The European intercomparison of in-vivo monitoring laboratories: the EIVIC-2020 project.

The EIVIC project was launched in 2020, and the main goal was the organisation of a European intercomparison of in-vivo monitoring laboratories dealing with direct measurements of gamma-emitting radionuclides incorporated into the body of exposed workers. This project was organised jointly by members of EURADOS Working Group 7 on internal dosimetry (WG7), the Federal Office for Radiation Protection (BfS, Germany) and the Radioprotection and Nuclear Safety Institute (IRSN, France). The objective was to assess the implementation of individual-monitoring requirements in EU Member States on the basis of in-vivo measurements and to gain insight into the performance of in-vivo measurements using whole-body counters. In this context, a total of 41 in-vivo monitoring laboratories from 21 countries, together with JRC (EC) and IAEA participated. The results were submitted in terms of activity (Bq) of the radionuclides identified inside phantoms that were circulated to all participants. The measured data were compared with reference activity values to evaluate the corresponding bias according to the standards ISO 28218 and ISO 13528. In general, the results of the different exercises are good, and most facilities are in conformity with the criteria for the bias and z-scores in the ISO standards. Furthermore, information about technical and organisational characteristics of the participating laboratories was collected to test if they had a significant influence on the reported results.

The consequences of the Odra flood (summer 1997) for the Odra lagoon and the beaches of Usedom: what can be expected under extreme conditions?

The exceptional flood of the river Odra in July/August 1997 caused severe damage, especially on the Polish side of the Odra valley. An additional 5 km3 of water were discharged during the flood. This represents about a third of the normal annual Odra discharge of 17 km3. Large agricultural and industrial areas were submerged, as well as towns and villages. However, as regards the Odra lagoon and the beaches of the Isle of Usedom, the substances transported, such as nutrients and pollutants, did not cause much damage, due to strong dilution. Hygienic investigations (human pathogenic bacteria and viruses) showed that the water had bathing quality during the whole flood.

When the body and appendage of the left atrium disagree: "Focal" atrial fibrillation-implications for atrial thrombus formation and risk of thromboembolism.

A case is presented of a man who had 5 hours of atrial fibrillation followed by spontaneous conversion and maintained sinus rhythm that persisted as shown by surface electrocardiography. Transesophageal echocardiography performed 24 hours after electrocardiographic conversion documented an atrial fibrillation pattern within the left atrial appendage, with a normal sinus Doppler pattern in the body of the left atrium. This apparent regional discrepancy in atrial function may partially explain the increased risk for "late" thromboembolism among patients with atrial fibrillation who appear to be successfully converted with sustained sinus rhythm.

Epigenetic transcriptional silencing and 5-azacytidine-mediated reactivation of a complex transgene in rice.

Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btt cryIIIA and bar transgenes in rice (Oryza sativa L.) that displayed aberrant segregation in R1 progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R1 plants had a DNA hybridization profile identical to that of the resistant R0 parent and R1 siblings. Genomic DNA analysis revealed substantial methylation of the Ubi1/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R2 progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubi1 promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

Characterization of a rice gene family encoding root-specific proteins.

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (> 70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5'-upstream sequence of RCg2 was translationally fused to a beta-glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5'-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.

Cyclophilins are encoded by a small gene family in rice.

cDNA clones were isolated and sequenced that encode two related but distinct rice cyclophilins, Cyp1 and Cyp2. The predicted amino acid sequences of each are 72% identical to human T-cell cyclophilin. Genomic DNA gel blot analysis suggests cyclophilins in rice are encoded by a small, 6-10-member gene family. Both Cyp1 and Cyp2 have seven extra amino acid residues in the N-terminal portion of the proteins that are not found in human or other non-plant cyclophilins, suggesting that this is a characteristic of plant cyclophilins. Cyp2 was expressed as 1000 nt transcripts in leaf and root tissues. Cyp1 was expressed as 800 and 900 nt transcripts. Whereas the 900 nt transcript was present in both root and leaf mRNA, the 800 nt transcript was only detectable in root mRNA. A genomic clone of Cyp2 was isolated, sequenced and shown to lack introns. A single transcriptional start site was identified 27 residues downstream of a putative TATA box. The 5' end of the transcript was shown to contain a region rich in adenyl residues (27 of 35). This region would not be conducive to secondary structure formation, which raises the possibility that Cyp2 might be preferentially translated during stress conditions.

Quantitation of chloramphenicol acetyl transferase in transgenic tobacco plants by ELISA and correlation with gene copy number.

A monoclonal antibody to chloramphenicol acetyl transferase (CAT) was used in an indirect competitive enzyme immunoassay (ELISA) for the quantitation of CAT in leaf extracts of eighteen transgenic tobacco plants containing the CAT gene fused to the cauliflower mosaic virus 35S promoter. The ELISA could be used to quantify CAT when present in extracts at 20 ng/ml. Enzymatic activity and electrophoretic mobility of CAT in these extracts was not different from CAT from Escherichia coli. Concentrations of CAT in these transgenic plants ranged from 79 to 732 ng CAT/mg protein. The average coefficient of variation among three replicate samples was 15%. All plants were sampled on two separate occasions. The CAT concentrations often varied between the two sampling dates. We determined the CAT gene copy number and the number of independently segregating loci in each plant by Southern blot analysis and progeny testing. We found no significant differences in CAT expression among all ten plants with a single CAT gene. We also found a significant correlation between CAT gene copy number and the level of CAT expressed in each plant, although plants with one gene copy sometimes had more CAT than plants with more than one gene copy. In this population, therefore, gene copy number contributed more to the variation in CAT expression than did position effects.

Direct analysis of RNA transcripts in electroporated carrot protoplasts.

We describe a method for direct analysis of RNA transcribed from DNA introduced into carrot cells by electroporation. Octopine synthase RNA transcribed from the plasmid p35SOcs was detected in total and poly A(+) RNA on Northern blots and in RNA protection assays. The highest level of octopine synthase transcript was detected at approximately 8 hrs post-electroporation, although RNA could still be detected after 48 hrs. This method allows detection of foreign gene expression in a plant system and bypasses the need for reporter genes.

Factors affecting PEG-mediated stable transformation of maize protoplasts.

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 1∶1 ratio to 65% at a 100∶1 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.

Specificity of Agrobacterium-mediated delivery of maize streak virus DNA to members of the Gramineae.

Parameters affecting the efficiency of agroinfection of maize streak virus (MSV) in maize have been determined. Monomeric units, cloned at a number of sites in the MSV genome were not infectious but multimeric units containing partial duplications were equally as infectious as complete tandem dimeric clones. Inoculation of tandem dimeric units conjugated into different strains of Agrobacterium showed that both A. tumefaciens and A. rhizogenes were able to transfer DNA to maize and this ability was Ti (or Ri) plasmid-specific. Nopaline strains of A. tumefaciens and both agropine and mannopine A. rhizogenes strains efficiently transferred MSV DNA to maize. A number of strains were capable of MSV DNA transfer to other members of the Gramineae, providing information which may be essential for Agrobacterium-mediated transformation of monocotyledonous plants.