Binucleate Research Articles

The analysis of the histomorphometric characteristics and ultrastructural features of the liver in response to environmental cycle desynchronization in the wild desert rodent (Gerbillus tarabuli)

The architecture of the hepatic tissue in the nocturnal wild desert rodent (Gerbillus tarabuli) subjected to a shifted LD (Light/Dark) cycle was investigated using transmission electron microscopy and histomorphometry. Investigation involved two groups: the control group (n = 5), exposed to the regular LD cycle (LD: 12:12), and the shifted group (n = 5), exposed to the LD regimen with a prolonged light phase of 8 h, in a day by day alternation with regular LD cycle (LD: 12:12 / LD: 20:4) during 12 weeks. Histological examination showed in the shifted group disorganization of hepatocyte trabeculae, sinusoidal dilatation, and inflammatory cell infiltration around the portal tracts. Morphometric results revealed hypertrophy of hepatocytes with a size of 29.83 ± 1.69 μm vs. 17.52 ± 0.81 μm and of their nuclei diameter with 8.69 ± 0.25 μm vs. 6.55 ± 0.15 μm. This increase is highly significant (p<0.001) and represents an increase of 70.17% and 32.67%, respectively. We also noticed a significant increase in the number of binucleated cells compared to the control group with an average of 9.73 ± 0.98 vs. 5.53 ± 0.38 binucleated hepatocytes per area unit, respectively. Ultrastructural analysis corroborated the histological findings, confirming the installation of inflammatory foci, characterized by portal and intraparenchymal infiltration of leukocytes, proliferation of Kupffer cells and stellate cells, as well as an expansion of collagen fiber deposits. Electron microscopy also revealed in the shifted group some hepatocytic lesional features represented by vacuolar or liquefied cytoplasm associated with degenerative mitochondria and a rarefaction of organelles in addition to an abundance of lysosomes. Additionally, strong glycogen depletion was observed at the metabolic level. This study showed that 12 weeks of lengthening the light phase of the LD cycle, corresponding to the gerbil's resting period, had a discernible impact on hepatocellular activity, making G. tarabuli an interesting model to study the influence of artificial light at night as a circadian rhythm disruptor.

Hazard assessment and cytogenotoxic effect of different concentrations of mercury chloride sterilant using the Allium cepa assay

The Allium cepa assay represents a crucial in vivo model for evaluating the cytotoxicity and genotoxicity of substances. This study investigated the cytogenotoxicity of mercury chloride (HgCl2), a laboratory disinfectant and catalyst, using the Allium cepa assay. Mitotic slides were prepared from onion root tip cells grown on media supplemented with different concentrations of HgCl2 (0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%). The slides were observed to evaluate cytogenotoxicity based on the assessment of the mitotic index, mitotic inhibition, clastogenic effect, and root length. The results showed that the concentrations used are harmful to the cell, leading to adverse impacts on the mitotic index, mitotic inhibition, root growth, and chromosome structure. Different chromosomal aberrations, such as bridge formation, fragmentation fragments, wandering chromosomes, stickiness, binucleate cells, and micronucleus formation, were detected depending on the concentration. Although lower concentrations (0.2–0.4%) had fewer effects on the cells, they still had a significant cytogenotoxic effect (p < 0.05) compared to that of the control (0.0%). The higher the concentration, the greater the effects on clastogenic changes. The abnormalities in both mitotic spread and root growth indicate that mercury chloride is cytotoxic even at low concentrations and can cause mito-depressive effects at higher concentrations. The results of this investigation can be used as a guide to guarantee sufficient safety precautions for people and organs during the regular use of HgCl2.

Synovial myxosarcoma in the scapulohumeral joint of a dog: a case report.

A 12-year-old, 3kg spayed female mixed-breed dog was evaluated to assess a 1-year history of intermittent right forelimb lameness that did not have adequate response to nonsteroidal anti-inflammatory drugs. The radiographic study performed under sedation showed multifocal radiolucent areas affecting both the right humerus and scapula with focal soft tissue swelling; a CT scan confirmed the existence of an aggressive and invasive soft tissue mass affecting the scapulohumeral joint. Fine needle aspiration results suggested a low-grade synovial sarcoma and therefore a scapulectomy was performed. The biopsy showed spindle to stellated cells immersed in a basophilic and mucinous (myxoid) matrix with mild to moderate anisocytosis, moderate anisokaryosis, some binucleated cells and sporadic multinucleated cells. These findings are consistent with low-grade synovial myxosarcoma, a not well described synovial neoplasm that can mimic other commonly seen joint tumors or even septic arthritis on radiographs. The purpose of this case report is to describe the first reported synovial myxosarcoma affecting the scapulohumeral joint of a small dog.

Evaluation of the Hepatotoxicity of Buprenorphine in Rat Pups Born to an Exposed Mother During Lactation.

This study aimed to evaluate the hepatotoxicity of buprenorphine in lactating rat pups of buprenorphine-injected mothers. Buprenorphine (BUP), a semisynthetic opioid, is increasingly administrated as a first-line standard maintenance treatment for opioid dependence due to its high safety and efficacy compared to other opioids. Numerous studies have confirmed the safety of BUP maintenance treatment in addicted patients. This study was designed to assess the effects of BUP on the activities of liver enzymes, oxidative parameters, and liver histopathological changes in pups born to a mother exposed to this drug during lactation. BUP at a dose of 0.5 or 0.1 mg/kg was subcutaneously administrated to lactating rats for 28 days. At the end of the experiment, the pups were anesthetized, and blood samples were obtained from their hearts for measuring liver enzymes. Then the livers of the animals were dissected to measure oxidative stress parameters. In addition, the liver samples were fixed for histopathological evaluation. The findings indicated a decrease in the activities of serum liver enzymes (ALT and AST) of the pups born to mothers exposed to 0.5 and 1 mg/kg of BUP during lactation. BUP could not change malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO) levels, nor superoxide dismutase (SOD) activity in the liver tissue of animals. Some vacuolated hepatocytes with dark, eccentric nuclei, necrosis with karyolytic nuclei, mitotic figures, and multiple binucleated cells were seen in the pups which received 1 mg/kg of BUP. In conclusion, BUP may induce liver dysfunction in pups born to mothers exposed to this drug during lactation.

Interspecies pregnancy between Ovis canadensis mexicana and Ovis aries with reduced placental P450scc expression and intrauterine growth restriction

The present study aimed to assess the steroidogenic process in the placenta of hybrids Ovis canadensis mexicana × Ovis aries, particularly, to determine the plasma lipid profile and the relative abundance of proteins related to the steroidogenesis, to elucidate in part the mechanism of progesterone deficiency in hybrids. Plasma lipoproteins and placental cholesterol were determined by dry chemical colorimetric assays. Steroidogenic acute regulatory protein (STARD1), StAR-related lipid transfer domain protein 3 (STARD3), cholesterol side-chain cleavage enzyme (P450ssc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were evaluated by Western blot. In addition, STARD3 was also investigated by immunohistochemical experiments in sheep placenta. Results indicated that cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and very low-density lipoprotein (VLDL) plasma levels were similar between hybrid and control pregnancy groups. Conversely, intracellular cholesterol was increased (P ˂ 0.05) in the placenta tissue of hybrids. No significant differences in the relative abundance of both STARD3, STARD1, and 3β-HSD enzyme were observed in the placenta of hybrids compared to control animals, while the relative abundance of cytochrome P450scc was lower in hybrids than in control animals (P = 0.0279). The presence of STARD3 was observed in the cytoplasmic and perinuclear regions of mononucleate and binucleate cells of the trophoblast in the sheep placenta. The significant decrease in the abundance of P450scc in hybrids’ placenta may explain the decrease in progesterone biosynthesis, which has been related to intrauterine growth restriction (IUGR) in these animals previously.

Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Bovine placental extracellular vesicles carry the fusogenic syncytin BERV-K1

Syncytins are endogenous retroviral envelope proteins which induce the fusion of membranes. A human representative of this group, endogenous retrovirus group W member 1 envelope (ERVW-1) or syncytin-1 is present in trophoblast-derived extracellular vesicles and supports the incorporation of these extracellular vesicles into recipient cells. During pregnancy, placenta-derived extracellular vesicles participate in feto-maternal communication. Bovine fetal binucleate trophoblast cells express the syncytin, bovine endogenous retroviral envelope protein K1 (BERV-K1). These cells release extracellular vesicles into the maternal stroma, but it is unclear whether BERV-K1 is included in these extracellular vesicles. Here, extracellular vesicles were isolated from bovine placental tissue using collagenase digestion, ultracentrifugation, and size exclusion chromatography. They were characterized with transmission electron microscopy, nanoparticle tracking analysis, immunoblotting and mass spectrometry. Immunohistochemistry and immunoelectron microscopy were used to localize BERV-K1 within the bovine placental tissue. The isolated extracellular vesicles range between 50 and 300 nm, carrying multiple extracellular vesicle biomarkers. Proteomic analysis and immunoelectron microscopy confirmed BERV-K1 presence on the isolated extracellular vesicles. Further, BERV-K1 was localized on intraluminal vesicles in secretory granules of binucleate trophoblast cells. The presence of BERV-K1 on bovine placental extracellular vesicles suggests their role in feto-maternal communication and potential involvement of BERV-K1 in uptake of extracellular vesicles by target cells.

Dynamics of cytomorphological indicators in armwrestlers during the training process and competitive activity

The issue of the functional state of the body of arm wrestlers is poorly studied. There are isolated research data on the improvement of the functional state of the body of arm wrestlers as a result of training. The study of the functional state of the body of these athletes is extremely important, as it determines the athlete's success during competitions and the growth of skill during many years of sports training. The purpose of this work was to determine the cytomorphological indicators of the buccal epithelium of arm wrestlers during the training process and during the performance at the championship of the Lviv region on February 10-11, 2024.&#x0D; As a result of research, we established that the cytomorphological indicators of the buccal epithelium of arm wrestlers, obtained by us during the training process, indicate the absence of discomfort in the body of athletes after training, which is due to the adequacy of physical exertion during training, which is not excessive for athletes of a given age.&#x0D; Changes in the cytomorphological indicators of the buccal epithelium during the Championship of the Lviv Region indicate a significant strain on the body's functional systems, signs of dehydration in the winner who played twelve matches (on both hands). In other athletes, there are certain signs of discomfort and strain on the body's functional systems (cytolysis, karyolysis, karyorrhexis, the presence of binucleated cells and leukocytes). The indicators we obtained during the athletes' performance at the Lviv Region Championship show gradual progressive changes in the athletes' bodies (the development of fatigue and dehydration) depending on the duration of their participation in the competition.

Genotoxicity and Cytotoxicity Activities of Stembark Extract of Mammea africana

Mammea africana, a medicinal plant, used in ethnomedicine for the treatment of malaria, diabetes, poisoning and inflammatory diseases was investigated for cytotoxic and genotoxic effects on the root meristem cells of Allium cepa. Onion bulbs were exposed to 2.5 mg/mL, 5mg/mL, and 10 mg/mL concentrations of the stembark extract for macroscopic and microscopic analysis. Tap water was used as a negative control and Methotrexate (0.1 mg/mL) as a positive control. There was statistically significant (p&lt; 0.05) inhibition of root growth depending on concentration by the extract when compared with the negative control group. All the tested concentrations of the extract were observed to have cytotoxic effect on cell division in A. cepa. When compared to the control group, the extract-induced chromosomal abnormalities and micronuclei (MNC) forms in A. cepa root tip cells were substantial (p&lt;0.05). Further inducing cell death, ghost cells, membrane damage, and binucleated cells was the extract treatment. These findings imply that the phytochemical components of Mammea africana root extract exert their cytotoxic and genotoxic actions on A. cepa.

Abstract 1650: Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells

Abstract Nucleoli are large nuclear sub-compartments where vital cellular processes, such as rRNA production and ribosome assembly, take place. Aggressive tumor types, such as Triple Negative Breast Cancer (TNBC), require increased ribosome biogenesis to support their enhanced proliferation. Therefore, it has been suggested that the nucleolus could be a valid anti-neoplastic target. However, we still have a limited understanding of the cancer-specific functions of the nucleolar components, preventing the effective development of therapeutic agents. The characterization of nucleolar proteins is technically challenging. Several nucleolar proteins are essential, and their complete abrogation cannot be achieved through conventional RNAi or CRISPR-KO approaches. Additionally, many nucleolar proteins display peculiar biophysical properties, and their functions are connected to their abundance. Therefore, artificial overexpression systems might not fully recapitulate their endogenous biological functions. To overcome these challenges, we implemented an Auxin Inducible Degron (AID) system to acutely abrogate endogenous nucleolin, one of the most abundant nucleolar proteins, and asses its impact on the proliferation of TNBC cell lines. We used CRISPR/Cas9 editing in TNBC cell lines to modify the endogenous NCL gene, fusing it with an AID tag. Then, we stably transduced the cells with a modified Oryza Sativa TIR1 (OsTIR1-F74G) E3 ubiquitin ligase. In response to the synthetic phytohormone phenyl-auxin, OsTIR1 leads to the proteasomal degradation of AID-containing endogenous nucleolin in less than 6h, sparing any other human protein. RNA sequencing analysis showed that nucleolin degradation significantly alters the expression of genes involved in cell cycle progression. Accordingly, nucleolin degradation reduces cancer cell proliferation in vitro, associated with increased propidium iodide staining, suggestive of a G2/M cell cycle arrest. However, immunofluorescence analyses revealed that nucleolin abrogation leads to the accumulation of bi-nucleated cells with actin patches. These findings are indicative of potential cytokinesis defects due to altered spindle tension during mitosis. Notably, nucleolin abrogation increased the efficacy of chemical inhibitors of the Anaphase Promoting Complex on suppression of cell proliferation. To the best of our knowledge, this study is the first reporting the acute abrogation of an endogenous, highly abundant nucleolar protein, using the AID system. This approach allowed the characterization of nucleolin biological functions with unprecedented temporal resolution, shedding light on new biological functions in the regulation of cell division. Finally, our results suggest that nucleolin inhibition could enhance the therapeutic activities of drugs affecting cell cycle progression in TNBC. Citation Format: Nastaran Samadi Rad, Joseph Mills, Anna Tessari, Damu Sunil Kumar, Vollter Anastas, Saranya Lamba, Ashley B Reers, Ilaria Cosentini, Zirui Zhu, Thomas J Magliery, Emanuele Cocucci, Lara Rizzotto, Dario Palmieri. Characterization of nucleolar biological functions via Auxin inducible degron-mediated acute depletion in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1650.

Cytogenotoxicity and inflammatory response in liver of rats exposed to different doses of cannabis nano emulsions.

Cannabis is the most used illicit substance for recreational purposes around the world. However, it has become increasingly common to witness the use of approved cannabis preparations for symptoms management in various diseases. The aim of this study was to investigate the effects of cannabis nano emulsion in the liver of Wistar rats, with different proportions of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). For this, a total of 40 male Wistar rats were distributed into 5 groups, as follows (n = 8 per group): Control: G1, Experimental group (G2): treated with cannabis nano emulsion (THC and CBD) at a dose of 2.5mg/kg, Experimental group (G3): treated with cannabis nano emulsion (THC and CBD) at a dose of 5mg/kg, Experimental group (G4): treated with cannabis nano emulsion (CBD) at a dose of 2.5mg/kg; Experimental group (G5): treated with cannabis nano emulsion (CBD) at a dose of 5mg/kg. Exposure to the nano emulsion was carried out for 21days, once a day, orally (gavage). Our results showed that cannabis nano emulsions at higher doses (5mg/kg), regardless of the composition, induced histopathologic changes in the liver (G3 and G5) in comparison with the control group. In line with that, placental glutathione S-transferase (GST-P) positive foci increased in both G3 and G5 (p < 0.05), as well as the immune expression of Ki-67, vascular endothelial growth factor (VEGF) and p53 (p < 0.05). Also, the nano emulsion intake induced an increase in the number of micronucleated hepatocytes in G5 (p < 0.05) whereas G3 showed an increase in binucleated cells (p < 0.05). As for metanuclear alterations, karyolysis and pyknosis had an increased frequency in G3 (p < 0.05). Taken together, the results show that intake of cannabis nano emulsion may induce degenerative changes and genotoxicity in the liver in higher doses, demonstrating a clear dose-response relationship.

Evaluation of Genotoxic and Cytotoxic Activities of Leaf and Seed Extracts of Telfairia occidentalis

Telfairia occidentalis Hook (cucurbitaceae) leaves and seeds, which are used as vegetable in the preparation of soups and as medicine traditionally to treat various diseases by the Ibibios were investigated for genotoxic and cytotoxic effects using Allium cepa test. The effects of the leaves and seeds extracts on the root meristem cells of Allium cepa were investigated using onion bulbs exposed to 2.5 mg/mL, 5 mg/mL, and 10 mg/mL concentrations of the extracts for macroscopic and microscopic analysis. Tap water was used as a negative control and Methotrexate (0.1 mg/ml) was used as a positive control. There was statistically significant (p&lt; 0.05) inhibition of root growth depending on concentration by the extracts when compared with the negative control group. All the tested extracts were observed to have cytotoxic effects on cell division in A. cepa. The extracts-induced chromosomal aberrations and micronuclei (MNC) formations in A. cepa root tip cells were significant (p&lt;0.05) when compared with control group. The extracts treatment further induced cell death, ghost cells, cells membrane damage, and binucleated cells. The seed extract was found to have a higher cytotoxic and genotoxic effects than the leaf extract. These results suggest that the leaf and seeds extract of Telfairia occidentalis possess cytotoxic and genotoxic effects on A. cepa.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Mobile phone specific radiation disturbs cytokinesis and causes cell death but not acute chromosomal damage in buccal cells: Results of a controlled human intervention study

Several human studies indicate that mobile phone specific electromagnetic fields may cause cancer in humans but the underlying molecular mechanisms are currently not known. Studies concerning chromosomal damage (which is causally related to cancer induction) are controversial and those addressing this issue in mobile phone users are based on the use of questionnaires to assess the exposure. We realized the first human intervention trial in which chromosomal damage and acute toxic effects were studied under controlled conditions. The participants were exposed via headsets at one randomly assigned side of the head to low and high doses of a UMTS signal (n = 20, to 0.1 W/kg and n = 21 to 1.6 W/kg Specific Absorption Rate) for 2 h on 5 consecutive days. Before and three weeks after the exposure, buccal cells were collected from both cheeks and micronuclei (MN, which are formed as a consequence of structural and numerical chromosomal aberrations) and other nuclear anomalies reflecting mitotic disturbance and acute cytotoxic effects were scored. We found no evidence for induction of MN and of nuclear buds which are caused by gene amplifications, but a significant increase of binucleated cells which are formed as a consequence of disturbed cell divisions, and of karyolitic cells, which are indicative for cell death. No such effects were seen in cells from the less exposed side. Our findings indicate that mobile phone specific high frequency electromagnetic fields do not cause acute chromosomal damage in oral mucosa cells under the present experimental conditions. However, we found clear evidence for disturbance of the cell cycle and cytotoxicity. These effects may play a causal role in the induction of adverse long term health effects in humans.

Pregnancy maintenance and fetal loss assessment in Holstein cows through analyzing pregnancy-associated glycoproteins in milk

Fetal loss (FL) from the 45th day of gestation until calving can impose a significant economic burden on dairy farmers, resulting in lost profits and increased production costs. Pregnancy-associated glycoprotein (PAG) is commonly used for detecting pregnancy in cows. PAG is secreted by binucleated trophoblast cells of the placenta and regulated by more than 24 genes. The purpose of this study was to determine the PAG threshold for FL and a probability of pregnancy maintenance until calving based on milk PAG levels. Our results reveal that primiparous and multiparous cows that maintained pregnancy until the 40th week exhibited higher PAG sample-negative (SN) values in their milk in the 6th week of gestation than did those that experienced FL later in gestation. Pregnant cows with higher PAG SN values in the 6th week of gestation were more likely to maintain their pregnancies. The area under the receiver operating characteristic curve for predicting the probability of pregnancy maintenance was 0.722 for our prediction model. On the other hand, a milk PAG SN value of <0.192 indicated 95 % confidence that FL would occur between the 7th and 40th weeks of gestation. Milk PAG testing is a noninvasive sampling technique that does not induce additional stress in lactating cows. The study reveals that PAG SN values increase significantly in Holstein cows during the 6th week of gestation. The predictive model developed was effective in forecasting pregnancy outcomes up to the 40th week of gestation or calving. The model's performance is moderately good for field application and could be a useful tool for dairy producers.

Nuclear Changes in Controlled and Uncontrolled Type-2 Diabetic Patients In Buccal Exfoliative Cytology Smears Using Pap And Giemsa Staining – A Comparative Study

Background: Diabetes is a third leading cause of mortality and morbidity in the world. Oral exfoliative cytology may be a more appropriate adjunctive diagnostic tool in conditions like diabetes mellitus, where the invasive techniques lose viability. Exfoliative cytology helps to detect cellular aging, cell atrophy, alterations in nucleus and cytoplasm, binucleation and occasional karyorrhexis in type-2diabetic patients which proves to be a valuable tool for early diagnosis of diabetes. The purpose of this research was to develop a new method for an early diagnosis of diabetes mellitus by exfoliative cytology using PAP stain and GIEMSA stains which is a nuclear specific stain. The aim of the study was to compare the exfoliative cytology of oral mucosa in controlled and uncontrolled type-2 diabetic patients using PAP stain and GIEMSA stain detect the nuclear and cytoplasmic changes. Materials And Methods: The smears were obtained from buccal mucosa of the mouth in total of 25 individuals out of which 10 are controlled (&lt;120 mg/dl) and 10 are uncontrolled (&gt;130 md/dl) diabetic patients and 5 normal individuals. These smear samples was obtained from the clinical lab of Saveetha Dental College. Results: The nuclear changes include nuclear bud, binucleate/micronucleate, karyolysis, increased nuclear cytoplasmic ratio &amp; hyperchromatism. The binucleated cells were found to have higher differences between controlled &amp; uncontrolled whereas karyorrhexis showed no significant changes between controlled &amp; uncontrolled diabetes. Conclusion: This is a preliminary study conducted to analyse the potential interaction between the nuclear changes and the amount of genetic damage caused in diabetes. However, our result clearly demonstrated that uncontrolled diabetic patients have significantly more genetic damage (in terms of MN frequency) compared to controlled diabetic patients. This indicates than MN may be a useful constituent in a panel of biomarkers for the risk of diabetes.

The Micronucleus Assay on Cryopreserved Whole Blood.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

Toxoplasma gondii Me49 and NED strains arrest host cell cycle progression and alter chromosome segregation in a strain-independent manner.

Toxoplasma gondii is an obligate intracellular parasite that modulates a broad range of host cell functions to guarantee its intracellular development and replication. T. gondii includes three classical clonal lineages exhibiting different degrees of virulence. Regarding the genetic diversity of T. gondii circulating in Europe, type II strains and, to a lesser extent, type III strains are the dominant populations, both in humans and animals. Infections with the type I strain led to widespread parasite dissemination and death in mice, while type III is considered avirulent. Previously, we demonstrated that primary endothelial cells infected with the T. gondii RH strain (haplotype I) were arrested in the G2/M-phase transition, triggering cytokinesis failure and chromosome missegregation. Since T. gondii haplotypes differ in their virulence, we here studied whether T. gondii-driven host cell cycle perturbation is strain-dependent. Primary endothelial cells were infected with T. gondii Me49 (type II strain) or NED (type III strain), and their growth kinetics were compared up to cell lysis (6-30 h p. i.). In this study, only slight differences in the onset of full proliferation were observed, and developmental data in principle matched those of the RH strain. FACS-based DNA quantification to estimate cell proportions experiencing different cell cycle phases (G0/1-, S-, and G2/M-phase) revealed that Me49 and NED strains both arrested the host cell cycle in the S-phase. Cyclins A2 and B1 as key molecules of S- and M-phase were not changed by Me49 infection, while NED infection induced cyclin B1 upregulation. To analyze parasite-driven alterations during mitosis, we demonstrated that both Me49 and NED infections led to impaired host cellular chromosome segregation and irregular centriole overduplication. Moreover, in line with the RH strain, both strains boosted the proportion of binucleated cells within infected endothelial cell layers, thereby indicating enhanced cytokinesis failure. Taken together, we demonstrate that all parasite-driven host cell cycle arrest, chromosome missegregation, and binucleated phenotypes are T. gondii-specific but strain independent.

Amelioration of Cytogenotoxic Damage in Drug Abusers Supplemented with Folic Acid.

Cytogenotoxic damage caused by the consumption of legal and illegal drugs in drug abusers has been demonstrated, primarily due to alterations in their antioxidant capacity, cellular repair mechanisms, and increased production of free radicals. Folic acid shows antioxidant activity by acting as a reducing agent, neutralizing present free radicals, and reducing genomic damage. The intervention involved administering 15 mg of folic acid, divided into three doses per day, to a group of 44 drug abusers. The frequency of nuclear abnormalities (NAs) was determined; micronuclei (MNs), nuclear buds (NBUDs), binucleated cells (BNs), abnormally condensed chromatin (CC), karyorrhexis (KX), pyknotic nuclei (PNs), and karyolysis (KL) were determined at different pre-treatment (baseline) and post-treatment time points at 15 and 30 days. Additionally, a group of 44 healthy individuals was used as the control group. We observed a statistically significant decrease in the frequency of NAs in the drug abuser group (28.45 ± 17.74 before supplementation vs. 11.18 ± 7.42 at 15 days and 9.11 ± 10.9 at 30 days of supplementation). Specifically, it decreased the frequency of NBUDs, BNs, CC, KX, and PNs (p < 0.05). Our study demonstrates a clear improvement in cytogenotoxic damage in drug abusers supplemented with folic acid.