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The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

BACKGROUND: Bone allografts can elicit immune responses which is correlated with the presence of Human Leukocyte Antigen (HLA) and cellular DNA. It also has risk of causing occult infection arising out of contamination during its processing and storage. The presence of immunogenic materials like cells, cellular remnants and DNA in a decalcified bone allograft during different phases of processing has never been studied. Present study was conducted to explore- the cell viability using routine Hematoxylin and Eosin, presence of DNA using Feulgen staining and etiology of contamination in decalcified bone allograft during procurement, demineralization and ethanol preservation. METHODS: The harvested bones from patients undergoing hemireplacement/THR/TKR were processed to prepare decalcified bone allografts. The samples during procurement (A), HCL treatment (B) and ethanol preservation (C) were sent for histopathological analysis (number of osteocytes in the maximum density field under 40x and the cells demonstrating presence of DNA on feulgen stain) and microbiological assessment (aerobic/anaerobic/fungal cultures). RESULTS: Histopathological study demonstrated the presence of osteocytes and other cells like bone marrow, adipocytes, endothelial cells in the decal bone allograft. The average number of osteocytes gradually decreased from 55.47, 9.6, 0.86 in sample A, B, C, respectively. Feulgen staining confirmed the presence of DNA in osteocytes and other cells which decreased both qualitatively and quantitatively in subsequent stages of processing. Rate of contamination demonstrated at the procurement was 6.67% (Staphylococcus aureus). After treatment with HCl (demineralisation), 7.14% of non-contaminated allografts were found contaminated (Staphylococcus epidermidis). None of the remaining 13 non-contaminated allografts showed contamination after storage in ethanol. Overall 13% of the patients had positive cultures on microbiological assessment. CONCLUSION: The population of osteocytes in the harvested bone reduced significantly after processing with HCl and ethanol preservation. Presence of DNA, demonstrated by using Feulgen staining, was observed in bone marrow cells, adipocytes along with osteocytes which showed quantitative reduction on processing. Hence, antigenicity, conferred by cells and their DNA, reduced significantly after processing of decal bone. Contamination rate of banked decalcified allograft was 13%. Thus, culture and sensitivity tests should be carried out at each step of processing of decal bone allograft.

Diagnostic and prognostic DNA-karyometry represents an automated computerized microscopical procedure, designed to improve cancer diagnostics at three different aspects: Screening for cancer cells, e.g. in body cavity effusions, urines or mucosal smears Specifying the risk of dysplasias or borderline lesions to progress to manifest cancer, e.g. of oral, bronchial or cervical mucosa, or the ovary. Grading the malignancy of certain tumors, like prostate cancer. It combines an automated diagnostic classification of Feulgen-stained nuclei with precise nuclear DNA-measurements. DNA-aneuploidy is used as a specific marker of malignancy and its degree for grading. All types of cytological specimens can be used after (re-)staining specific for DNA according to Feulgen. Histological specimens are subjected to enzymatic cell separation before Feulgen-staining. A video-slide scanner is used for automated scanning of microscopical slides. Diagnostic nuclear classifiers have tissue-specifically been trained by an expert-cytopathologist (A. B.), based on Random Forest Classifiers, applying 18 different morphometric features. They achieve an overall accuracy of 91.1% to differentiate 8 differents types of objects/nuclei. Nuclear DNA-measurements of diploid nuclei achieve a CV of 1% morphometrically abnormal nuclei. Time needed for loading, scanning and validation of results per slide is about 10 minutes. Results of digital diagnostic nuclear classification can be verified by a cytopathologist, using image galleries. Likewise automated diagnostic interpretation of nuclear DNA-distributions can be checked on the monitor, before a pathologists validated diagnoses are issued. Screening-results are presented for body cavity effusions and urines. Evaluations of dysplasias are reported for oral, bronchial and cervical smears. Results of grading malignancy are shown for prostate cancers.

Made available in DSpace on 2021-06-25T11:11:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-04-01 Objective: The aim of this study was to investigate cytogenetic and cytotoxic damage through the evaluation of micronuclei (MN) and metanuclear anomalies in the oral mucosa of electronic cigarette (e-cig) users. Study Design: The patients were recruited into 4 groups: e-cig users, smokers, former smokers, and nonsmokers (control). The samples were collected by means of exfoliative cytology of the lateral region of the tongue and the floor of the mouth. The smears obtained were fixed and stained by the Feulgen method for investigation of MN and metanuclear anomalies. Results: A significant difference was observed for MN frequency only between the smoker and control groups. As for metanuclear anomalies, significant differences were observed: karyolysis between: smokers and control, e-cig and control, as well as former smokers; karyorrhexis: between smoker and control; binucleation: between e-cig and former smoker, as well as control; broken eggs: between e-cig and all other groups; nuclear bud: between e-cig and former smokers, as well as control. Conclusions: E-cig and alcohol users presented genotoxicity and cytotoxicity in the oral mucosa cells. The use of e-cigs and alcohol by former smokers can cause more damage to the cells of the oral mucosa compared to those who have not used e-cigs. Department of Biosciences and Diagnosis Institute of Science and Technology São Paulo State University (Unesp) School of Dentistry Universidade Mogi das Cruzes School of Medicine Universidade Anhembi Morumbi Outpatient Smoking Treatment Program of the Heart Institute (Incor) Faculty of Medicine University of São Paulo (FMUSP) Department of Biosciences and Diagnosis Institute of Science and Technology São Paulo State University (Unesp)

Genome size, known also as the C-value, has been proposed as an important determinant of life-history variation in numerous animal taxa. We assessed the relationships between genome size and fitness-related life-history traits in six species of interstitial marine annelids of the genus Ophryotrocha. Life-history traits and genome size data obtained from 18 additional annelid species were included in our analyses to have a broader phylogenetic scope. Unexpectedly, genome sizes assessed here by flow cytometry in four Ophryotrocha species were three times larger than previously reported values obtained using Feulgen densitometry. This has implications for the hypothesis that harsh interstitial habitats select for small genomes in meiofaunal annelids. Within the genus Ophryotrocha, significant and positive relationships were found between genome size and nucleus size, and between genome size, age at first egg mass deposition, body size and lifespan. These relationships held up in the broader phylogenetic comparison. Our study provides evidence for the important role played by genome size in the evolution of life-history traits in annelids.

Paspalum cromyorrhizon Trin. ex Döll is a species adapted to low and humid fields, river and stream banks, throughout S Brazil, Uruguay and NE Argentine. Diploids (2n = 2x = 20) and tetraploids (2n = 4x = 40) from natural populations of P. cromyorrhizon from Corrientes (Argentina) were used to analyse the karyotype morphometry of both cytotypes. The chromosome count was performed using classical Feulgen staining. The karyotype of diploid P. cromyorrhizon has 20 metacentric chromosomes, while the tetraploid has a karyotype of 40 metacentric chromosomes. The total length of the chromosomal complement in diploids is 32.5 μm and in tetraploids 63.5 μm, the total monoploid chromosome length is 15.48 μm in diploids and 15.21 μm in tetraploids and both karyotypes are symmetrical. Karyotype analysis confirms the autopolyploid origin proposed for tetraploids.

A retrospective examining of cytology specimens obtained and verified by a fine-needle aspiration biopsy from patients with autoimmune thyroiditis and benign thyroid gland (cyst and goiter) formations allowed to note that in thyroid lobes they coincided in various combinations, thus rising a question about their potential etiological relation. In particular, a hemosporidian (blood parasitic) infection was found while analyzing cytology specimens from patients with autoimmune thyroiditis and benign thyroid gland (cyst and goiter) tumors prestained by Romanowsky-Giemsa dye. An evolution of developing intra-thyrocyte hemosporidia was tracked during a long-term detailed analysis of cytology specimens noted above. A panel of select specimens was stained (re-stained) with Schiff reagent according to the Feulgen method to clarify position of thyrocyte DNA and hemosporidian pathogens. Owing to an absorption approach, Romanovsky-Giemsa method allowed to repeatedly use specimens pre-stained with Schiff reagent according to the Feulgen method, wherein fuchsine was incorporated into DNA molecules after they were hydrolyzed by hydrochloric acid to stain specimens into magenta-lilac color. It allowed to identify a parasitic DNA inside developing hemosporidia most probably at exoerythrocytic stage and some erythrocytes cyst-based medusiform structures. Such technique used to stain specimens from patients with autoimmune thyroiditis allowed to localize the thyrocyte nuclear DNA as well as punctate and diffuse cytoplasmic inclusions of parasitic DNA, including magenta-lilac nuclei of different sizes inside erythrocytes. Thyrocyte nuclear DNA as well as punctate and diffuse hemosporidian DNA were distinguished in nodular goiter. Moreover, hemosporidian DNA was identified in a form of magenta-lilac multi-size nuclei inside erythrocytes. In contrast, unstained hemosporidian protoplasm was revealed as light-colored band around erythrocyte nuclei. The intra-erythrocyte nuclear hemosporidian material of different sizes may evidence about various species and/or pathogen generations. Intra-thyrocyte development of hemosporidian infection in patients with goiter results in marked cytoplasmic hyperplasia and its vacuolization associated with thyrocyte nuclear deformation, vacuolization, decreased size and degradation (with highly probability of mutations and deletions), reaching a pre-neoplastic level.

Aim Waterpipe tobacco smoking (WTS) has become a global epidemic, especially among youth. WTS has not been studied like cigarette smoking. There is a dire need to study and document health effects of waterpipe smoking in general and specifically on the oral cavity. Materials and methods A total sample size of 400 was studied. One hundred exclusive shisha smokers, 100 exclusive cigarette smokers, 100 subjects smoking both cigarette and shisha and 100 non-smokers. We recorded and associated socio-demographical data pertaining to WTS and cigarette smoking in UAE and examining their toxic effects on the oral mucosa at a cytogenetic level by studying the micronuclei (MN) stained by Feulgen and Acridine Orange (DNA specific stains). Results A significant association was observed between age distribution and groups. Majority of subjects were males. Arabic nationalities were consuming a higher percentage of Waterpipe and Cigarette separately, and Indian nationality was consuming a higher percentage of waterpipe and cigarette together. Comparison of Micronuclei in Feulgen and Micronuclei in Acridine Orange group between four groups was significant. Mean micronuclei in feulgen was highest for Waterpipe and Cigarette group followed by shisha group and least for Control group. Similarly, Mean micronuclei in acridine orange was highest for waterpipe and cigarette group followed by shisha group and least for control group. Conclusion Further epidemiological studies should be undertaken to determine whether WTS is associated with the incidence of lung cancer/oral cancerous lesions. Clinical significance The findings of this study could be used to spread awareness that waterpipe smoking, like cigarette smoking, has the potential to cause genotoxic effects and could eventually lead to carcinogenicity based on duration and frequency.

Nuclear size and shape are important components in the diagnosis of pathological specimens. However, a qualitative evaluation is typically applied rather than a quantitative evaluation technique. In the present study, we sought to evaluate the nuclear morphological characteristics of lung adenocarcinoma using whole-slide imaging (WSI) and computer-assisted image analysis (IA). We evaluated the nuclear characteristics of 106 cases of surgically resected lung adenocarcinoma according to Feulgen staining and immunohistochemistry (IHC) for the inner nuclear membrane protein emerin. According to the Feulgen reaction, although the nuclear area (size) of the carcinoma cells was correlated with the nuclear perimeter (NP) (R=0.8973), the nuclear staining intensity of carcinoma cells was not correlated with the nuclear area. Using emerin IHC, we used IA software that was able to detect both the NP and the emerin-stained nuclear membrane length (ENML) in the nucleus, and found that the more nuclei exhibited a longer ENML relative to the NP, the more nuclear grooves and intranuclear cytoplasmic inclusions were present. In addition, the nuclear area was correlated with the percentage of nuclei that had a longer ENML compared to the NP against the total nuclei (R=0.7759). Furthermore, the emerin low expression group showed an enlarged nuclear area (P=0.0264), elongated NP (P=0.0091), and lower shape factor (P=0.0486) compared with the normal emerin expression group. Our data indicated the usefulness of WSI and IA for pathological specimen analysis. In addition, this study is the first to report that the low expression of emerin in cancer cell results in an oval shape of nuclei and nuclear enlargement in clinical samples.

Aim: The present study aims to assess the genotoxic effect of various forms of tobacco users on buccal mucosa and nuclear changes as biomarkers. Materials and Methods: The study involves 150 cases, they were divided into three groups (two study groups and one control group). The buccal cytological smears were taken from three groups: Group I – 50 smokers, Group II – 50 nonsmokers (smokeless tobacco), and Group III – 50 control group. The buccal cells were transferred into a test tube containing Tris-ethylenediaminetetraacetic acid buffer (pH = 7) and was centrifuged (Remi® 1500 revolution/min [rpm]). Cell suspensions were transferred to the slides and fixed. The slides were stained using PAP and Feulgen stain. The MN and other nuclear abnormalities were studied and compared. Results: Nonsmokers (smokeless tobacco) had significantly increased frequency of all nuclear anomalies compared to smokers and healthy controls. Binucleation, karyorrhexis, micronuclei (MN), karyolysis, broken egg nuclei, and prominent nucleoli in nonsmokers (smokeless tobacco) and condensed chromatin in smokers were the most frequent anomalies. Binucleation and karyorrhexis were significantly more frequent in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear abnormalities were not statistically significant in smokers and nonsmokers (smokeless tobacco). Conclusion: Numerous studies have stated that MN and other nuclear anomalies were present in higher frequency in smokers and nonsmokers. In our study, we found binucleation and karyorrhexis were statistically significant in nonsmokers (smokeless tobacco) compared to smokers. The other nuclear anomalies showed insignificant results. In order to further validate the significance of this study, a larger sample size has to be studied. On comparing the staining efficacy of smokers and nonsmokers using PAP and Feulgen stain, both the stains showed positive results. In the present study, DNA-specific Feulgen stain shows better staining of nuclear anomalies compared to DNA nonspecific PAP stain, which was found to be statistically significant.

Introduction: Establishing one’s sexual identity has a critical role in medical conditions like ambiguous genitalia and in crime investigations. There are various investigations to determine the sexual identity of a person and Barr body evaluation in buccal smears forms the first line of investigation. Barr body estimation in buccal mucosal scrapes has been demonstrated by using different stains like Papanicolaou, Aceto-Orcein(AO), Feulgen, Guard, Cresyl violet, Carbol fuchsin and fluorescent staining methods, but with varying efficacy. The current study is done to evaluate the efficacy of Papanicolaou and AO stains in demonstration of Barr bodies. Materials and Methods: A total of 207 medical students were included in the study. Two buccal smears were collected from each student and subsequently, one was stained with Aceto-Orcein by squash technique and the other with Papanicolaou stain. Both slides were evaluated for percentage of Barr bodies using 1000x magnification and cytomorphological features in 400x. Results: The percentage of Barr bodies in AO stained slides ranged from 5-18 among females and 0-8 in males, while with Papanicolaou stain the ranges recorded were 4 – 12 in females, 0 – 2 in males. The accuracy of AO and Papanicolaou stains for detecting sex accurately were 97% and 91% respectively. Evaluation of the buccal smears was better in AO stained smears because of the clean background and better cytoplasmic and nuclear contrast in comparison to PAP stain. Conclusion: Aceto-Orcein staining method is rapid, economical, accurate, reproducible and comparable to Papanicolaou staining, for the detection of Barr body in buccal mucosal smears. Keywords: Aceto-Orcein, Papanicolaou, Buccal smears, Barr body.

Chromosome numbers, karyotypes, and genome sizes of 14 Iris L. (Iridaceae Juss.) species in Korea and their closely related taxon, Sisyrinchium rosulatum, are presented and analyzed in a phylogenetic framework. To date, understanding the chromosomal evolution of Korean irises has been hampered by their high chromosome numbers. Here, we report analyses of chromosome numbers and karyotypes obtained via classic Feulgen staining and genome sizes measured using flow cytometry in Korean irises. More than a two-fold variation in chromosome numbers (2n = 22 to 2n = 50) and over a three-fold genome size variation (2.39 pg to 7.86 pg/1 C) suggest the putative polyploid and/or dysploid origin of some taxa. Our study demonstrates that the patterns of genome size variation and chromosome number changes in Korean irises do not correlate with the phylogenetic relationships and could have been affected by different evolutionary processes involving polyploidy or dysploidy. This study presents the first comprehensive chromosomal and genome size data for Korean Iris species. Further studies involving molecular cytogenetic and phylogenomic analyses are needed to interpret the mechanisms involved in the origin of chromosomal variation in the Iris.

Cervical cancer is the second most common cancer type in women around the world. In some countries, due to non-existent or inadequate screening, it is often detected at late stages, making standard treatment options often absent or unaffordable. It is a deadly disease that could benefit from early detection approaches. It is usually done by cytological exams which consist of visually inspecting the nuclei searching for morphological alteration. Since it is done by humans, naturally, some subjectivity is introduced. Computational methods could be used to reduce this, where the first stage of the process would be the nuclei segmentation. In this context, we present a complete pipeline for the segmentation of nuclei in Feulgen-stained images using Convolutional Neural Networks. Here we show the entire process of segmentation, since the collection of the samples, passing through pre-processing, training the network, post-processing and results evaluation. We achieved an overall IoU of 0.78, showing the affordability of the approach of nuclei segmentation on Feulgen-stained images. The code is available in: https://github.com/luizbuschetto/feulgen_nuclei_segmentation., 7 pages, 8 figures (Figure 2 with "a" and "b"), conference paper accepted for presentation in XI Computer on the Beach (https://www.computeronthebeach.com.br/)

Compared with small model plants like Arabidopsis containing ovules with few cell layers, embryo sac and embryo development of model crop plants such as maize and other grasses are difficult to image. Multiple layers of tissue usually surround the deeply embedded embryo sac and developing embryo. Moreover, reliable cell biological marker lines labeling, for example, nuclei, plasma membrane, cell walls, or cells of a specific identity are often not available. The introduction of markers to study mutants is difficult and time-consuming and may require several generations of backcrosses. In this chapter, we therefore present an easy protocol to image maize ovaries and developing embryo sacs before and after fertilization allowing also high-throughput mutant analysis. The laborious embedding of samples and preparation of thin sections are omitted in this fixing-Feulgen staining-clearing (FFC) method. Optical sectioning through multiple layers of tissue is possible allowing 3D reconstructions of the whole embryo sac if necessary. The advantage of staining cell nuclei using the FFC method described here compared, for example, with DAPI staining is a wide range of Schiff's type reagents available for the Feulgen reaction. Depending on the reagent of choice, various conditions such as different excitation/emission filters or even white light can be applied for imaging. Moreover, in order to better visualize cell division, nuclei polarity as well as cell extent and integrity, periodic acid staining (PAS) of cell walls can be combined with Feulgen staining.

This study focused on a method based on the capacity of cationic dyes to stain only the nucleus and chromosomes in cells subjected to either acid or alkaline hydrolysis. The method and the squash were optimized for the Characeae and were described in detail. Nuclei from vegetative shoot apices and antheridial filament cells of unfixed, fixed and herbarium material of Nitella opaca were investigated using the Azure A or Toulidine Blue stains. Comparisons with some other staining methods, used in the previous studies, were also reported. The Azure A/Toulidine Blue method is useful to obtain clear images of chromosome morphology comparable or higher to that obtained with Feulgen or Aceto-Orcein. It requires little time and a less complicated procedure in comparison with other staining methods.

Quantitation of cell DNA content, DNA ploidy, has been established as a research and prognostic technique for decades. A variety of instruments have been used although only a few commercially available systems have established quality assurance and published outcome data. The aim of this study was to compare two automated systems.Nuclear monolayers were obtained from 112 oral biopsies by enzyme digestion and Feulgen staining. These were scanned on both the Fairfield and the Ploidy Work Station (PWS) systems. The overall ploidy diagnosis, number of epithelial nuclei, coefficient of variation (CV) and 5c exceeding rate (5CER) were compared by quantile-quantile plots, t test, Wilcoxon and Spearman's tests.The PWS system identified more nuclei (p 0.0001) at a lower CV (p 0.0001). Using the PWS system, fewer samples were classified as indeterminate. No difference between 5CER was found between systems (p 0.54). There was complete concordance between the two systems in terms of DNA ploidy diagnosis.The PWS system is comparable to the Fairfield system for determination of DNA ploidy and has advantages that may lead to improved performance.

Introduction: Micronucleus/micronuclei) have the potential to serve as an important biomarker in exfoliated cell. Exfoliated buccal mucosal cells can be evaluated using MN assay which can be applied to individuals practicing tobacco habits and are at risk of developing oral cancer. Material and Methods: 80 tobacco users and 20 non tobacco users as control were included in the study. Smears were taken and stained with H&E, feulgen, acridine orange. MN was counted using criteria given by Tolbert et al. Data was analyzed using SPSS software. Results: MN higher in tobacco users. The values obtained for the MN count in tobacco chewers using these stains were statistically significant (P

Context: A huge number of people carry dental fillings which contain either mercury-based amalgam and/or the recently introduced methacrylate-based resins. It has been shown that both these materials are known to be leached into the oral cavity and induce genotoxic alterations in the buccal mucosal cells. Because of its low cost and ease of manipulation, dental amalgam is still widely used as a restorative material in developing countries. The health risks associated with the components of this restorative material has always been a matter of concern. The present study was designed to assess the frequency of micronuclei (MN) in oral mucosal cells as it is a promising tool for studying the genotoxic effect of clastogenic agents on them. Aims: The aim of this study is to evaluate the genotoxic effects of silver amalgam and composite restorations by measuring the mean number of MN in oral exfoliated cells. Materials and Methods: The present study was a prospective cohort study which includes a study group consisting of 110 participants. The study sample was equally divided into 55 participants requiring only amalgam restoration and 55 participants requiring only composite restoration in any permanent molar teeth. The same participants before the restoration formed the control group. Smears were obtained from each patient before and 10 days after restoration and were stained with DNA-specific Feulgen stain. The number of cells containing MN out of 500 cells were counted and recorded. After the evaluation of the slides, the results were compiled and subjected to statistical analysis. Results: There was a statistically significant (P < 0.01) variation in the mean number of MN after the restoration in both amalgam (5.41 ± 1.25) and composite (2.83 ± 0.85) restorations when compared to before the restoration. However, the mean number of MN in composite restoration was significantly less when compared to amalgam restoration. There was also a statistically significant difference in the mean number of MN in subjects with single restoration when compared with multiple restorations in both amalgam and composite restorations. Conclusions: The observations from the present study showed the genotoxic effect of amalgam and composite restorations on the oral cavity. However, composite restorations were least cytotoxic when compared to amalgam restoration. Future research and technical advancements are needed for developing safer materials for use in humans.

Electromagnetic fields (EMF) are classified as "possibly carcinogenic" by the International Agency for Research on Cancer (IARC). Some publications have reported associations between EMF exposure and DNA damage, but many other studies contradict such findings. Cytomorphological changes, such as micronuclei (MN), indicative of genomic damage, are biomarkers of genotoxicity. To test whether mobile phone-associated EMF exposure affects the MN frequency in exfoliated buccal cells, we obtained cells smears from the left and right inner cheeks of healthy mobile phone users, aged 18-30 (n=86), who also completed a characterization survey. MN frequencies were tested for potential confounding factors and for duration of phone use and preferential side of mobile phone use. No relationship was observed between MN frequency and duration of mobile phone use in daily calls. Cells ipsilateral to mobile phone use did not present a statistically significantly higher MN frequency, compared to cells contralateral to exposure. A highly statistically significant (p

Objective: The purpose of this study is to determine the mitotic chromosome number, morphometric parameters, and karyotypes of Xanthium strumarium and make contributions to other multidisciplinary studies on the genus. Material and Methods: The seeds were germinated on moist filter paper in Petri dishes at 25°C. Actively growing root tips were pre-treated with aqueous paradichlorobenzene for 4h at room temperature. Then, the root tips were fixed with acetic alcohol (1:3 glacial acetic acid–absolute ethanol) for at least 24 h at 4°C, hydrolysed in 1 N HCl at 60°C for 3 min, then rinsed in tap water for 3–5 min. Finally, they were stained in Feulgen for 1 h and mounted in 45% acetic acid. Digital microphotographs from at least five well-spread metaphase plates were taken using an Olympus BX51 microscope and were recorded with an Olympus Camedia C-4000 digital camera. Results: The chromosome number is determined as 2n = 36 for this taxon. The karyotype consists of 16 median region (m) and 2 submedian region (sm) chromosomes. The metaphase chromosome length ranges from 2.30 to 4.03 μm, longest to shortest chromosome ratio is 1.7:1.1, total karyotype length (TKL) 54.76 mm and the karyotype symmetry is type 1A. Conclusion: The results of this study showed that the chromosome number of Xanthium strumarium is 2n=36. Satellites were not observed in the karyotype of this species.Identifying the chromosome number of this species in this study provides a base for biosystematic studies.

The Feulgen reaction has been proposed by Robert Feulgen and Heinrich Rossenbeck for the identification of DNA nearly a hundred years ago. Since then, many other applications of this cytochemical/topochemical procedure at qualitative and quantitative level have been proposed in relation to DNA and its role in chromatin in human, animal and plant cells. In this article, we briefly review some fundamental aspects of the Feulgen reaction and current applications of such a method in studies of altered chromatin texture, including its association with or preceding changes in transcriptional activities and effect on epigenetic marks. Further perspectives on the use of the Feulgen reaction will depend of the proposal of innovative biological questions in which its reveals appropriate.

Background To evaluate HIV infection-induced alterations in the oral mucosa by comparing inflammation, cell maturation and cytomorphometric changes in oral mucosal cells between HIV-infected patients undergoing highly active antiretroviral therapy (HAART) and non-HIV-infected patients. \ Methods Thirty HIV-infected patients undergoing HAART and 30 non-HIV-infected patients were studied. Four smears were obtained from the lateral border of the tongue and floor of the mouth with a cytobrush. One sample was stained by the Papanicolaou technique and three samples were processed for Feulgen staining. Papanicolaou-stained smears were analyzed by light microscopy and the cytoplasmic (CA) and nuclear (NA) area was measured with the Axion Vison 4.7 program (Carl Zeiss). Results The Wilcoxon signed-rank test showed a significant difference in intermediate epithelial cell types between the HIV-infected and non-HIV-infected groups (p=0.005). However, this difference was not observed for superficial epithelial cell types with (p=0.672) and without a nucleus (p=0.069). Comparative analysis revealed no significant difference in CA (p=0.604), NA (p=0.298) or NA/CA (p=0.456) between the HIV-infected and non-HIV-infected groups. Keratohyalin granules were more frequent in the non-HIV-infected group (p=0.0001). Conclusions The results showed alterations in cell maturation in HIV-infected patients undergoing HAART with undetectable viral load, but no morphometric changes were observed. This article is protected by copyright. All rights reserved.

Background Approximately 5 million workers are estimated to be occupationally exposed to welding fumes worldwide. Nickel and chromium are genotoxic metals found in welding fumes, therefore welders are exposed to these metals at the workplace. Objective The objective of the present study was to investigate the frequency of cytotoxic and genetic damage in cells harvested from the oral mucosa of welders and also from a group of workers not exposed to metallic fumes. Methods A total of 44 individuals, divided into 2 groups - welders and non-welders - were compared using the micronucleus assay technique and cell death (pyknosis, karyorrhexis and karyolysis) on buccal mucosa cells of welding workers. The examined cells were stained with Feulgen/Fast Green. Results: Welders exhibited higher frequency (p

Background: Exfoliative cytology is an easy, economical, noninvasive, and feasible method for early detection and screening program of any premalignant or malignant lesion. In case of routine cytological procedure, classical Papanicolaou (PAP) stain is widely used while Romanowsky stains are sparingly used. Leishman-Giemsa (LG) cocktail, being a easier, cost effective staining technique, has not been used in exfoliative cytology. Therefore, this pilot study was carried out to compare and contrast the role of LG stain in routine cytological procedure which is very cost-effective, less time-consuming and requires less infrastructural support. Aim and Objectives: To study the diagnostic efficiency of LG cocktail in comparison with PAP stain and Feulgen stain in mucosal cells for evaluating cellular changes of oral squamous cell carcinoma (OSCC). Materials and Methods: Three cytological smears were prepared from 10 healthy controls and 10 patients clinically diagnosed with OSCC, and they were stained with LG cocktail stain, PAP stain, and Feulgen stain. The stained smears were evaluated for cytologic diagnosis and the staining characteristics such as nuclear and cytoplasmic details were recorded as per criteria by Sujathan et al. Statistical Analysis: The data were statistically evaluated with analysis of variance test using SPSS 15 software for windows. Results: The results from the cases diagnosed as OSCC by PAP and LG cocktail were almost identical and superior to Feulgen stain both in diagnostic ability and in staining characteristics. Conclusion: The one-step LG cocktail is easy, very cost-effective, less time-consuming with less infrastructural support as compared to PAP stain; however, it warrants further evaluation for screening of oral cancer as a potential aid.

Objective: In this study the somatic chromosome numbers and detailed morphometric properties of Tulipa pulchella (Fenzl ex Regel) Baker was examined. Material and Methods: The seeds were germinated on moist filter paper in petri dishes at 25°C. Actively growing root tips were pretreated with 0.05 M colchicine for 6 h at room temperature. Then, the root tips were fixed with acetic alcohol (1:3 glacial acetic acid–absolute ethanol) for at least 24 h at 4°C, hydrolyzed in 1 N HCl at 60°C for 1-2 min, then rinsed in tap water for 3–5 min. Finally, they were stained in Feulgen for 1 h and mounted in 45% acetic acid. Digital microphotographs from at least five well-spread metaphase plates were taken using an Olympus BX51 microscope X40 objective and were recorded with an Olympus Camedia C-4000 digital camera. Results: The chromosome number of Tulipa pulchella is determined as 2n = 24 for this taxon. The karyotype consists of 8 submedian region (sm) and 4 subterminal (st) chromosomes. The metaphase chromosome length ranges from 9.35 to 16.48 μm, longest to shortest chromosome ratio was 1.9:4.1, total karyotype length (TKL) was 150.99 um and the karyotype symmetry was type 1A. Conclusion: The basic chromosome number of Tulipa pulchella is x=12. The majority of tulip species and cultivars is diploid (2n=2x=24), however, encounters to triploids (2n=3x=36), tetraploids (2n=4x=48) and even some pentaploids (2n=5x=60) and hexaploid (2n=6x=72). Identifying the chromosome number of this species in this study would provide a base for biosystematic studies.

Background Due to increased formaldehyde exposure, carcinogenic to humans, several researches have been studying the potential toxicity and the safe levels for human beings. The aim of this study was to investigate mutagenicity and cytotoxicity in buccal epithelial exfoliated cells (BEC) of students subjected to formaldehyde (FA) during anatomy classes. Material and Methods BEC were collected periodically from 17 volunteers of undergraduate programs, who had participated in practical anatomy classes, before and after FA exposure. Cells were stained according to Feulgen method and then micronucleus test was applied. A total of 1,500 cells were assessed per individual in this study for the micronucleus frequency and other parameters of cytotoxicity. Results There was statistically significant increase in number of micronucleated BEC after FA exposure (after 1 month p=.034 and after 3.5 months p=.017). However, FA exposure caused no significant increase in other nuclear alterations closely related to cytotoxicity (p≥.05). Conclusions FA induced mutagenicity during anatomy classes. Cell death increased, but it was not statistically significant. Efforts have to be made to improve air quality and reduce exposures during anatomy classes. Key words:Carcinogens, formaldehyde, micronucleus tests, mutagenicity tests.

Chromosome number changes and karyotype evolution play an important role in plant genome diversification and eventually in speciation. The genus Ajuga L. (Lamiaceae) has approximately 50 species distributed in temperate to subtropical regions. Four of these species are currently recognized in Korea (A. decumbens Thunb., A. multiflora Bunge, A. nipponensis Makino and A. spectabilis Nakai). Understanding the karyotype evolution in Ajuga has been hampered by the small size of their chromosomes and symmetrical karyotypes. Here we used classic Feulgen staining to establish chromosome numbers and construct karyotypes of the four species of Ajuga recognized in Korea and flow cytometry was used to study their variation in genome. The chromosome number of all investigated plants was 2n = 32. Still, the 2C DNA content ranged from 2.18 pg (A. decumbens) to 4.53 pg (A. multiflora). While the chromosome numbers were the same for all investigated species, the genome size variation could potentially be used as a taxonomic marker.

Objective: To evaluate the histochemical alterations in DNA and total carbohydrates, in colorectal cancer cells. Methods: This study was carried out on 48 colorectal carcinoma and 10 adenoma specimens. Hematoxylin and Eosin staining was carried out for histopathological examination to confirm the diagnosis and to evaluate the histopathological characteristics of tumor. Histologic grade and pathologic stage was assessed according to TNM staging system. Staging was also assessed according to original Dukes’ staging system. DNA was demonstrated by Feulgen method and carbohydrates were demonstrated by periodic acid Schiff’s reaction. Results: Adenoma cases showed that the cells lining the glands of the polyp have more crowded, irregular and darker nuclei (hyperchromatic), anisonucleosis, abnormal mitotic figures with prominent nucleoli and variability in the size and shape of nuclei. Colorectal carcinoma cases showed a condensation and reduction in the size of a cell nucleus associated with hyperchromatosis, pyknotic nuclei, abnormal mitotic figures, anisonucleosis, irregular nuclear membrane and inequality in the size of the nuclei (Pleomorphosis). There was a statistical significant differences between adenoma and carcinoma regarding number of mitotic cells (P = 0.03) that was in favour of malignant group. Adenoma and colorectal carcinoma cases showed periodic acid Schiff’s reactivity with different degree. Conclusions: These histochemical alterations can be so characteristic of a given tumor type and stage that they are used in cancer diagnosis and might also be related to the altered functional properties of cancer cells. Article history

Introduction: macrolide antibiotics are a class of potent and well established antimicrobials that also possess anti-inflammatory and/or immunomodulatory properties. Because of their size, lower levels of macrolides are able to reach the developing fetuses. Materials and method: the pregnant rats were orally administered with clarithromycin at early and late gestational periods. The 20 day-old fetuses were dissected for excision of the kidney. Half of the kidney was processed and stained with H & E, PAS, Masson’s trichrome and Feulgen techniques then followed by morphometric measurements and statistical study. The other half of the kidney was preserved for DNA fragmentation assay.Results: This study revealed that clarithromycin administration to pregnant rats showed different histopathological, histochemical and DNA changes in the kidneys of their fetuses. Conclusion: Administration of the antimicrobial agent; clarithromycin at early and late gestational periods exhibits nephrotoxicity in the developing fetuses.

For this study, we used leaves of fossilized Metasequoia nathorstii from the Middle Eocene sediments of the Axel Heiberg Island of the Canadian Arctic Archipelago, and the leaves of modern M. glyptostroboides collected from the garden of the Komarov Botanical Institute RAS. Feulgen staining of the Metasequoia fossil leaves revealed the preservation of Feulgen-positive, heterogeneously stained nuclear structures in the cells, similar to those in the cells of the modern plant. The nuclei were brown-red-stained, and the chromocenters were stained more intensely, suggesting that the general morphology of the cell nucleus was preserved and the aldehyde groups of the deoxyribose that were available to react with the Feulgen's basic fuchsin were preserved in the nuclear chromatin of the fossil plants. The additional staining of the preparations with the alcian blue and Ehrlich's hematoxylin displays the cell walls and the adjacent intracellular material that is colored from light to dark blue. Staining the preparations with dA + dT-specific fluorochrome DAPI induced a weak fluorescence of the fossil cell nuclei, indicating the presence of some dsDNA molecules in the Metasequoia fossil leaves.

Fixed orthodontic appliances made of metal alloys are routinely used in dental practise to correct malocclusion. Oral environment favours the biodegradation of metal alloys. The discharged metal ions accumulate cumulatively on the adjacent tissues and results in toxic effects on cellular metabolism and DNA stability. The study was designed to evaluate localized genetic and cytokinetic toxicity due to fixed orthodontic appliance by measuring micronuclei frequency and other nuclear abnormalities in the buccal mucosal cells. Subjects with orthodontic appliance were selected as exposure cohort (n=20) and subjects without orthodontic appliance were selected as comparative cohort (n=20). Buccal smears were collected and DNA specific feulgen stain was used. Micronuclei, nuclear bud and binucleate cells frequency were recorded and compared between the study groups. We found a significant increase in the frequency of cells with micronuclei (p

Objectives: To evaluate the genotoxic and cytotoxic effects of Haas appliances through micronuclei test and cytogenetic damage analysis in buccal mucosa epithelial cells of patients undergoing orthodontic treatment. Materials and Methods: Twenty-eight patients, 6–12 years of age and of both genders, who required a Haas appliance for the correction of a posterior crossbite were included. Epithelial cells from the mucosa were collected by gently scraping the inside of both the right and left cheeks. The cells were collected before the insertion of the appliance (T0), 1 month after the device was installed (T1), and again 3 months after the appliance was immobilized (T2). The cells were processed to obtain slides. Feulgen/Fast Green was used as the staining method, and the number of normal, karyolytic, pyknotic, nuclear buds, bi/trinucleated, and micronucleus cells were counted under light microscopy. Cellular abnormalities were evaluated with parametric and nonparametric tests for comparison of the means by analysis of variance testing, Tukey posttest, or the Kruskal-Wallis test and then by Dunn's posttest. The significance level was 5%. Results: There were no statistically significant changes in the micronuclei in the evaluated periods (P > .05). Nuclear buds increased at T1 (P < .05), returning to baseline levels at T2. Other abnormalities (cariolytic, pyknotic, and bi/trinucleated cells) showed a significant increase at T1 and T2 (P < .0001). Conclusions: The Haas appliance did not cause an increase in micronuclei in cells of the buccal mucosa. However, statistically significant increases in cariolytic, pyknotic, and bi/trinucleated cells were observed during treatment, suggesting possible DNA damage.