Nucleolar Organizer Research Articles

Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

Ribosomal DNA (rDNA) repeats harbor ribosomal RNA (rRNA) genes and intergenic spacers (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding rRNA components of ribosomes. IGS-associated Pol II prevents Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli and rRNA production. Here, compartment-enriched proximity-dependent biotin identification (compBioID) revealed the TATA-less-promoter-binding TBPL1 and transcription-regulatory PAF1 with nucleolar Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I-associated ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the nucleolar interactome of Pol II and show that its regulation by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and function.

What is the True Karyotype of <i>Bufotes latastii</i> (Amphibia, Anura, Bufonidae)?

The karyotype is one of the most important characteristics of an animal species and can be very useful for identifying taxa. The chromosome sets of the West Himalayan endemic Ladakh toad, Bufotes latastii, were previously described in several papers, but differed greatly from each other. To deal with these contradictions, we re-examined the karyotype of B. latastii from India and compared it with the sets of chromosomes of the most morphologically similar toad species inhabiting East Asia. It turned out that the diploid set of B. latastii consists of 22 chromosomes (2n = 22; NF = 44), of which six pairs are large and five are small. As a rule, the 4th, 5th, and 7th pairs of chromosomes are submetacentric and all the others are metacentric. The chromosomal formula is 2n = 16 m + + 6 sm = 22. The nucleolar organizers (NOR) in B. latastii are located on the long arms of submetacentric chromosomes of the 5th pair, which greatly distinguishes this species from most diploid species of the genus Bufotes. Karyotypes of two other studied species (Duttaphrynus stomaticus and Strauchbufo raddei), which can be morphologically similar to B. latastii, also consist of 22 chromosomes (2n = 22; NF = 44) of which six pairs are large and five are small, but there is only a pair of submetacentric chromosomes (2n = 20 m + 2 sm = 22). The most notable differences between these three species were in the position of NORs. In B. latastii they were located in the telomeric region of the long arm of the 5th pair, in D. stomaticus in the submedial region of the short arm of the 7th pair, and in S. raddei in the telomeric region of the short arm of the 4th pair. Discriminant analyses of the relative length of chromosomes and centromeric indices also revealed significant differences between the karyotypes of these three species. Our study made it possible to clearly distinguish B. latastii from D. stomaticus and S. raddei by karyotype characteristics. This may be important if it is necessary to identify East Asian morphologically similar toad species.

The value and significance of nucleolar organizer region proteins as markers of malignancy in breast cancer patients.

To assess argyrophilic nucleolar organizer regions (AgNORs) in 60 patients with primary breast carcinoma and evaluated their association with clinical prognostic parameters of breast cancer. Argyrophilic nucleolar organizer regions were stained in paraffin sections of the tissues using Ploton's silver method. For each sample, the number of AgNORs within the nuclei of 100 tumor cells was counted. The average number of AgNORs per nucleus was calculated, and the results were expressed as mean. The number of AgNORs was significantly higher in breast invasive ductal carcinoma (6.6) compared to benign breast tumors (fewer than 2.0). However, differences in AgNOR counts across different age groups were not statistically significant. This study suggests that AgNOR counts could be used as a potential procedure for estimating proliferation characteristics in histopathological sections of benign and malignant breast lesions. Argyrophilic nucleolar organizer region counts may also be valuable for identifying high-risk patients and indicating tumor aggressiveness. A larger study with an increased sample size that incorporates both AgNOR numbers and Ki67 scores for assessing cell kinetics is needed to confirm our findings.

LINE1 and PRC2 control nucleolar organization and repression of the 8C state in human ESCs.

The mechanisms that ensure developmental progression in the early human embryo remain largely unknown. Here, we show that the family of long interspersed nuclear element 1 (LINE1) transposons prevents the reversion of naive human embryonic stem cells (hESCs) to 8-cell-like cells (8CLCs). LINE1 RNA contributes to maintenance of H3K27me3 levels, particularly at chromosome 19 (Chr19). Chr19 is enriched for key 8C regulators, H3K27me3, and genes derepressed upon LINE1 knockdown or PRC2 inhibition. Moreover, Chr19 is strongly associated with the nucleolus in hESCs but less in 8CLCs. Direct inhibition of PRC2 activity induces the 8C program and leads to a relocalization of Chr19 away from the nucleolus. LINE1 KD or PRC2 inhibition induces nucleolar stress, and disruption of nucleolar architecture is sufficient to de-repress the 8C program. These results indicate that LINE1 RNA and PRC2 maintain H3K27me3-mediated gene repression and 3D nuclear organization to prevent developmental reversion of hESCs.

Mapping and engineering RNA-controlled architecture of the multiphase nucleolus.

Biomolecular condensates are key features of intracellular compartmentalization. As the most prominent nuclear condensate in eukaryotes, the nucleolus is a layered multiphase liquid-like structure and the site of ribosome biogenesis. In the nucleolus, ribosomal RNAs (rRNAs) are transcribed and processed, undergoing multiple maturation steps that ultimately result in formation of the ribosomal small subunit (SSU) and large subunit (LSU). However, how rRNA processing is coupled to the layered nucleolar organization is poorly understood due to a lack of tools to precisely monitor and perturb nucleolar rRNA processing dynamics. Here, we developed two complementary approaches to spatiotemporally map rRNA processing and engineer de novo nucleoli. Using sequencing in parallel with imaging, we found that rRNA processing steps are spatially segregated, with sequential maturation of rRNA required for its outward movement through nucleolar phases. Furthermore, by generating synthetic de novo nucleoli through an engineered rDNA plasmid system in cells, we show that defects in SSU processing can alter the ordering of nucleolar phases, resulting in inside-out nucleoli and preventing rRNA outflux, while LSU precursors are necessary to build the outermost layer of the nucleolus. These findings demonstrate how rRNA is both a scaffold and substrate for the nucleolus, with rRNA acting as a programmable blueprint for the multiphase architecture that facilitates assembly of an essential molecular machine.

Description of the complete rDNA repeat unit structure of Coturnixjaponica Temminck et Schlegel, 1849 (Aves).

Ribosomal RNA (18S, 5.8S, 28S) gene clusters in genomes form regions that consist of multiple tandem repeats. They are located on a single or several pairs of chromosomes and play an important role in the formation of the nucleolus responsible for the assembly of ribosome subunits. The rRNA gene cluster sequences are widely used for taxonomic studies, however at present, complete information on the avian rDNA repeat unit structure including intergenic spacer sequence is available only for the chicken (Gallusgallusdomesticus Linnaeus, 1758). The GC enrichment and high-order repeats peculiarities within the intergenic spacer described for the chicken rDNA cluster may be responsible for these failures. The karyotype of the Japanese quail (Coturnixjaponica Temminck et Schlegel, 1849) deserves close attention because, unlike most birds, it has three pairs of nucleolar organizer bearing chromosomes, two of which are microchromosomes enriched in repeating elements and heterochromatin that carry translocated terminal nucleolar organizers. Here we assembled and annotated the complete Japanese quail ribosomal gene cluster sequence of 21166 base pairs (GenBank under the registration tag BankIt2509210 CoturnixOK523374). This is the second deciphered avian rDNA cluster after the chicken. Despite the revealed high similarity with the chicken corresponding sequence, it has a number of specific features, which include a slightly lower degree of GC content and the presence of bendable elements in the content of both the transcribed spacer I and the non-transcribed intergenic spacer.

Epigenetic control and inheritance of rDNA arrays.

Ribosomal RNA (rRNA) genes exist in multiple copies arranged in tandem arrays known as ribosomal DNA (rDNA). The total number of gene copies is variable, and the mechanisms buffering this copy number variation remain unresolved. We surveyed the number, distribution, and activity of rDNA arrays at the level of individual chromosomes across multiple human and primate genomes. Each individual possessed a unique fingerprint of copy number distribution and activity of rDNA arrays. In some cases, entire rDNA arrays were transcriptionally silent. Silent rDNA arrays showed reduced association with the nucleolus and decreased interchromosomal interactions, indicating that the nucleolar organizer function of rDNA depends on transcriptional activity. Methyl-sequencing of flow-sorted chromosomes, combined with long read sequencing, showed epigenetic modification of rDNA promoter and coding region by DNA methylation. Silent arrays were in a closed chromatin state, as indicated by the accessibility profiles derived from Fiber-seq. Removing DNA methylation restored the transcriptional activity of silent arrays. Array activity status remained stable through the iPS cell re-programming. Family trio analysis demonstrated that the inactive rDNA haplotype can be traced to one of the parental genomes, suggesting that the epigenetic state of rDNA arrays may be heritable. We propose that the dosage of rRNA genes is epigenetically regulated by DNA methylation, and these methylation patterns specify nucleolar organizer function and can propagate transgenerationally.

Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Terrein Exhibits Anti-tumor Activity by Suppressing Angiogenin Expression in Malignant Melanoma Cells.

Malignant melanoma is a tumor with a poor prognosis that can metastasize distally at an early stage. Terrein, a metabolite produced by Aspergillus terreus, suppresses the expression of angiogenin, an angiogenic factor. However, the pharmacological effects of natural terrein have not been elucidated, because only a small amount of terrein can be extracted from large fungal cultures. In this study, we investigated the antineoplastic effects of terrein on human malignant melanoma cells and its underlying mechanisms. Human malignant melanoma cell lines were cultured in the presence of terrein and analyzed. Angiogenin production was evaluated using ELISA. Ribosome biosynthesis was evaluated using silver staining of the nucleolar organizer region. Intracellular signaling pathways were analyzed using western blotting. Malignant melanoma cells were transplanted subcutaneously into the backs of nude mice. The tumors were removed at 5 weeks and analyzed histopathologically. Terrein inhibited angiogenin expression, proliferation, migration, invasion, and ribosome biosynthesis in malignant melanoma cells. Terrein was shown to inhibit tumor growth and angiogenesis in animal models. This study demonstrated that terrein has anti-tumor effects against malignant melanoma. Furthermore, chemically synthesized non-natural terrein can be mass-produced and serve as a novel potential anti-tumor drug candidate.

Karyotypic description and comparison of Litoria (L.) paraewingi (Watson et al., 1971), L.ewingii (Duméril et Bibron, 1841) and L.jervisiensis (Duméril et Bibron, 1841) (Amphibia, Anura).

The karyotype of Litoria (L.) paraewingi (Watson et al., 1971) (Big River State Forest, Victoria) is described here for the first time. It is prepared following tissue culture of toe clipping macerates, cryopreservation, reculture and conventional 4',6-diamidino-2-phenylindole (DAPI) staining. The L.paraewingi karyotype is then compared to similarly processed IUCN (International Union for the Conservation of Nature) least concern members L.ewingii (Duméril et Bibron, 1841) (southern Victoria) and L.jervisiensis (Duméril et Bibron, 1841) (Myall Lakes National Park, New South Wales), all members of the same L.ewingii complex/group. The L.paraewingi diploid number is 2n = 26, the same as for the other two species. Litoriaparaewingi chromosomes 1, 2, 6 and 7 are submetacentric, chromosomes 3 and 5 are subtelocentric and the remainder are metacentric. No secondary constriction or putative nucleolus organiser region (NOR) was readily identifiable following conventional DAPI staining in any scored L.paraewingi metaphase spread. Conversely, a putative NOR was readily identifiable on the long arm of chromosome 1 in all examined metaphase spreads for the other two species. The karyotypes of L.ewingii and L.jervisiensis here further differ from L.paraewingi with chromosome 1 being metacentric and chromosomes 8 and 10 being submetacentric for both former species. The L.jervisiensis karyotype differs from those of L.ewingii and L.paraewingi by DAPI staining with: (i) apparent relative length inversion of subtelocentric chromosome 3 and metacentric chromosome 4 and (ii) chromosome 6 being metacentric rather than submetacentric. All three species have a highly conserved chromosome morphology with respect to chromosomes 2, 5, 7, 9, 11, 12 and 13. The greatest gross morphological difference karyotypically is observed between L.paraewingi and L.jervisiensis. These karyotype data support the previous phylogenetic separation of these three species based upon genetic compatibility and behavioural, biochemical and molecular genetic analyses.

Structure and Evolution of Ribosomal Genes of Insect Chromosomes.

Currently, clusters of 45S and 5S ribosomal DNA (rDNA) have been studied in about 1000 and 100 species of the class Insecta, respectively. Although the number of insect species with known 45S rDNA clusters (also referred to as nucleolus-organizing regions, or NORs) constitutes less than 0.1 percent of the described members of this enormous group, certain conclusions can already be drawn. Since haploid karyotypes with single 45S and 5S rDNA clusters predominate in both basal and derived insect groups, this character state is apparently ancestral for the class Insecta in general. Nevertheless, the number, chromosomal location, and other characteristics of both 45S and 5S rDNA sites substantially vary across different species, and sometimes even within the same species. There are several main factors and molecular mechanisms that either maintain these parameters or alter them on the short-term and/or long-term scale. Chromosome structure (i.e., monocentric vs. holokinetic chromosomes), excessive numbers of rRNA gene copies per cluster, interactions with transposable elements, pseudogenization, and meiotic recombination are perhaps the most important among them.

Melatonin Level and Activity of Nucleolar Organizer Regions in the Prognosis of Infertility in Women with Ovarian Endometriosis

Introduction. Endometriosis is a multifactorial disease, affecting about 10 % of women of reproductive age. The purpose of the work is predicting the development of infertility in women with ovarian endometriosis by determining urine melatonin levels and activity indicators of nucleolar organizer regions in endometrioid ovarian heterotopias.Materials and methods. In the Clinic of South Ural State Medical University from 2018 to 2020, 2 groups of women with ovarian endometriosis were studied: group 1 — with infertility (n = 22); group 2 — without it (n = 27). The level of urinary 6-SMT and the activity of ribosomal synthesis were determined.Results. The level of 6-SMT is reduced in patients with stage I ovarian endometriosis. At stagesII and III, the level of 6-SMTis lower in women with infertility than without it: Stage II — (38.82±1.49) ng/ml and (93.04±3.64) ng/ml (p < 0.001); Stage III — (42.84±1.85) ng/ml and (58.06±1.65) ng/ml (p = 0.340). The activity of ribosomal synthesis in epithelial cells of endometrioid heterotopias increases at stage III. In fibroblasts of the stroma of endometrioid heterotopias, the activity of ribosomal synthesis is higherin women with stage III infertility, and in histiocytes— in women with stage II infertility. A logistic stepwise regression analysis was carried out and the most important predictors influencing the development of infertility were identified.Discussion. The decrease in melatonin in women with ovarian endometriosis, suffering from infertility, as well as in stage I ovarian endometriosis is consistent with the research of M. I. Yarmolinskaya et al. (2019). In the presence of infertility, activation of connective tissue histiocytes begins in the early stages. The results obtained may reflect the immunomodulatory effect of melatonin, which is expressed inincreased functional activity of connective tissue histiocytes and fibroblasts.Conclusion. Using mathematical analysis, the most important indicators that influence the reproductive prognosis in women with ovarian endometriosis (activity of nucleolar organizer regions, urine 6-SMT level) were determined, which makes it possible to create a model for calculating an individual reproductive prognosis.

Pseudoendemism in Mediterranean black flies (Diptera: Simuliidae): a new record for Africa

The first record of a European black fly, Simulium ichnusae Rivosecchi & Contini, is documented for Africa, where it was found at elevations above 1000 m in the Djurdjura Mountains of Algeria. Considered an endemic species of Sardinia for 60 years, S. ichnusae must now be regarded as pseudoendemic. Larvae, pupae, females, and males from the Djurdjura Mountains compare well with those in the original description of the species. Chromosomally, S. ichnusae differs from the standard banding sequence of the S. vernum group by a transposed nucleolar organizer—the only known member of the group with this condition—and three fixed inversions, all of which are shared by other members of this species group. The addition of S. ichnusae to the Algerian fauna increases the number of nominal species of black flies known from the country to 34. The discovery of S. ichnusae suggests that additional trans-Mediterranean species await discovery in the coastal Mediterranean mountains and emphasizes that claims of endemism depend on thorough surveys and taxonomic accuracy.

FOXA1 regulates ribosomal RNA transcription in prostate cancer.

Ribosome biogenesis is excessively activated in tumor cells, yet it is little known whether oncogenic transcription factors (TFs) are involved in the ribosomal RNA (rRNA) transactivation. Nucleolar proteomics data and large-scale immunofluorescence were re-analyzed to jointly identify the proteins localized at nucleolus. RNA-Seq data of five prostate cancer (PCa) cohorts were combined and integrated with multi-dimensional data to define the upregulated nucleolar TFs in PCa tissues. Then, ChIP-Seq data of PCa cell lines and two PCa clinical cohorts were re-analyzed to reveal the TF binding patterns at ribosomal DNA (rDNA) repeats. The TF binding at rDNA was validated by ChIP-qPCR. The effect of the TF on rRNA transcription was determined by rDNA luciferase reporter, nascent RNA synthesis, and global protein translation assays. In this study, we reveal the role of oncogenic TF FOXA1 in regulating rRNA transcription within nucleolar organization regions. By analyzing human TFs in prostate cancer clinical datasets and nucleolar proteomics data, we identified that FOXA1 is partially localized in the nucleolus and correlated with global protein translation. Our extensive FOXA1 ChIP-Seq analysis provides robust evidence of FOXA1 binding across rDNA repeats in prostate cancer cell lines, primary tumors, and castration-resistant variants. Notably, FOXA1 occupancy at rDNA repeats correlates with histone modifications associated with active transcription, namely H3K27ac and H3K4me3. Reducing FOXA1 expression results in decreased transactivation at rDNA, subsequently diminishing global protein synthesis. Our results suggest FOXA1 regulates aberrant ribosome biogenesis downstream of oncogenic signaling in prostate cancer.

The development of chicken cerebellar cortex and the determination of AgNOR activity of the Purkinje cell nuclei

In this study, the embryonic and post-hatching development of the chicken cerebellar cortex, and the number, size and area of the silver-staining nucleolus-organizer regions (AgNORs) were determined in cerebellar Purkinje cells in layer-hen (Babcock-B380) embryos and chickens. The mean transverse diameter of the Purkinje cells and their nuclei, and the mean area of the Purkinje cell nucleus and AgNOR area increased during the entire experimental period. The mean ratio of the AgNOR area to the Purkinje cell nucleus area reached its highest value at the 15th day of incubation. There was no difference between the mean AgNOR numbers per nucleus. These findings provide reference data for future studies in this and related research topics, for example for establishing AgNOR parameters and we also hope that our data will be supplemented with other techniques such as PCNA and TUNEL in the near future.

In-section Click-iT detection and super-resolution CLEM analysis of nucleolar ultrastructure and replication in plants

Correlative light and electron microscopy (CLEM) is an important tool for the localisation of target molecule(s) and their spatial correlation with the ultrastructural map of subcellular features at the nanometre scale. Adoption of these advanced imaging methods has been limited in plant biology, due to challenges with plant tissue permeability, fluorescence labelling efficiency, indexing of features of interest throughout the complex 3D volume and their re-localization on micrographs of ultrathin cross-sections. Here, we demonstrate an imaging approach based on tissue processing and embedding into methacrylate resin followed by imaging of sections by both, single-molecule localization microscopy and transmission electron microscopy using consecutive CLEM and same-section CLEM correlative workflow. Importantly, we demonstrate that the use of a particular type of embedding resin is not only compatible with single-molecule localization microscopy but shows improvements in the fluorophore blinking behavior relative to the whole-mount approaches. Here, we use a commercially available Click-iT ethynyl-deoxyuridine cell proliferation kit to visualize the DNA replication sites of wild-type Arabidopsis thaliana seedlings, as well as fasciata1 and nucleolin1 plants and apply our in-section CLEM imaging workflow for the analysis of S-phase progression and nucleolar organization in mutant plants with aberrant nucleolar phenotypes.

Macromolecular condensation organizes nucleolar sub-phases to set up a pH gradient

Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. Invitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs invitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.

First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand

The first chromosome study of Trigonostigma somphongsi (Meinken,1958) from Thailand. Specimens were collected from Pak Phli District, Nakhornnayok Province, central Thailand. The mitotic chromosomes were directly prepared from kidney tissues of two males and two females. Conventional staining and Ag-NOR banding techniques were applied to stain the chromosomes. The results shown that diploid chromosome number of T. somphongsi was 2n=50, the fundamental numbers (NF) were 92 in both male and female. The types chromosomes consisted of 10 metacentric, 20 submetacentric, 12 acrocentric and 8 telocentric chromosomes. The result also exhibited that the interstitial nucleolar organizer regions (NORs) were clearly observed at the short arm of chromosome pair 2. The karyotype formula could be deduced as: 2n (diploid) 50=10m+20sm+12a+8t.

Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek (Trigonella foenum-graecum L.): a neglected legume

Fenugreek or Trigonella foenum-graecum L. is a commercially important yet neglected crop of the family Fabaceae, with potent medicinal applications, and can treat several diseases as well. Conventional breeding studies for higher yields of commercial crops largely depend on chromosomal information of the particular species. Despite a number of cytological research being conducted on T. foenum-graecum, a complete characterization of its chromosomes has not been achieved due to the limitations of traditional karyotype analysis methods. A range of chromosomal markers are advantageous to characterize at full extent and identify individual chromosomes rather than relying on only physical metrics. Thus, in this study, in addition to giemsa staining, other approaches like fluorochrome and silver staining were used for the precise karyomorphological analysis of this species. Enzyme maceration and air drying (EMA) based fluorochrome banding with GC-specific stain Chromomycin A3 (CMA), and AT-specific stain 4’,6-diamidino-2-phenylindole (DAPI) applied for the first time for chromosome characterization. The results showed 2n = 16 chromosomes in metaphase cells, with karyotype formula of 2m+6sm. The unique banding pattern observed in the CMA/DAPI and AgNOR staining highlights the AT and GC-rich regions as well as the nucleolar organizer regions (NORs). All this crucial information can further assist in conducting breeding studies of more precision with simultaneously encouraging similar studies that need to be done in other unexploited species of importance.

Computer-Assisted Morphometric Comparative Analysis of Argyrophilic Nucleolar Organizer Regions (AgNORs) in Leukoplakia With Dysplasia and Oral Squamous Cell Carcinoma.

Background Oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL) with dysplasia are closely linked conditions in the oral cavity, with the latter often indicating precancerous changes, underscoring the urgency of early detection and intervention. Histopathological confirmation is crucial for accurate diagnosis. The nucleolar organizer region (NOR), specifically analyzed through silver-staining (argyrophilic NORs), provides insights into nuclear changes associated with the lesion. Computer-assisted morphometric analysis enhances precision and objectivity in evaluating AgNOR-related parameters. Aim To conduct a computer-assisted morphometric comparison of AgNORs using various NOR-related parameters in cases of OSCC and leukoplakia with dysplasia and to evaluate their diagnostic significance. Materials and methods A computer-assisted morphometric analysis was conducted using various NOR-related parameters, such as nuclear profile area, single AgNOR profile area per nucleus, total AgNOR profile area per nucleus, and number of AgNOR profiles per nucleus on a total sample of 90 specimens, which includes leukoplakia with dysplasia (30), OSCC (30), and a control group, including 30 samples of normal oral mucosa. A comparison was conducted on the morphometric values between the groups under investigation. Tukey's multiple comparison tests and ANOVA were used to analyze the data and determine the differences between the groups. Results The present investigation revealed a significant difference in all four AgNOR-related parameters between leukoplakia and OSCC in comparison to the control group (normal oral mucosa). Comparing OL (41.78 ± 0.46) and OSCC (62.78 ± 0.47) to the control group (35.93 ± 0.99), the mean value of nuclear profile area (A Nuc) was significantly greater. In comparison to the control group (3.40 ± 0.09), the mean value of a single AgNOR profile area per nucleus (A NOR) was found to be relatively lower in both research groups, OL (2.00 ± 0.02) and OSCC (1.39 ± 0.01). The total AgNOR profile area per nucleus (TA NOR) had a mean value of 10.61 ± 0.69 in OL and 12.05 ± 0.28 in OSCC, respectively, compared to 7.82 ± 0.38 in the control group. The study found that there was more number of profiles of AgNORs per nucleus (n NOR)in the study groups of OL (5.30 ± 0.29) and OSCC (8.69 ± 0.19) than in thecontrol group (2.32 ± 0.11). Conclusion The parameters linked to the NOR are biologically informative and easy to check regularly in a pathology lab. Additionally, AgNORs give us important information that enables us to study the range of nuclear changes in malignant and potentially malignant lesions.