The surface detector of the Pierre Auger Observatory is a 1600 water Cherenkov tank array on a triangular 1.5 km grid. The signals from each tank are read out using three 9'' photomultipliers and processed at a sampling frequency of 40MHz, from which a local digital trigger efficiently selects shower candidates. GPS signals are used for time synchronization and a wireless communication system connects all tanks to the central data acquisition system. Power is provided by a stand-alone solar panel system. With large ambient temperature variations, that can reach over 20 degrees in 24 hours, high salinity, dusty air, high humidity inside the tank, and remoteness of access, the performance and reliability of the array is a challenge. Several key parameters are constantly monitored to ensure consistent operation. The Surface Array has currently over 750 detectors and has been in reliable operation since January 2004. Good uniformity in the response of different detectors and good long term stability is observed.

A computer-assisted assay based on the quantitative analysis of DNA methylation in individual interphase nuclei by indirect immunolabelling with anti-5-methylcytosine antibodies was recently developed in our laboratory. In situ analyses were performed on individual nuclei from normal and experimentally hypo- or hypermethylated cultured cells as well as on human peripheral blood B-lymphocytes from normal and chronic lymphoid leukemia (CLL) samples. We present the results obtained on cells from patients affected by different degrees of preneoplastic or neoplastic changes of the uterine cervix as compared to normal controls. The analysis of DNA methylation in individual cells from cytofuge samples was performed as follows: within each nucleus the eu- and heterochromatin methylation levels were quantified in the grey scale range by dedicated software in terms of numbers, areas and optical densities (ODs) of the immunolabeled dense heterochromatic regions ("spots"), and of the optical density of nuclear background, i.e., of nuclear euchromatin. Analogously, in randomly chosen microscope fields of tissue sections from paraffin-embedded samples, progressive tissue demethylation was observed in dysplastic and cancer cells as compared to normal ones. Both methods showed significant and progressive DNA hypomethylation in dysplastic and cancer cells as compared to control specimens.

The Pierre Auger Observatory will study cosmic rays with energy exceeding 10(19) eV. The experiment will measure properties of extensive air showers with a hybrid detector consisting of a surface array and an atmospheric fluorescence telescope.This paper presents the characteristics and performance of the optical system for the fluorescence detector prototype. The system adopts a Schmidt camera design with a diaphragm to remove coma aberration. A large spherical mirror (approximately 3.6 x 3.6 in, with radius of curvature 3.47 in), segmented in 49 trapezoidal elements, is supported by a mechanical structure, where segments are positioned on the meridians of the spherical cap. At the diaphragm, an absorption filter is installed to preferentially transmit the nitrogen fluorescence light.The construction technique, image characteristics, and results of tests performed on the system are discussed.

We present the characteristics and performance of the optical system for the fluorescence detector prototype of the Pierre Auger Project. The system adopts a Schmidt camera design with a mirror collecting, on a spherical surface, the light incoming through a limited aperture diaphragm. An absorption filter is installed in the diaphragm to preferentially trasmit the nitrogen fluorescence light.

The diagnostic management of solitary thyroid nodules or dominant nodules in a multinodular goiter is still controversial. Although different imaging and laboratory examinations are available, often the decision to submit a patient to surgery relies upon the surgeon's individual clinical judgement. Fine needle aspiration biopsy (FNAB) is probably the most commonly performed diagnostic procedure, but its value is hampered by a low but definite rate of indeterminate or false-negative results. A possible way to enhance the diagnostic accuracy of thyroid cytology is to consider also cytogenetic and molecular features in addition to the usual morphological findings. Thyroid-specific transcription factors like PAX8 playa decisive role in the determination of cellular phenotype, activating thyroglobulin, thyroperoxidase and thyrotropin receptor gene transcription. Recent reportsl-? have shown that the expression of thyroidspecific genes is lost in cells from follicular, papillary and anaplastic tumors. Furthermore, a chromosomal translocation t(2;3)(q13;p25) resulting in fusion of the PAX8 binding domains to domains A to F of the peroxisome proliferator-activated receptor (PPAR) gamma 1 has been found in follicular carcinomas but not in follicular adenomas or papillary carcinomas, suggesting an oncogenic role for the PAX8-PPAR gamma 1 fusion protein", Finally, an overexpression of P53 and Bcl2 genes has been reported in anaplastic tumors'. DNA methylation and acetylation profiles are altered during neoplastic progression in many lymphomas and leukemias and in some solid tumors". Hence, the correlation of the differentiation phenotype with various molecular markers and with DNA methylation would help the understanding of thyroid carcinogenesis as well as combine and direct all the reported cytogenetic approaches to a more precise clinical diagnosis. The aim of this preliminary study was to evaluate DNA methylation levels and PAX8, P53 and Bcl2 expression on cytologic smears from resected thyroids and correlate the findings to clinical diagnosis.

32D is an IL-3-dependent mast cell line whose cells are often defined multipotent because they generate at low frequency (1 every 105 cells) cells which dependent for growth on factors other than IL-3. using growth factor as selective pressure, differentiation mutant cell lines were isolated from the 32D cells which were dependent on either erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF) or granulocyte-monocyte colony stimulating factor (GM-CSF) and had, consistently, either erythroid, granulocytic or monocytic phenotype. The 32D cell line, but none of its subclones, express Mpl (the receptor for thrombopoietin, TPO) by RT-PCR and clone at low frequency in response to TPO under serum-deprived conditions. Using TPO as selective pressure, TPO-dependent subclones were isolated and characterized. In contrast with the diploid karyotype of the original 32D cells, the TPO-dependent subclones have a nearly tetraploid karyotype (model chromosome number 66). A fraction of the TPO-dependent cells (5–10%) have twice the normal cell size and appear multinucleated although, when carefully analyze,d all the nuclei are interconnected. These giant cells have a chromosome number higher than 120. The 32D TPO cells express high levels of GpIIb–GpIIIa on their surface and release pro-platelets in the supernatant. These cells express high levels of megakaryocytic genes (Mpl, GpIIb and von Willebrand factor) and do not express detectable levels of erythroid (EPO receptor, α and β-globin) genes by RT-PCR. Interestingly, the previously isolated erythroid 32D Epo cell lines did not express megakaryocytic gene detectable by RT-PCR. In conclusion, the newly isolated differentiation mutant 32D TPO cells are dependent for growth on TPO and have a pure megakaryocyte morphology. Since 32D TPO and 32D EPO are the only cell lines isolated up to now which are “pure” respectively, for the megakaryocytic and erythroid lineages, they may represent a valuable tool to dissect lineage-specific events in the megakaryocytic and erythroid pathway.

The Fluorescence Detector of the Pierre Auger Cosmic Ray Observatory will provide a measurement of the parameters of Extensive Air Showers with energy >10/sup 19/ eV. The mirror of the Fluorescence Detector prototype has dimension /spl sim/3.6/spl times/3.6 m/sup 2/ and is composed of 49 trapezoidal segments of approximately 50/spl times/50 cm/sup 2/. Each segment is curved using a steel mold in which the glass sags in a controlled environment. The glass is then aluminized under vacuum and coated with oxide (Al/sub 2/O/sub 3/). The oxide thickness is chosen to maximize the reflectivity in the wavelength range of fluorescence light (300 nm </spl lambda/<400 nm). The 49 mirror segments plus 10 spare ones are now ready for installation. In this paper we discuss the production technique of the segments for the Torino prototype mirror.

Background-Global hypomethylation of DNA is frequently observed in human tumours. This alteration is detected in early adenomas in colorectal tumorigenesis. Information is currently acquired after extraction of DNA from tissues, digestion with nucleases, and analysis by reverse phase chromatography, or treatment with restriction enzymes followed by gel electrophoresis analysis and Southern hybridisation with radiolabelled probes.Aims-The purpose of our work was to evaluate the global methylation status of DNA in malignant lesions without loosing the histopathological features of the samples.Patients-The investigation was performed on paired normal-tumour tissues from 13 patients undergoing surgical resection of colorectal adenocarcinomas.Methods-Antibodies raised against 5-methylcytidine can be used to label methyl rich regions in interphase nuclei. This technique was adapted to the study of paraffin embedded tissues and an immunohistochemical method was developed to assess the global methylation status of individual nuclei while preserving cell morphology and tissue architecture. Computer assisted quantification of the staining intensity was performed on malignant and normal zones of human colon tissues to test the correlation between the immunolabelling signal and the respective histological patterns observed.Results-Qualitative and quantitative differences were observed and measured between the normal and malignant part of each sample. Morphologically altered nuclei displayed densely labelled spots within faintly labelled areas whereas normal nuclei were darker and uniformly stained. Image analysis allowed calculation of the average integrated optical density of the nuclei in both types of tissues, demonstrating a constant and significantly lower intensity for the former type of cells.

In tumors, DNA is often globally hypomethylated compared to DNA extracted from normal tissues. This observation is usually made after extraction and exhaustive digestion of DNA followed by analysis of nucleosides by chromatography or digestion with restriction enzymes, gel analysis, and hybridization. This approach provides an average value which does not give information on the various cell subpopulations included in heterogeneous samples. Therefore an immunochemical technique was set up with the aim of demonstrating, in a population of mixed cells, the possibility of detecting the presence of individual nuclei containing hypomethylated DNA, on a cell-by-cell basis. Monoclonal antibodies to 5-methylcytidine were used to label cells grown in vitro. Under appropriate fixation and permeabilization conditions, interphase nuclei were labeled. Quantitative differences in the labeling were detected between Epstein-Barr virus-transformed cells and normal peripheral blood monocytes by flow cytometry analysis. Similar differences were observed by fluorescence microscopy. Both results were confirmed by Southern transfer and hybridization of DNA fragments generated by restriction enzyme digestion. This observation, which is in accordance with the occurrence of global DNA hypomethylation in tumors as established by chromatography, opens the field for the analysis of fresh tumor samples by flow cytometry and microscopy.

The unmethylated status of the CpG islands is important for gene expression of correlated housekeeping genes since it is well known that their methylation inhibits transcription process. An interesting question that has been discussed but not solved is how the CpG islands maintain their characteristic unmethylated status even though they are rich in CpG dinucleotides. Our previous in vitro and in vivo research has shown that poly(ADP-ribosyl)ation is involved in protecting CpG dinucleotides from full methylation in genomic DNA and that a block of poly(ADP-ribosyl)ation is also involved in modifying the methylation pattern in the promoter region of Htf9 housekeeping gene. In this study we locked for cytological evidence that in the absence of an active poly(ADP-ribosyl)ation the DNA methylation pattern in L929 and NIH/3T3 mouse fibroblast cell lines is altered. For this purpose, differences in the methylation levels of interphase nuclei from control and treated cultures of two murine cell lines preincubated with 2 mM 3-aminobenzamide, an inhibitor of poly(ADP-ribosyl)ation, were measured in individual cells after indirect immunolabeling with anti-5MeC antibodies. The quantitative analysis allowed us to demonstrate that blocking of the poly(ADP-ribosyl)ation results in a higher number, size, and density of antibody binding regions in treated cells when compared to the controls. Analogously, sequential Giemsa staining and indirect immunolabeling of the same slides showed the heterochromatic regions colocalized with the extended methyl-rich domains.

CytometryVolume 36, Issue 2 p. 157-159 Letter to the EditorFree Access Methylation levels of normal and chronic lymphocytic leukemia B lymphocytes: computer-assisted quantitative analysis of anti-5-methylcytosine antibody binding to individual nuclei A. de Capoa, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorC. Grappelli, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorF.R. Febbo, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorA. Spanò, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorA. Niveleau, CNRS UPRES-A 5082 Département de Virologie, Faculté de Medicine Université Joseph Fourier Grenoble, FranceSearch for more papers by this authorA. Cafolla, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorI. Cordone, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorR. Foa, Dipartimento di Scienze Biomediche ed Oncologia Umana Università di Torino, Torino, ItalySearch for more papers by this author A. de Capoa, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorC. Grappelli, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorF.R. Febbo, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorA. Spanò, Dipartimento di Genetica e Biologia Molecolare, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorA. Niveleau, CNRS UPRES-A 5082 Département de Virologie, Faculté de Medicine Université Joseph Fourier Grenoble, FranceSearch for more papers by this authorA. Cafolla, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorI. Cordone, Dipartimento di Biotecnologie Cellulari ed Ematologia, Università “La Sapienza”, Roma, ItalySearch for more papers by this authorR. Foa, Dipartimento di Scienze Biomediche ed Oncologia Umana Università di Torino, Torino, ItalySearch for more papers by this author First published: 17 May 1999 https://doi.org/10.1002/(SICI)1097-0320(19990601)36:2<157::AID-CYTO10>3.0.CO;2-KCitations: 6AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume36, Issue21 June 1999Pages 157-159 ReferencesRelatedInformation

This paper demonstrates that (a) differences in the methylation levels of interphase nuclei can be measured on a cell-by-cell basis, (b) the binding sites of beta-satellite DNA and 5-methylcytosine (5MeC)-rich regions can be localised in interphase nuclei and metaphase chromosomes by sequential in situ hybridization and indirect immunolabelling, and (c) quantitative differences in the relative extensions of beta-satellite DNA and anti-5MeC antibody binding areas can also be measured. This goal was achieved by indirect immunolabelling by anti-5MeC antibodies (Reynaud et al.: Cancer Lett. 61:255-262, 1991) of control and 5-azacytidine-treated human cell cultures. A quantitative analysis of the number, total, and mean areas of labelled heterochromatic regions and the optical densities of euchromatin and heterochromatin was performed for the cells on microscope slides. Dedicated software was used to select and measure the areas of cytological interest. In additional experiments, DAPI-stained slides from control cultures were sequentially treated by in situ hybridization with beta-satellite DNA probe and indirect immunofluorescent labelling with anti-5MeC antibodies. Fluorescent signals of probe and antibodies were pseudocoloured and merged on digital images. The relative locations of probe- and antibody-positive areas were analysed on metaphases and nuclei, and their extensions were quantified in interphase nuclei. Our results show that (a) our analysis can successfully detect different levels of DNA methylation within individual nuclei, (b) in metaphase chromosomes the antibody binding sites are mostly coincident with the hybridisation sites, and (c) in interphase nuclei a quite different picture is consistently observed.

This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.

Ribosomal gene activity and levels of DNA methylation were investigated by cytochemical and immunological methods in the nucleolar organizer regions (NORs) of individually recognised acrocentric chromosomes. Mendelian inheritance of ribosomal gene activity in a three generation family was demonstrated, together with consistent behaviour of individual gene clusters in different carriers, even when environmental conditions were changed. For most chromosomes, an inverse relationship between gene activity and the level of DNA methylation was observed. Exceptions were the two chromosomes 15 and chromosomes 13cp and 22p, all being strongly chromomycin-A3-positive in their short arms. These chromosomes bound to anti-5-MeC antibodies with differential frequencies in the different carriers. The possibility of involvement of repetitive GC-rich DNA in this behaviour is discussed.

The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of 3H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.

rRNA gene activity was evaluated by cytologic methods in cultured human cells from two different tissues grown under controlled experimental conditions. The modal and average numbers of silver positive nucleolus organizers (NOs) per cell as well as the distribution of cells with different numbers of silver positive NOs and different combinations of D-plus G-group silver stained chromosomes, were evaluated. Statistically significant differences in the average number of silver positive NOs per cell between leukocytes and fibroblasts grown under standard experimental conditions have been demonstrated. The observed differences became sharper in cells cultured under more restrictive conditions. Also, differences in the frequency of silver positivity of specific chromosomal NOs located on individually indentified chromosomes were observed in cells from the same tissue. Furthermore, differences in the frequency of activation of rDNA clusters located on the same chromosome were also observed between cells from the two tissues. The possible biologic meanings of these findings are discussed.

The results of a detailed analysis of DNA replication in a late replicating tX/X chromosome (qter → p221::p223 » qter) are reported. The chronology of DNA replication has been analyzed by comparing (a) the replication patterns of each of the two moieties of the translocation chromosome in different cells and (b) the two moieties with each other in the same cell. The study has been done on leukocyte and fibroblast cultures after BUdR incorporation. A comparison with the late replication pattern of the normal X chromosome has also been done.

The frequency of major and minor chromosome variants is studied in a random sample of newborns in Central Italy. Special attention is paid to the objective criteria used to evaluate minor variants. In our sample, the frequency of acrocentric chromosome variants is found to be unusually high compared with previous studies. Also, the distribution of C-band sizes differs from that reported for other populations, while the frequency of major chromosome variants is found to be the same.