Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions.

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

Quantitative analysis of AgNOR counts and pap stain in exfoliative cytology specimens of oral mucosa in bidi smokers and nonsmokers.

Background: Bidi smoking is a serious health hazard which is common throughout South Asia and parts of the Middle East. It has been strongly implicated to various benign and malignant lesions of oral cavity and oropharynx. These tobacco-filled leaves deliver more than three times the amount of nicotine, carbon monoxide, and tar as cigarettes which exert injurious effects on cells reflected in terms of accelerated proliferative activity in normal oral mucosal cells. Aim: This study aimed to compare the exfoliated cells from the oral mucosa of bidi smokers and nonsmokers, with emphasis on proliferative activity. Materials and Methods: Exfoliative smears were obtained from the oral mucosa of forty participants (twenty nonsmokers and twenty smokers) with age group ranging from 30-80 years, in and around Barwala (Haryana). The cytologic smears were evaluated using Papanicolaou (PAP) stain and AgNOR in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Only PAP Class I and Class II smears were observed. Results: Comparison of the mean number of AgNORs showed a significant difference between nonsmokers and smokers. Inflammatory alterations were found in 70% of smokers and 55% of nonsmokers. A significant difference in proliferative activity was observed between smokers and nonsmokers classified as PAP Class II. Conclusion: A significant difference of AgNORs/nucleus was observed between bidi smokers and nonsmokers.

RésuméContexte: Bidi fumeurs est un grave danger pour la santé qui est commune dans toute l'Asie du Sud et certaines parties du Moyen-Orient. Il a été fortement impliqué dans diverses lésions bénignes et malignes de la cavité buccale et l'oropharynx. Ces feuilles de tabac offrent plus de trois fois la quantité de nicotine, monoxyde de carbone et de goudron que les cigarettes qui exercent des effets préjudiciables sur les cellules reflétés sous la forme d' une accélération de l'activité proliférative des cellules de la muqueuse buccale normale. Objectif: Cette étude visait à comparer les cellules exfoliées de la muqueuse orale des bidis fumeurs et non fumeurs, avec l'accent sur l'activité proliférative. Matériels et méthodes: frottis Exfoliative ont été obtenus à partir de la muqueuse orale de 40 participants (20 non-fumeurs et fumeurs) avec 20 Groupe d'âge allant de 30-80 ans, dans et autour de Barwala (Haryana). Le frottis cytologique ont été évalués à l'aide de la coloration de Papanicolaou (PAP) et d'AgNOR afin d'évaluer la présence d' altérations cytologiques évocateurs d'infl ammation, dysplasie, la kératinisation, et l'activité proliférative des cellules épithéliales. PAP uniquement les catégories I et II de Papanicolaou n'a été observé. Résultats: comparaison du nombre moyen d'AgNORs ont montré une différence entre les non-fumeurs et les fumeurs. Des modifications ont été trouvés infl ammatory dans 70% des fumeurs et 55% des non-fumeurs. Une différence dans l'activité proliférative a été observée entre les fumeurs et les non-fumeurs PAP, le niveau d'emploi de la classe II. Conclusion: une différence de AgNORs/noyau a été observée entre fumeurs et non-fumeurs bidi. Mots-clés: AgNOR, frottis exfoliative, les fumeurs.

Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing.

Despite the key role of the human ribosome in protein biosynthesis, little is known about the extent of sequence variation in ribosomal DNA (rDNA) or its pre-rRNA and rRNA products. We recovered ribosomal DNA segments from a single human chromosome 21 using transformation-associated recombination (TAR) cloning in yeast. Accurate long-read sequencing of 13 isolates covering ∼0.82 Mb of the chromosome 21 rDNA complement revealed substantial variation among tandem repeat rDNA copies, several palindromic structures and potential errors in the previous reference sequence. These clones revealed 101 variant positions in the 45S transcription unit and 235 in the intergenic spacer sequence. Approximately 60% of the 45S variants were confirmed in independent whole-genome or RNA-seq data, with 47 of these further observed in mature 18S/28S rRNA sequences. TAR cloning and long-read sequencing enabled the accurate reconstruction of multiple rDNA units and a new, high-quality 44 838 bp rDNA reference sequence, which we have annotated with variants detected from chromosome 21 of a single individual. The large number of variants observed reveal heterogeneity in human rDNA, opening up the possibility of corresponding variations in ribosome dynamics.

Integrating the genomic architecture of human nucleolar organizer regions with the biophysical properties of nucleoli.

Nucleoli are the sites of ribosome biogenesis and the largest membraneless subnuclear structures. They are intimately linked with growth and proliferation control and function as sensors of cellular stress. Nucleoli form around arrays of ribosomal gene (rDNA) repeats also called nucleolar organizer regions (NORs). In humans, NORs are located on the short arms of all five human acrocentric chromosomes. Multiple NORs contribute to the formation of large heterochromatin-surrounded nucleoli observed in most human cells. Here we will review recent findings about their genomic architecture. The dynamic nature of nucleoli began to be appreciated with the advent of photodynamic experiments using fluorescent protein fusions. We review more recent data on nucleoli in Xenopus germinal vesicles (GVs) which has revealed a liquid droplet-like behavior that facilitates nucleolar fusion. Further analysis in both XenopusGVs and Drosophila embryos indicates that the internal organization of nucleoli is generated by a combination of liquid-liquid phase separation and active processes involving rDNA. We will attempt to integrate these recent findings with the genomic architecture of human NORs to advance our understanding of how nucleoli form and respond to stress in human cells.

Morphological Studies of Nucleologenesis in Giardia lamblia.

The nucleolus is a nuclear organelle involved in ribosome biogenesis. In most eukaryotes this structure disperses during prophase through anaphase and reorganizes at telophase by a process known as nucleologenesis. This process involves new transcription of ribosomal DNA at the nucleolar organizer region and the formation of prenucleolar bodies fusing to it. In Giardia lamblia, for a long time considered the only anucleolated eukaryote, a very small nucleolus has been recently described. In order to evaluate whether nucleologenesis is also present in Giardia, we analyzed the distribution of nucleolar material during telophase using different light and electron microscopy techniques including silver staining for the nucleolar organizer. Results indicate that in G. lamblia, nucleolar elements persist mainly as an intranuclear peripheral organelle during all stages of division, including telophase, however, no prenucleolar bodies are detected in the nucleoplasm. Therefore, in the parasite, nucleolar material is present throughout cell division including telophase and formation of prenucleolar bodies may not be required for nucleologenesis.

Decreased argyrophilic nucleolar organiser region (AgNOR) expression in Purkinje cells: first signal of neuronal damage in sudden fetal and infant death.

AIMS: The nucleolus is an important cellular component involved in the biogenesis of the ribosome. This study was performed in order to validate the introduction of the argyrophilic nucleolar organiser region (AgNOR) stain technique, specific for the nucleoli detection, in neuropathological studies on sudden fetal and infant death. METHODS: In a wide set of fetuses and infants, aged from 27 gestational weeks to eight postnatal months and dead from both known and unknown causes, an in-depth neuropathological study usually applied at the Lino Rossi Research Center of the Milan University was implemented by the AgNOR method. RESULTS: Peculiar abnormalities of the nucleoli, as partial or total disruption above all in Purkinje cells (PCs), were exclusively found in victims of sudden fetal and infant death, and not in controls. The observed nucleolar alterations were frequently related to nicotine absorption in pregnancy. CONCLUSIONS: We conclude that these findings represent early hallmarks of PC degeneration, contributing to the pathophysiology of sudden perinatal death.

Effect of Hydroxyapatite Nanoparticles on the Growth Potential of Hepatoma Cells in Nude Mice.

AIM: To evaluate the activity of hydroxyapatite (HAP) nanoparticles against the proliferation of hepatoma cells. METHODS: HAP nanoparticles were prepared by homogeneous precipitation. The size distribution and morphology of these nanoparticles were determined by laser particle analysis and transmission electron microscopy, respectively. Xenograft tumor models of human hepatoma cells (Bel-7402) implanted in nude mice under the right scruff skin were established and divided into two groups: treatment and control. Once the xenograft tumor grew to a diameter of 0.8 cm, 0.2 ml HAP nanoparticle suspension was injected into the tumor every day for 2 weeks. The long and short diameters of the tumors were measured before and after HAP injection, and the inhibition rate of tumor growth was calculated. Paraffin tissue sections were prepared from xenograft tumors treated as above for 2 weeks, histologically stained for DNA and agyrophilic nucleolar organizer region (AgNORs), and immuno-histologically stained for proliferating cell nuclear antigens (PCNAs). The stained sections were examined by microscopy. Images of these sections were recorded and analyzed by image analysis system and relevant software for DNA content, AgNOR intensity, and PCNA expression in the nucleus, nucleoli, and hepatoma cells, respectively. RESULTS: The HAP nanoparticles were uniformly distributed, with a size of 44.6 nm to 86.8 nm. Upon the local injection of the tumor with the HAP nanoparticles, the average volumes of the tumors were significantly reduced compared with those of the control group, which had a tumor inhibition rate of 51.32%. The DNA content, AgNOR intensity, and PCNA expression in the hepatoma cells were all significantly reduced (P < 0.01) compared with those in the control group. CONCLUSION: HAP nanoparticles inhibit the proliferation of hepatoma cells in vivo.

Ellagic Acid-Changed Epigenome of Ribosomal Genes and Condensed RPA194-Positive Regions of Nucleoli in Tumour Cells.

We studied the effect of ellagic acid (EA) on the morphology of nucleoli and on the pattern of major proteins of the nucleolus. After EA treatment of HeLa cells, we observed condensation of nucleoli as documented by the pattern of argyrophilic nucleolar organizer regions (AgNORs). EA also induced condensation of RPA194-positive nucleolar regions, but no morphological changes were observed in nucleolar compartments positive for UBF1/2 proteins or fibrillarin. Studied morphological changes induced by EA were compared with the morphology of control, non-treated cells and with pronounced condensation of all nucleolar domains caused by actinomycin D (ACT-D) treatment. Similarly as ACT-D, but in a lesser extent, EA induced an increased number of 53BP1-positive DNA lesions. However, the main marker of DNA lesions, γH2AX, was not accumulated in body-like nuclear structures. An increased level of γH2AX was found by immunofluorescence and Western blots only after EA treatment. Intriguingly, the levels of fibrillarin, UBF1/2 and γH2AX were increased at the promoters of ribosomal genes, while 53BP1 and CARM1 levels were decreased by EA treatment at these genomic regions. In the entire genome, EA reduced H3R17 dimethylation. Taken together, ellagic acid is capable of significantly changing the nucleolar morphology and protein levels inside the nucleolus.

[Analysis of fragments of intergenome spacers of human body observed in chromosomes containing no nuclear organization].

Tandem repetitions of rDNA provide so-called nuclear organizations (NOR). On the other hand, rDNA-structures are observed in some NOR chromosomes. It was demonstrated that, in addition to ribosome biogenesis, nucleoli provided a number of functions: cell cycle regulation, stress-induced response, transcription regulation, which often induced cell cascades. The mechanisms of the induction of rDNA segments in NOR chromosomes are obscure and require further research. About 1/3 repetitions are associated with nucleoli and SINE/Alu repetitions, homogeneous repetition, and tandem repetition. Perhaps, relative position of nucleoli and chromosomes may facilitate/prevent interaction of chromosomes with rDNA clusters. The variability of two larger repetitions in the central part of rMGS, LR1, and LR2 similar by -90% and separated by several hundred pairs of bases from each other was studied in our previous works. This work was devoted to the search for the LR1-LR2 segments in other chromosomes, characterization of their terminal tips at rupture points and genome areas of incorporation of the LR1-LR2 segments.

Argentophilic nucleolus organizer region as a proliferation marker in cervical intraepithelial neoplasia grade 1 of the uterine cervix.

AIM: p16INK4a and argentophilic nucleolus organizer region (AgNOR) can be used as markers for progression of cervical intraepithelial neoplasia grade 1 (CIN1) of the uterine cervix. Our objective was to study the predictive value of the AgNOR technique as a progression marker of CIN1 and its correlation with p16INK4A. MATERIAL AND METHODS: One uterine cervix biopsy from each of 75 patients with diagnosis of CIN1 was selected. All of these patients underwent a second biopsy, and these were also used for the study. RESULTS: The second biopsies showed: regression (20 patients), persistent CIN1 (38 patients), progression to CIN2 (10 patients) and progression to CIN3 (seven patients). p16INK4A showed reactivity in 67 of the 75 first CIN1 biopsies: 12 of the 20 cases that cleared the lesions and the 55 cases with persistent or progressive lesions were positive for p16INK4a (specificity: 40%; sensitivity: 100%; positive predictive value [PPV]: 82%; negative predictive value [NPV]: 100%). Samples with AgNOR areas less than 3.0 µ(2) returned in all cases, but patients whose lesions persisted or progressed to CIN2/CIN3, showed AgNOR areas greater than 3.0 µ(2) in 50/55 cases (specificity: 100%; sensitivity: 91%; PPV: 100%; NPV: 80%). CONCLUSIONS: p16INK4a is expressed in a high percentage of returning lesions. AgNOR might be a better marker of proliferation of CIN1 than p16INK4a (PPV = 100%), which means that a value greater than 3.0 µ(2) indicates the persistence or progression of the lesion. As its NPV is 80%, a value of AgNOR area less than 3.0 µ(2) in CIN1 leaves a margin of doubt about the future behavior of the lesion.

Long-term analysis to objectify the tumour grading by means of automated microscopic image analysis of the nucleolar organizer regions (AgNORs) in the case of breast carcinoma.

BACKGROUND: Apart from a number of cases of inaccurate prognosis in regard to individual patients, the inter- and intra-observer variability of the classical, histological prognosis parameters have been under repeated discussion. For this reason, a long-term analysis was carried out in regard to overall survival by means of automated microscopic image analysis of the nucleolar organizer regions (AgNORs) to objectify tumour grading in the case of breast carcinoma.This consists of a selective representation of argyrophilic proteins that are associated with the nucleolus organising regions. METHODS: The evaluation included 244 female patients with an average age of 59.3 years. The characterisation of the histological sections was carried out on the basis of the AMBA/R system. With this software the histometric characterisation level was evaluated in terms of the nucleolus organizer regions. The post-observation data were obtained from the clinical register and were complemented by mortality data from the cancer registers and by data supplied by the residents' registration office of Berlin. RESULTS: The average post-observation period was 106.6 months. With the Cox-Regression the influence of the co-variables (conventional prognosis parameters and AgNOR parameters) were examined. In the model, only the parameters pN, G and various AgNOR parameters remain present. CONCLUSION: There is a strong correlation between survival and selected AgNOR parameters. These could replace the conventional grading as the standard measure for the mitosis rate together with the pleomorphism level. Instead of the-time consuming AMBA/R system originally used, a new implementation of AgNOR quantification with modern VM systems could be applied. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1449591192859058.

Exfoliative cytological assessment of apparently normal buccal mucosa among quid chewers using argyrophilic nucleolar organizer region counts and Papanicolaou staining.

OBJECTIVE: Quid chewing is associated with an increased risk of oral cancer. This study aims to analyze argyrophilic nucleolar organizer region (AgNOR) counts along with Papanicolaou (PAP) staining in exfoliative smears of quid chewers and non-chewers to correlate quid chewing habits with possible early cytological changes in apparently normal buccal mucosa. STUDY DESIGN: Exfoliative smears were obtained from normal buccal mucosa of 30 male quid chewers and non-chewers. The smears were stained using the AgNOR silver staining technique to evaluate the proliferative activity and PAP for cytological appearance. RESULTS: Statistically higher AgNOR counts were observed in chewers as compared to non-chewers. The difference in the mean percentage of nuclei having ≥5 AgNORs in both groups was statistically significant (p = 0.001). In chewers, PAP showed 77% with class I and the remaining 23% were class II, while the non-chewers showed only class I cytology. AgNOR counts between chewers and non-chewers having class I cytological appearance demonstrated a greater mean AgNOR count in chewers (p = 0.0001). CONCLUSION: Quid chewing seems to have a definite role in promoting proliferative activity of apparently normal buccal mucosal cells. Exfoliative cytological assessment of a combination of AgNOR counts and PAP has the potential for prediction of early quid-associated cellular changes before the appearance of clinical premalignant and malignant lesions.

Comparative cytogenetics of opisthorchid species (Trematoda, Opisthorchiidae).

In the present study karyotypes and chromosomes of five species of the family Opisthorchiidae (Opisthorchis felineus (Rivolta, 1884), O. viverrini (Poirier, 1886), Metorchis xanthosomus (Creplin, 1846), M. bilis (Braun, 1893), and Clonorchis sinensis (Cobbold, 1875)) were compared. Karyotypes of O. felineus, M. xanthosomus, M. bilis and C. sinensis consist of two pairs of large meta- and submetacentrics and five pairs of small chromosomes (2n = 14). The karyotype of O. viverrini is 2n = 12, which indicates a fusion of two chromosomes of opisthorchid ancestral karyotype. Analysis of mitotic and meiotic chromosomes was performed by heterologous in situ hybridization of microdissected DNA probes obtained from chromosomes 1 and 2 of O. felineus and chromosomes 1 and 2 of M. xanthosomus. Results of chromosome staining (C- and AgNOR-banding) and FISH of telomeric probes and ribosomal DNA probe on opisthorchid chromosomes were used for chromosome comparison. Data on chromosome number in opisthorchid species were also discussed.

A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae).

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 µm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 µm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 µm long in F. hepatica while subtelocentric and 4.7 µm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

Chromosomal localization of rDNA genes and genomic organization of 5S rDNA in Oreochromis mossambicus, O. urolepis hornorum and their hybrid.

In this study, classical and molecular cytogenetic analyses were performed in tilapia fishes, Oreochromis mossambicus (XX/XY sex determination system), O. urolepis hornorum (WZ/ZZ sex determination system) and their hybrid by crossing O. mossambicus female x O. u. hornorum male. An identical karyotype ((2n = 44, NF (total number of chromosomal arms) = 50) was obtained from three examined tilapia samples. Genomic organization analysis of 5S rDNA revealed two different types of 5S rDNA sequences, 5S type I and 5S type II. Moreover, fluorescence in situ hybridization (FISH) with 5S rDNA probes showed six positive fluorescence signals on six chromosomes of all the analysed metaphases from the three tilapia samples. Subsequently, 45S rDNA probes were also prepared, and six positive fluorescence signals were observed on three chromosome pairs in all analysed metaphases of the three tilapia samples. The correlation between 45 rDNA localization and nucleolar organizer regions (NORs) was confirmed by silver nitrate staining in tilapia fishes. Further, different chromosomal localizations of 5S rDNA and 45S rDNA were verified by two different colour FISH probes. Briefly, the current data provide an insights for hybridization projects and breeding improvement of tilapias.

Quantitative analysis of argyrophilic nuclear organizer regions in giant cell lesions of jaws.

BACKGROUND: Giant cell lesions of the jaws are considerably similar according to histopathologic characteristics yet show different clinical behaviors. These lesions include central giant cell granuloma (CGCG), aneurysmal bone cyst, Cherubism, and Brown tumor associated with hyperparathyroidism. The present study aimed to investigate AgNORs count in these lesions as a proliferative marker and to determine whether it can be used to discriminate between them or not. METHODS: Forty-one cases of giant cell lesions of jaws were retrived from Oral Pathology Department (1987-2007). They included 21 cases of CGCG, eight cases of aneurysmal bone cyst (ABC), six cases of Cherubism, six cases of Brown tumor. The mean AgNORs count was calculated for all cases. To compare mean AgNORs in groups of lesions, ANOVA test was performed. RESULTS: Mean AgNOR counts were: (0/85 +/- 0/29) in CGCG, (0/76 +/- 0/32) in ABC (0/87 +/- 0/10) in Cherubism and (0/82 +/- 0/16) in Brown tumor. A significant difference was not observed in AgNOR counts among these groups of lesions. CONCLUSIONS: Jaws giant cell containing lesions have no acceptable differences in mean AgNORs.

Redefining the Scl-70 indirect immunofluorescence pattern: autoantibodies to DNA topoisomerase I yield a specific compound immunofluorescence pattern.

OBJECTIVES: To report on a novel IIF pattern specifically associated with antibodies to DNA topo I. METHODS: A novel compound IF pattern, designated Scl-70 pattern, was characterized in routine ANA-HEp-2 IIF screening. Within the last 3 years, all serum samples presenting the Scl-70 pattern at the ANA-HEp2 IIF screening were tested for anti-topo I reactivity. Conversely, 16 serum samples with known anti-topo I reactivity and affinity-purified anti-topo I antibody preparations were tested for the Scl-70 pattern. RESULTS: The Scl-70 pattern comprised the staining of five cellular regions: nucleus, nucleolus and cytoplasm in interphase cells; nucleolar organizing region (NOR) and chromosomes in mitotic cells. All 81 serum samples selected as Scl-70 pattern reacted with topo I. All 16 anti-topo I samples and antibody preparations reproduced the Scl-70 pattern. This compound IF pattern was consistently observed in different commercial HEp-2 cell slides and in home-made slides with HEp-2 cells and human fibroblasts fixed with alternative protocols. Double IIF experiments demonstrated the co-localization of topo I and human upstream binding factor at the NOR. CONCLUSIONS: The Scl-70 pattern belongs to the group of compound IF patterns that hold strong association with the respective autoantibody specificities, such as that observed with centromere protein F (CENP-F) and nuclear mitotic apparatus-1 (NuMA-1) protein. The identification of the Scl-70 pattern at routine ANA-HEp-2 IIF screening may lead to implementation of specific tests for the identification of anti-topo I antibodies. In addition, the Scl-70 pattern outlines cellular domains other than those previously reported for topo I, which is of interest for further understanding the roles of this enzyme in cell biology.

Toward a multimodal cell analysis of brush biopsies for the early detection of oral cancer.

BACKGROUND: This report describes what to the authors' knowledge is the first clinical application of semiautomated multimodal cell analysis (MMCA), a novel technique for the early detection of cancer for cases with a limited number of suspicious cells. In this clinical study, MMCA was applied to oral cancer diagnostics on brush biopsies. The MMCA approach was based on the sequential application of multiple stainings of identical, slide-based cells and repeated relocalizations and measurements of their diagnostic features, resulting in multiparametric features of individual cells. Data integration of the variously stained cells increased diagnostic accuracy. The implementation of MMCA also enabled fully automatic, adaptive image preprocessing, including registration of multimodal images and segmentation of cell nuclei. METHODS: In a preliminary clinical trial, 47 slides from brush biopsies of suspicious oral lesions were analyzed. The final histologic diagnoses included 20 squamous cell carcinomas, 7 hyperkeratotic leukoplakias, and 20 lichen planus mucosae. RESULTS: The stepwise application of 2 additional approaches (morphology, DNA content, argyrophilic nucleolar organizer region counts) increased the specificity of conventional cytologic diagnosis from 92.6% to 100%. This feasibility study provided a proof of concept, demonstrating efficiency, robustness, and diagnostic accuracy on slide-based cytologic specimens. CONCLUSIONS: The authors concluded that MMCA may become a sensitive and highly specific, objective, and reproducible adjuvant diagnostic tool for the identification of neoplastic changes in oral smears that contain only a few abnormal cells.

[Study of nucleolar organizer regions in prostate cancer]. / Studiul regiunilor organizatoare nucleolare în tumorile prostatice.

AIM: To compare the qualitative and quantitative AgNOR scores per nucleus of histological grades. MATERIAL AND METHODS: Thirty-five needle biopsies from patients with prostate cancer (PC) aged 60 to 75 years were investigated. The morphologic parameters examined on histological specimens were: microscopic patterns, Gleason's histological grades (GLG), and AgNOR scores by automated image analyzer and quantitative AgNOR counts according to the classification, fine granules, large granules, and total granule counts. RESULTS: Our data suggest a significant correlation between GLG, AgNOR scores, and the percentage of both AgNOR fine and large granules. In HPIN, AgNOR count was evidently higher (5.56) as compared to normal acinar epithelium, slightly exceeding the level found in well-differentiated carcinoma (5.3). In our cases, the highest AgNOR counts were found in poorly differentiated carcinomas (9.81). AgNOR density in ductal carcinomas (7.61) was close to that in moderately differentiated carcinomas (7.35). CONCLUSION: This simple method may aid diagnostic judgement when interpreting needle biopsies of malignancy and act as a prognostic marker useful for identifying PC of low and high malignancy.

Digital image analysis of mean AgNOR area objectively discriminates between reactive plasmacytosis and multiple myeloma in cytologic smears from bone marrow aspirates.

OBJECTIVE: To differentiate between reactive plasmacytosis (RP) and multiple myeloma (MM) in bone marrow aspirates by assessment of silver-stainable nucleolar organizer regions (AgNORs). METHODS: Archival cytologic smears (n = 69) from bone marrow aspirates were retrospectively investigated. Twenty cases had RP and 49 cases MM (24 low-grade, 22 intermediate-grade and 3 high-grade MM). Diagnoses were confirmed by evaluation of the corresponding bone marrow trephine biopsies. Cytologic smears were subjected to AgNOR staining using a standardized silver-staining protocol and assessed by two independent clinical histopathologists using digital image analysis. AgNOR count and area were analyzed in 100 plasma cell nuclei per sample. RESULTS: The mean AgNOR area was found to be significantly (p < 0.001) higher in low-grade MM (mean: 23.4 microm(2); range: 19.2-26.7 microm(2)) than in RP cases (mean: 6.2 microm(2); range: 3.8-8.4 microm(2)). In contrast, mean AgNOR count failed to disclose significant differences between low-grade MM (mean: 5.36; range: 1-7) and RP (mean: 3.68; range: 1-6). CONCLUSION: Digital image analysis of the mean AgNOR area might be used as a diagnostically useful adjunct to aid objectively in the challenging differentiation between RP and MM in bone marrow aspirates.