-effect levels and/or lowest-observed-adverse-effect levels and no risk characterisation was possible. For salmonids, the CONTAM Panel considers the risk for adverse effects to be low., The Panel wishes to thank the following for the support provided to this scientific output: Giovanna Boschin and Elena Rovesti. The Panel wishes to acknowledge all European competent institutions, Member State bodies and other organisations that provided data for this scientific output.

13 p.-7 fig.-5 tab., Piwi-interacting RNAs (piRNAs) are a subclass of the small non-coding RNAs (sncRNAs). Their main reported function was to exert control over transposable elements (TEs) in mammalian germline. In this study undertaking a deeper bioinformatics analysis of piRNAs present in mouse oocytes, sperm cells and zygotes, we first elaborated a new piRNA database based on sequences identified as piRNAs by immunoprecipitation with PIWI proteins. Our bioinformatics analysis revealed that, at least in gametes and zygotes, piRNAs could encompass multifunctional cell-dependent regulatory molecules. Indeed, genome analysis of the piRNA mapping density (reads/kb) evidenced in all samples an enrichment of intron-derived piRNAs. Further, piRNA population was classified into sequences not associated to TEs or repeats (NRapiRNAs) and associated to repetitive genome elements (RapiRNAs). In oocytes most of the NRapiRNAs mapped to the 5′UTRs of coding mRNAs, while higher proportion of NRapiRNAs was detected in sperm cells associated to the 3′UTRs of mRNAs. This piRNA complementarity to mRNA UTRs suggests key post-transcriptional regulatory roles over mRNAs such as those encoding MHC genes. In addition, a striking association of RapiRNA with long non-coding RNAs (lncRNAs) was identified. piRNAs associated with relevant lncRNAs such as: Rab26os and GAS5 and key mRNAs, were particularly assessed., This research was supported by grants from the Ministerio de Ciencia Innovación y Universidades (BFU2013-42164-R and BFU2017-87095-R), Spain.

4 p.-4 fig., Ran is a small GTP-binding protein involved in several essential roles for cell viability. This observation implies Ran might be ubiquitously expressed during development. However, Ran shows a differentiated expression pattern that is restricted to specific tissues from embryo to adult. At early embryonic stages of mouse development we found persistent Ran expression in proliferating neural tissue, neural crest derived dorsal root ganglions and sensory pits. We also showed an accumulation of Ran transcripts in main embryonic haematopoietic tissues: blood islands first and then hepatic bud. In advanced stages of development Ran is also expressed in other tissues showing a high cell turnover., This work wassupported by grants from the CICYT: (BMC2000-1127) to J.M. and (PM-97-0138) to D.B.K.

9.-5 fig., Murine erythroleukemia (MEL) cells undergo erythroid differentiation in vitro when treated with hexamethylene bisacetamide (HMBA).To identify genes involved in the commitment of MEL cells to differentiate, we screened a cDNA library constructed from HMBA-inducedcells by differential hybridization and isolated GTPaseRanas a down-regulated gene. We observed thatRanwas expressed in a biphasicmode. Following a decrease in mRNA level during the initial hours of induction,Ranre-expressed at 24–48 h, and gradually declinedagain. To investigate the role ofRanduring MEL differentiation we constructed MEL transfectants capable to express or blockRanmRNAproduction constitutively. No effects were observed on cell growth and proliferation. Blockage of Ran, however, interfered with MEL celldifferentiation resulting in a decrease of cell survival in the committed population., Thiswork was supported by grant PM97-0138 (Spanish Ministry of Education), grant 08.1/0067.1/2001.1 (CAM, Regional Government of Madrid) and grant 99/0850 (FIS, Spanish Ministry of Health). JM was supported by grants from DGI-CYT (PB95-0119), EU (PL-960183) and CAM (07B/0022).

6 p.-3 fig.-1 tab., Replacement-variant H3.3 histones have been isolated and sequenced in different eukaryotes, but no functional H3.3A gene has been characterized in the mouse so far. We have cloned an H3.3A cDNA from a mouse fetal ovary library, differentially screened with testis versus somatic cDNA probes. We showed this gene contains a region homologous to the reverse and complementary alpha-globin gene. We believe such a structure could have been generated by retroposition during the evolution of both globin and histone gene families. The sequence coding for H3.3A is 76.6% homologous to the mouse H3.3B gene at the nucleotide level and differs in only one amino acid at the protein level. The high degree of homology between these genes and the H3.3 variant histones from other eukaryotes reveals the conservation of these replication-independent class of histones throughout evolution. Analysis of gene expression reveals a developmental regulation concurrent with meiotic progression, with the highest level of transcript detection coincident with meiotic onset during both oogenesis and spermatogenesis., These studies were supported by grants from the DGICYT (PB92-0063) and CAM (C077/91) to J.M. and from FIS (9301/61) and CAM (AE 00118/94).

4 p.-5 fig., Two isoforms of Ran-GTPase have been described: Ran/M1 and Ran/M2. Ran/M2 is testis specific, whereas the Ran/M1 isoform is also expressed in somatic tissues. Here we show that both mRNAs, differing in 35 of the 648 nucleotides included in the ORFs, are developmentally regulated during spermatogenesis. Real-time RT-PCR experiments demonstrated that the expression of Ran/M1 and Ran/M2 increased in pachytene spermatocytes with progressive transcript accumulation until they reached the round spermatid stage, in the seminiferous epithelium of adults. In the testis, the expression of both isoforms was found to be restricted to germ cells. An expression window from early pachytene spermatocytes to late round spermatids was detected by in situ hybridization., This work was supported by grants from the CICYT (BMC2000-1127), EU (QLK4-C7- 2002-02403) and PROFIT (FIT-010000-2002-43) to J.M. and (PM-97-0138) to D.B. K.

5 p.-8 fig., Cloning and characterization of H3.3A variant histone expression has recently been reported to be associated with meiotic development in mouse testis and ovary. Using Northern analysis and in situ hybridization, the pattern of H3.3A expression was studied during the development of different tissues. In addition to the differential expression detected in male and female meiosis, H3.3A was found to be highly expressed in preantral follicles of adult ovaries and in the basal regions of seminiferous epithelium corresponding to spermatogonia. Different patterns of expression were observed in somatic tissues, which also differed with respect to the developmental stage of the tissue. The lowest expression was detected in adult skeletal muscle. High expressions were found in foetal liver and spinal cord. These different expressions might reflect a possible function of H3.3A in cell differentiation as detected in MEL cells., This work was supported by grants from the DGICYT (PB92-0063,PB9S-0119) and The European Commission PL 960183 to JM. and from the FIS (93/0161) and CAM (AE 001 18/94) to DK.