Micronuclei and Nuclear Buds Induced by Cyclophosphamide in Crocodylus moreletii as Useful Biomarkers in Aquatic Environments

Micronuclei (MN) are used to assess genotoxic exposure, whereas nuclear buds (NBs) have been linked to genotoxic events. Crocodylus moreletii was studied to identify MN and NBs. Three groups were formed: Group 1 (water) and groups 2 and 3 (7 or 10 mg/kg of cyclophosphamide). A drop of blood was obtained daily from the claw tip at 0 to 120 h. Spontaneous micronucleated erythrocytes (MNEs) and erythrocytes with nuclear buds (NBEs) were counted. The frequencies of micronucleated young erythrocytes (MNYEs) and NB young erythrocytes (NBYEs) were evaluated, including the ratio of young erythrocytes (YE)/1000 total erythrocytes. No significant differences were observed in the YE proportion on sampling days; group 1 did not show differences for any parameter, whereas group 2 showed significant differences in MNEs and NBEs, and group 3 showed differences in NBEs and NBYEs. Some mitotic activity in circulation was observed in YEs. In conclusion, NBEs could be a more sensitive biomarker to genotoxic damage than MNEs. The identification of these biomarkers leads us to propose Crocodylus moreletii as a possible environment bioindicator because these parameters could be useful to analyze the in vivo health status of these reptiles and for biomonitoring genotoxic pollutants in their habitats.

All eyes on a phosphatase in glioma stem cells

In this issue of JEM, Zhang et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202669) identify a dependency of glioma stem cells on tyrosine phosphatase activity of EYA2 and a new role for this phosphatase at the centrosome, offering a new therapeutic approach to target mitotic activity.

PSX-B-26 Mitotic activity of immune cells in the intestines of piglets in the treatment of gastrointestinal pathology

The study aimed to evaluate the effect of a drug based on the granulocyte-macrophage colony-stimulating factor (GM-CSF) on intestinal immune cells’ mitotic activity in treating gastrointestinal pathology. Two groups of 40 sick piglets with diarrhea at the age of 5–7 days were formed. The 1st group was the control. Animals of the 2nd group received an intramuscular injection of “Quinokol”(enrofloxacin) at a dose of 0.5 ml / 10 kg once a day for 5 days. The 3d - “Quinokol” at the same dose combined with GM-CSF at a dose of 0.1 ml/kg twice with an interval of 48 hours. From forcedly slaughtered piglets (by Directive 93/119 / EC) with the diarrheal syndrome (n = 4) and from animals of the experimental groups (n = 4), duodenum sites were taken for research. Immunohistochemical study of Ki-67 expression with additional staining with hematoxylin Karratsi was carried out according to the method described by V. Stafford. Cell counting and morphometric changes were performed using the ImageJ software. In preparations from sick piglets and piglets, which were used “Quinokol”, the main mitotic activity was 0.0036 ± 0.00047 and 0.0041 ± 0.00054 mitoses / μm2, respectively (fig 1 - A). Single Ki-67-positive cells were found in the stroma of the villi, vascular walls, and submucosa. In some areas, the bean or rod-shaped nucleus of Ki-67 positive cells was distinguished. At the samples from the 3d group (fig 1 - C, D), a sharp increase in the mitotic activity of immune cells in the stroma of the villi was noted - 0.0067 ± 0.00072 mitoses/μm2 and significantly more than in the 1st and 2nd groups by 64% and 86%, respectively (P < 0.001). The combined use of “Quinokol” with GMC-SF increased cell hyperplasia in the villous stroma, which indicates the activation of local intestinal immunity.

A case of NTRK-rearranged Spindle cell tumor in a Pediatric patient

Introduction/Objective Recent studies continue to demonstrate that NTRK fusions occur more frequently in pediatric than in adult patients involving a broader panel of fusion partners as well as a wider range of pediatric tumors than previously recognized. The identification of these NTRK fusions has facilitated precision cancer diagnosis and TRK inhibitor targeted therapy. With the recent FDA approval of larotrectinib and entrectinib for the treatment of adult and pediatric NTRK-positive, unresectable solid tumors, identification of these fusions directly impacts patient care. Methods/Case Report Our patient, a 10 year old female presented with a large right sided buttock mass and pressure effects from the tumor. An incisional biopsy showed a moderately moderately cellular tumor with a collagenous and partially myxoid stroma. The atypical cells had ovoid nuclei with vesicular chromatin, minimal to no atypia, and rare mitotic activity (<2/30 high-power fields), as well as fibrous tissue that appeared as ropy collagen. Some of the blood vessels were rimmed by a hyalinized cuff. A mild inflammatory component, namely scattered lymphocytes and fewer plasma cells were noted. Immunohistochemistry showed: SMA(faint+), S100(+), CD34(+), CD31(+), FLI1(+), NTRK(+). Negative for ALK1, desmin, SOX10, EMA, keratin AE1/3, CAM5.2, D2-40, myogenin, MUC4, TLE1, STAT6, BCOR, ERG. Both INI1 and H3K27me3 were retained. Proliferative rate by Ki-67 was low, showing <2% positivity. Next generation sequencing revealed the following: LMNA-NTRK1 fusion; CD36 N53fs*24 and CDKN2A/B CDKN2A loss exon 1. Thus, the histologic, immunophenotypic, and molecular findings together supported a diagnosis of NTRK-rearranged spindle cell tumor. This entity has alternately been termed lipofibromatosis-like tumor. Following confirmation of NTRK fusion, she was treated with oral TRK inhibitor with near total response. With this NTRK-rearranged spindle cell tumor’s minimal mitotic activity, absence of necrosis, and low cellularity, the behavior of this tumor was expected to be indolent rather than aggressive. However, the patient was presented for assessment and management at a recent tumor board about 8 months after her initial diagnosis as she had residual/recurrent tumor. Results (if a Case Study enter NA) NA Conclusion Our case highlights the clinical utility of screening for NTRK fusions in all pediatric tumors.

Comparative clinicopathological and immunohistochemical study of micronodular thymoma and micronodular thymic carcinoma with lymphoid stroma

AimTo elucidate the clinicopathological and immunohistochemical characteristics of micronodular thymomas (MNTs) and micronodular thymic carcinomas (MNCs) with lymphoid stroma.MethodsWe examined four cases of MNTs and three cases of MNCs pathologically and immunohistochemically.ResultsThere were prominent cystic changes infive of the seven cases. The neoplasms contained epithelial tumour cells arranged in a micronodular growth pattern lined by cystic walls and separated by abundant lymphoid stroma. Only the tumour cell component of MNCs showed signs of malignancy characterised by cytological atypia and increased mitotic activity. Neoplastic MNC epithelial cells showed strong positivity for CD5 and CD117. However, no immature lymphocytes (TdT-positive and CD99-positive) were present in and around the tumour nodules. None of the patients died or suffered from disease due to MNTs or MNCs.ConclusionMNTs and MNCs are rare and less aggressive forms of thymic tumours and can be differentially diagnosed by immunohistochemistry.

Abstract P448: Role For Btg1 And Btg2 In Postnatal Cardiomyocyte Cell Cycle Arrest And Maturation

Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

The bystander effect formation in the meristem of Allium cepa L. plants with different genotype

Aim. Obtaining and analysis of cytogenetic parameters in meristem cells of Allium cepa L. seedling sprouting from unirradiated seeds, while germinating them with seeds, that were exposed to γ-radiation in different doses. Methods. Cytogenetic analysis included analysis of mitotic activity, anaphase method of chromosome mutations, nucleolar analysis. The F-test was used to compare the control and experimental samples. Results. The formation of the bystander effect is shown in the case of joint germination of irradiated and non-irradiated seeds. The severity of bystander effect increases with increasing irradiation dose. Conclusions. Irradiation of seeds with a dose of gamma radiation of 40 Gy causes the formation of radiation hormesis by the criterion of mitotic activity in the root meristem cells of seedlings, and the irradiation with a dose of 10 Gy does not affect the level of cell proliferation. The bystander effect formation occurs during the joint germination of intact and irradiated seeds as an increase in the level of mitotic activity, and an increase in the number of chromosomal mutations. Keywords: bystander effect, γ-radiation, meristem, mitotic activity, chromosomal abnormalities, micronuclei.

RADIOSENSITIVITY OF SOFT WHEAT SEEDS TO GAMMA RADIATION

The study of the effect of different doses of $\gamma$-irradiation on the growth and development of seedlings of soft wheat seeds, resistance, and germination of seeds to irradiation has been carried out. The data show that the seeds of common wheat are resistant to ionizing radiation and up to 80% of the seeds remained viable. Under the influence of ionizing radiation, the functional and mitotic activity of the cell nucleus is disrupted, which has a significant effect on the growth parameters of the seedlings of irradiated seeds.

Ki67 Protein expression and correlation with the histological grade and pTNM stage of colorectal carcinoma

: Colorectal cancer, is third most frequently encountered malignancy worldwide. Histological grade, stage, and proliferative index act as vital prognostic markers, playing a decisive role in patient care and prognosis. While histopathological examination can determine grade and stage, Ki67 protein expression by immunohistochemistry represents the proliferative capacity of the malignant cell population. : This Study was conducted to immunohistochemically analyze expression of Ki67 protein and its change with respect to different grades and stage of colorectal adenocarcinoma. : A total of 66 histologically diagnosed cases of colorectal carcinoma underwent histopathological examination followed by immunohistochemistry for Ki67 protein. : A statistically significant (p=0.04) correlation was obtained between Ki67 labelling index and histopathological grade, with higher values of Ki67 labelling index in poorly differentiated carcinomas (43.2±1.7). The Ki67 labelling index value was lowest in stage IV disease (11.4±2.4) with metastatic burden, with higher values in lower stage diseases, however this correlation was not found to be significant statistically (p=0.07). : The rate of cell division and proliferation measured by Ki-67 antibody is related to histological grade of the malignancy, demonstrating higher mitotic activity with loss of differentiation and anaplasia. Stage IV disease have lower mitotic activity, thus may be less amenable to cytostatic chemotherapy drugs, and require multimodality treatment with addition of radiotherapy and other drug regimes.

Effect of Heracleum sosnowskyi extract aqueous solution on the Allium cepa root meristem

Heracleum sosnowskyi (Apiaceae) contains a lot of useful chemical ingredients that can be used in industry, medicine and other fields as plant component extracts and as chemical compounds that have been extracted in different ways, which requires the last to be tested for chemical safety, including a genotoxic test in vivo. In the present paper, the 96-hour effect of the H. sosnowskyi extract aqueous solution at concentrations of 0.01, 0.05, 0.10, and 0.50 mL/L on the genetic apparatus and mitotic activity of the cells of the Allium cepa (Alliaceae) root meristem is discussed. Distilled water was applied as a negative control, and hydrogen peroxide 1% as a positive one. The extract was prepared from the plant’s fresh leaves by soaking them in acetone. It was then distilled at 57 ºС and diluted with distilled water to obtain the experimental concentrations. As extract content in the aqueous solution increased, a statistically significant decrease in mitotic activity, an increase in aberrant cell percentage and a concentration-dependent inhibition of root growth were observed. In the 0.5 mL/L solution, if compared against the other experimental concentrations, an increase in the metaphase, anaphase and telophase indices along with a decrease in the prophase index were observed. The most common aberrations for all the concentrations were lagging and sticking chromosomes, anaphase bridges, ring chromosomes and nuclear buds. The same solution and the positive control produced membrane damage; giant and ghost cells. The results of the experiment performed have demonstrated the extract’s aneugenic effect that causes spindle disturbance, mitodepression and inhibits the cells of the Allium cepa root meristem, prevails over its clastogenic effect.