Ultrastructural and Immunohistochemical Detection of Hydroxyapatite Nucleating Role by rRNA and Nuclear Chromatin Derivatives in Aortic Valve Calcification: In Vitro and In Vivo Pro-Calcific Animal Models and Actual Calcific Disease in Humans.

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.

In vitro spermicidal effect of Thevetia Peruviana leaves on human spermatozoa.

An in vitro spermicidal effect of aqua-methanolic (2:3) extract of Thevetia peruviana leaves on human spermatozoa was evaluated in a dose-dependent manner (20, 40, 80 and 160 mg/ml) at a 1:1 ratio. Sperm motility, viability, hypo-osmotic swelling (HOS) and acrosomal status and function tests were performed immediately (20 s), and after 5 and 10 min of exposure of the spermatozoa to the extract of Thevetia peruviana leaves at different dose concentrations. Nuclear chromatin decondensation (NCD) test, DNA fragmentation test and sperm revival test were also evaluated. The sperm motility was affected immediately at a dose of 20 mg/ml and reduced gradually at doses of 40 and 80 mg/ml of Thevetia peruviana extract. Complete immobilisation of spermatozoa was observed at 160 mg /ml dose of this extract treatment within 5 min. 50% immobilisation of spermatozoa (EC50) was observed at 28 mg/ml dose of Thevetia peruviana extract within 20 s. The sperm viability decreased significantly at a higher concentration of extract, and all spermatozoa were found to be non-viable after 10 min when treated with 160 mg/ml dose of Thevetia peruviana extract. HOS and NCD of spermatozoa also reduced gradually at a higher concentration of extract administration. The percentage of DNA damage in spermatozoa was four times greater than in the control group. The findings indicate that the hydro-methanolic extract of Thevetia peruviana leaves possesses appreciably potent spermicidal activity through an in vitro model, which may explore an effective vaginal contraceptive.

A Paradigm Shift in Nuclear Chromatin Interpretation: From Qualitative Intuitive Recognition to Quantitative Texture Analysis of Breast Cancer Cell Nuclei.

Assessing the pattern of nuclear chromatin is essential for pathological investigations. However, the interpretation of nuclear pattern is subjective. In this study, we performed the texture analysis of nuclear chromatin in breast cancer samples to determine the nuclear pleomorphism score thereof. We used three different algorithms for extracting high-level texture features: the gray-level co-occurrence matrix (GLCM), gray-level run length matrix (GLRLM), and gray-level size zone matrix (GLSZM). Using these algorithms, 12 GLCM, 11 GLRLM, and 16 GLSZM features were extracted from three scores of breast carcinoma (Scores 1-3). Classification accuracy was assessed using the support vector machine (SVM) and k-nearest neighbor (KNN) classification models. Three features of GLCM, 11 of GLRLM, and 12 of GLSZM were consistent across the three nuclear pleomorphism scores of breast cancer. Comparing Scores 1 and 3, the GLSZM feature large zone high gray-level emphasis showed the largest difference among breast cancer nuclear scores among all features of the three algorithms. The SVM and KNN classifiers showed favorable results for all three algorithms. A multiclass classification was performed to compare and distinguish between the scores of breast cancer. Texture features of nuclear chromatin can provide useful information for nuclear scoring. However, further validation of the correlations of histopathologic features, and standardization of the texture analysis process, are required to achieve better classification results. © 2021 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.

Effects of T-2 mycotoxin on in vitro development and chromatin status of mouse embryos in preimplantation stages.

T-2 toxin is a mycotoxin produced by phytopathogenic fungi of the Fusarium genus and has many well-studied deleterious effects on mammalian cells and reproductive tract. Despite the wide scale studies, the effects on preimplantation stage embryos are lacking. The aim of our study was to investigate the impact of T-2 on the cleavage stage of mouse embryos with regard to development to blastocysts and nuclear chromatin status.Six-weeks-old BDF1 female mice were superovulated and placed together overnight with mature males. Zygotes were flushed 20 h after human chorionic gonadotropin injection and divided randomly into treated (supplemented with 0.5, 0.75, and 1 ng/ml T-2) and nontreated (control) groups. Embryos were cultured in vitro for 96 h. Developmental stage was evaluated in the 72(nd)- and 96(th)-h for assessment of development dynamics. At the end of culture period, blastocysts from treated and control groups with normal morphology were selected for nuclear chromatin analysis. Blastocysts were categorized (grade A, B, and C) depending on the proportion of blasomeres with micronuclei and/or lobulated nuclei.Our data show significant decrease in the proportions of blastocysts in the 0.75 and 1 ng/ml toxin-supplemented groups compared with the control group. Blastocyst rate did not differ in embryos treated with 0.5 ng/ml T-2 but 24 h delay was found in blastocoel formation in all the treated groups. Only grade A (21.1%) and B (78.9%) blastocysts were found in low-toxin-contaminated group similar to the control ones (50-50%). Grade C embryos appeared in the 0.75 ng/ml (10%) treated group and the rate increased significantly (33.3%) in the highest contaminated group.T-2 mycotoxin has a harmful effect on early embryo development which results in decreased blastocyst proportion, delayed blastulation, and increased rate of chromatin damage.

T-2 mycotoxin slows down the development of mouse blastocysts, decreases their blastomere number and increases chromatin damage.

The mycotoxin T-2 has many harmful effects on mammalian cells and reproductive functions. In the present study, the in vitro effect of T-2 toxin on mouse blastocysts was examined. Embryos were cultured in media supplemented with 0.5, 0.75 and 1 ng/ml T-2. Different exposure times were applied [96 h (treatment I) or 24 h following 72 h in toxin-free media (treatment II)]. Blastomere number, nuclear chromatin status and blastocoel formation were investigated in blastocysts. Our data show that the effect of T-2 toxin may vary depending on the stage of the embryo at the start of exposure. At 96 h of exposure, the blastocysts had blastomeres with normal chromatin quality but their developmental potential was decreased. After 24 h of exposure applied following a 72-h culture, blastomeres had a higher level of chromatin damage, although their developmental potential was the same as in the control embryos. In both cases, decreased mitotic rate was found, which resulted in decreased blastomere number even at low toxin concentration.

Translocation of nuclear chromatin distribution to the periphery reflects dephosphorylated threonine-821/826 of the retinoblastoma protein (pRb) in T24 cells treated with Bacillus Calmette-Guérin.

The standard treatment for non-muscle-invasive bladder cancer is intravesical Bacillus Calmette-Guérin (BCG) therapy, which is considered the only intravesical therapy that reduces the risk of progression to muscle-invasive cancer. BCG unresponsiveness, in which intravesical BCG therapy is ineffective, has become a problem. It is thus important to evaluate the effectiveness of BCG treatment for patients as soon as possible in order to identify the optimal therapy. Urine cytology is a noninvasive, easy, and cost-effective method that has been used during BCG treatment, but primarily only to determine benign or malignant status; findings concerning the efficacy of BCG treatment based on urine cytology have not been reported. We investigated the relationship between BCG exposure and nuclear an important criterion in urine cytology, i.e., nuclear chromatin patterns. We used three types of cultured cells to evaluate nuclear chromatin patterns and the cell cycle, and we used T24 cells to evaluate the phosphorylation of retinoblastoma protein (pRb) in six-times of BCG exposures. The results revealed that after the second BCG exposure, (i) nuclear chromatin is distributed predominantly at the nuclear periphery and (ii) the dephosphorylation of threonine-821/826 in pRb occurs. This is the first report of a dynamic change in the nuclear chromatin pattern induced by exposure to BCG. Molecular findings also suggested a relationship between this phenomenon and cell-cycle proteins. Although these results are preliminary, they contribute to our understanding of the cytomorphological changes that occur with BCG exposure.

Evaluation of in vitro sperm nuclear chromatin decondensation among different subgroups of infertile males in Mysore, India.

BACKGROUND: Infertility is a condition associated with multiple etiologies. Sperm nuclear chromatin decondensation is one of the important events that occur during fertilization. Abnormal spermatogenesis leads to improper protamine package and chromatin condensation. The aim of the study was to analyze and understand the levels of fertilization capacity and nuclear stability of the spermatozoa in different infertile subgroups. MATERIAL AND METHODS: A total of 65 infertile males and 24 fertile males were employed in the study. Infertile subjects were classified into different groups according to the World Health Organization (WHO) protocol. In this study, in vitro nuclear chromatin decondensation status was assessed in different subgroups of infertile males. The obtained data was then statistically analyzed. RESULTS: Decreased sperm chromatin decondensation was observed in different infertile subgroups compared to the control group (p < 0.05). Spermatozoa with swollen head indicated a positive response and unswollen head indicated a negative response. CONCLUSION: This study asserts that abnormal nuclear decondensation is a potential factor that diminishes the fertilizing capacity of the sperms among different subgroups of infertile males.

Sperm nuclear maturity and chromatin stability in subfertile patients: Density gradient centrifugation is fair but non-discriminative in selecting the right population.

Multiple technologies exploring chromatin structure anomalies have been applied during the last decade to evaluate fertility disorders and to increase the predictive value of sperm analysis for procreation in vivo and in vitro. Our aim was to implement sperm nuclear maturity and nuclear chromatin stability as a functional test for male infertility diagnosis and to compare it with a fertile group. As semen processing is an integral part of assisted reproductive technologies the impact of density gradient centrifugation in selecting sperm based on nuclear maturity and stability was also analyzed. Flow cytometry combined with fluorescent dyes exhibiting affinity for DNA was implemented. Both nuclear parameters correlated significantly with semen parameters. The control fertile group had significantly higher mean condensed population and a significantly lower hypocondensed and hypercondensed fractions as compared to the subfertile study group. Density gradient centrifugation succeeded in selecting the condensed population in both the control and study groups, while reducing the hypocondensed percentage. The hypercondensed population which was ten-fold higher in the study group remained unchanged after selection, in both the control and the study groups. Sperm nuclear maturity and chromatin stability appears to be homogenous in the fertile sperm donors and heterogeneous in subfertile patients. Sperm preparation for assisted reproduction should aim to minimize the risk of abnormal spermatozoa being used for fertilization.

Evaluation of nuclear chromatin using grayscale intensity and thresholded percentage area in liquid-based cervical cytology.

OBJECTIVES: Development of computerized image analysis techniques has opened up the possibility for the quantitative analysis of nuclear chromatin in pathology. We hypothesized that the features extracted from digital images could be used to determine specific cytomorphological findings for nuclear chromatin that may be applicable for establishing a medical diagnosis. METHODS: Three parameters were evaluated from nuclear chromatin images obtained from the liquid-based cervical cytology samples of patients with biopsy-proven high-grade squamous intraepithelial lesion (HGSIL), and compared between non-neoplastic squamous epithelia and dysplastic epithelia groups: (1) standard deviation (SD) of the grayscale intensity; (2) difference between the maximum and minimum grayscale intensity (M-M); and (3) thresholded area percentage. Each parameter was evaluated at the mean, mean-1SD, and mean-2SD thresholding intensity levels. RESULTS: Between the mean and mean-1SD levels, the thresholded nuclear chromatin pattern was most similar to the chromatin granularity of the unthresholded grayscale images. The SD of the gray intensity and the thresholded area percentage differed significantly between the non-neoplastic squamous epithelia and dysplastic epithelia of HGSIL images at all three thresholding intensity levels (mean, mean-1SD, and mean-2SD). However, the M-M significantly differed between the two sample types for only two of the thresholding intensity levels (mean-1SD and mean-2SD). CONCLUSIONS: The digital parameters SD and M-M of the grayscale intensity, along with the thresholded area percentage could be useful in automated cytological evaluations. Further studies are needed to identify more valuable parameters for clinical application.

Role of mammalian sperm nuclear structure in fertilization and embryo development.

Although intact spermatozoon is successfully collected from infertile patients, repeated implantation failure or pregnancy loss are often experienced. Sperm nuclear defects have been thought to be one of the most important reasons for repeated assisted reproductive technology failure. In comparison with other mammalians, characteristic heterogeneity has been found in each mature human sperm nuclei, therefore it is necessary to investigate the significance between fertilization failure, developmental disability and structural abnormality of human sperm nuclei. Furthermore, if close relationships between the heterogeneity of human ejaculated sperm nuclei and DNA fragmentation are defined by analyzing sperm nucleoproteins, it would be clearly shown that impaired sperm chromatin leads to failure of embryo development in vitro or in vivo, so called late paternal effect on embryo development. It will be necessary in the near future to study the strategy for more novel methodology than those previously reported in terms of sperm selection. The present report reviews the roles of mammalian sperm nuclear structure, especially in humans, in fertilization and embryo development after the insemination procedure. (Reprod Med Biol 2006; 5: 161-168).

Evaluating IPMN and pancreatic carcinoma utilizing quantitative histopathology.

Intraductal papillary mucinous neoplasms (IPMN) are pancreatic lesions with uncertain biologic behavior. This study sought objective, accurate prediction tools, through the use of quantitative histopathological signatures of nuclear images, for classifying lesions as chronic pancreatitis (CP), IPMN, or pancreatic carcinoma (PC). Forty-four pancreatic resection patients were retrospectively identified for this study (12 CP; 16 IPMN; 16 PC). Regularized multinomial regression quantitatively classified each specimen as CP, IPMN, or PC in an automated, blinded fashion. Classification certainty was determined by subtracting the smallest classification probability from the largest probability (of the three groups). The certainty function varied from 1.0 (perfectly classified) to 0.0 (random). From each lesion, 180 ± 22 nuclei were imaged. Overall classification accuracy was 89.6% with six unique nuclear features. No CP cases were misclassified, 1/16 IPMN cases were misclassified, and 4/16 PC cases were misclassified. Certainty function was 0.75 ± 0.16 for correctly classified lesions and 0.47 ± 0.10 for incorrectly classified lesions (P = 0.0005). Uncertainty was identified in four of the five misclassified lesions. Quantitative histopathology provides a robust, novel method to distinguish among CP, IPMN, and PC with a quantitative measure of uncertainty. This may be useful when there is uncertainty in diagnosis.

Nuclear image analysis study of neuroendocrine tumors.

BACKGROUND: There is a subjective disagreement about nuclear chromatin in the field of pathology. Objective values of red, green, and blue (RGB) light intensities for nuclear chromatin can be obtained through a quantitative analysis using digital images. METHODS: We examined 10 cases of well differentiated neuroendocrine tumors of the rectum, small cell lung carcinomas, and moderately differentiated squamous cell lung carcinomas respectively. For each case, we selected 30 representative cells and captured typical microscopic findings. Using an image analyzer, we determined the longest nuclear line profiles and obtained graph files and Excel data on RGB light intensities. We assessed the meaningful differences in graph files and Excel data among the three different tumors. RESULTS: The nucleus of hematoxylin and eosin-stained tumor cells was expressed as a combination of RGB light sources. The highest intensity was from blue, whereas the lowest intensity was from green. According to the graph files, green showed the most noticeable change in the light intensity, which is consistent with the difference in standard deviations. CONCLUSIONS: The change in the light intensity for green has an important implication for differentiating between tumors. Specific features of the nucleus can be expressed in specific values of RGB light intensities.