Binucleate Research Articles

50 Human Hazard Assessment of Nanomaterials: Supporting Risk Decision Making Through Interlaboratory Trial Data

Abstract The prevalent application of engineered nanomaterials (ENMs) has led to an extensive effort to develop tools facilitating their risk assessment and management. This interlaboratory trial (encompassing two laboratories) sought to investigate the genotoxic impact of ENMs using the hypoxanthine phosphoribosyltransferase (HPRT) forward mutation assay and in vitro cytokinesis-blocked micronucleus (CBMN) assay, utilising harmonised protocols. In the CBMN assay, human lymphoblast (TK6) cells were exposed to zinc oxide (ZnO), titanium dioxide (TiO2) and tungsten carbide-cobalt (WC/Co) for 24-hours after which 1000 binucleated cells were scored for micronuclei (n=2). For the HPRT assay, TK6 and mouse fibroblast (V79) cells were exposed to ZnO, TiO2, WC/Co, CuO and Nanocyl-CNTs for 24-hours after which 600 wells were scored for point mutations (n=2). Significant cytotoxicity and micronuclei frequency was reported following ZnO exposure (20µg/ml). Significant micronuclei induction was reported following WC/Co exposures (2-fold over control, 100µg/ml). No significant mutagenicity was detected with ZnO or TiO2; both laboratories observed significant cytotoxicity following exposure to ZnO (20µg/ml; 37% reduction in cell viability). In the second interlaboratory trial CuO induced significant cytotoxicity (36% reduction in viability: 0.5µg/ml), and a 32-fold increase in mutagenicity. The Nanocyl induced a 6-fold increase in mutagenicity. The data generated here has shown good statistical concordance between laboratories and has highlighted a promising route forward in supporting risk decision making for ENMs. The authors would like to acknowledge this research has received funding from the European Union’s Horizon 2020 research and innovation program for the RiskGONE project, grant agreement #814425.

Review: Initial increase in pregnancy-specific protein B in maternal circulation after artificial insemination is a key indicator of embryonic survival in dairy cows.

Lactating dairy cows have reduced chances for pregnancy survival following 1st parturition. It appears that timing of conceptus attachment, defined as an initial daily rise in pregnancy-specific protein B (PSPB) postartificial insemination (AI), is key to the survival of the embryo. PSPB is a protein produced from binucleate cells of the trophectoderm of the conceptus. Once the conceptus has attached to the uterine epithelial cells, this large protein is detectable in maternal circulation. Daily sampling of serum allows for detection of the initial increase of PSPB. A percent increase threshold for PSPB was determined from results from each study using a conservative three-day minimum increase. Thus far, a 10 or 12.5% daily increase for three consecutive days had the greatest sensitivities and specificities for studies reported in this review. Nulliparous heifers receiving AI following estrus appear to have a reduced time to conceptus attachment compared to lactating cows that received either Ovsynch or AI following estrus. Increasing progesterone post-AI did not reduce time to conceptus attachment in lactating cows. Multiparous cows treated post-AI with human chorionic gonadotropin had a reduced percent with conceptus attachment in addition to greater time to conceptus attachment. Increased time to conceptus attachment increased the chances of multiparous cows having embryonic death before 35 d post-AI. Concentrations of PSPB are consistently reduced from conceptus attachment until d 28 post-AI in cows with pregnancy loss compared to cows that maintain pregnancy on d 34 post-AI. This novel model to determine timing of conceptus attachment provides a new perspective on percent of cows pregnant around d 20-23 post-AI and the potential for a greater understanding of subsequent pregnancy loss.

In vitro micronucleus assay: Method for assessment of nanomaterials using cytochalasin B.

The in vitro micronucleus (MNvit) assay is used to evaluate the aneugenic and clastogenic potential of a test material based upon its ability to induce micronuclei in the cells. This protocol is provided for testing of nanomaterials (NM) with standard cell lines in the absence of metabolic activation. The use of cytochalasin B (CytoB) and the analysis of binucleated cells in the cytokinesis-block version of the micronucleus assay ensures that cells analyzed have undergone cell division, which is required for expression of DNA damage and micronucleus formation. Issues specific to NM that were problematic with standard test methods are addressed, including test system choice, dose selection, test material exposures, CytoB timing, cytotoxicity determination, and DNA damage expression time. A step-by-step protocol for in vitro micronucleus assessment of NM is provided.

Determination of Disease Severity of Rhizoctonia solani Kühn (Telemorph: Thanatephorus cucumeris (Frank) Donk) Isolates from Bean, Sugar Beet and Potato Planting Areas in Konya

This study was carried out to determine the disease severity of Rhizoctonia solani isolates isolated from plant samples collected from bean, sugar beet and potato cultivation areas in Konya in 2020 and the anastomosis groups of the most virulent isolates. A total of 40 R. solani isolates were obtained as a result of the isolations made from 86 plant samples (36 beans, 25 sugar beets and 25 potatoes) showing root rot symptoms in general appearance. The number of R. solani isolates obtained from these plants, respectively; 10 isolates from beans, 15 from potatoes and 15 from sugar beets. Nine of the bean isolates and 14 of the potato and sugar beet isolates were determined as multinucleic. One isolate each isolated from the bean, sugar beet and potato was determined as binucleic. As a result, a total of 37 multinucleate (MN) and 3 binucleate (BN) isolates were obtained from all plants. As a result of pathogenicity tests, 3 R. solani isolates with the highest disease severity for each plant were amplified using ITS1F and ITS4B primers and anastomosis groups were determined. Accordingly, the anastomosis groups of Fa 3.2 (97%), Fa 2.2 (89%) and Fa 1 (86%) in beans were characterized as AG 4HGI. The isolates with the highest disease severity in potatoes (Pa 10, Pa 12.1 and Pa 15.2) were determined as AG 3 group. Disease severity was determined as 50% of Pa 15.2, 44% of Pa 10 and 42% of Pa 12.1. The disease severity of 9 of the isolates obtained from sugar beet was determined as 100%. The anastomosis group of 3 randomly selected isolates from these isolates was characterized as AG 2-2.

Morphological characteristics of the placenta of Balami and Yankasa ewes at different stages of gestation in Maiduguri, Nigeria

The morphological characteristics of the placenta of Yankasa and Balami ewes were evaluated. Uteri of 20 pregnant Yankasa and Balami ewes were collected as abattoir waste from Maiduguri Municipal abattoir and were placed in containers containing 10% neutral buffered formalin. Using the crown-rump length (CRL) method of estimating the ages of the foetuses, they were grouped into three gestational stages. In both breeds, 50% of pregnancy occurred in the left uterine horn; in the Yankasa, 20% and 30% occur in the right uterine horn, and both (twining), respectively. While in the Balami, 40% and 10% occurred in the right and both (twinning) uterine horns, respectively. The results showed that the placenta-maternal contact in both breeds was fragile in the first stage, firm in the second stage, and loose in the third stage. The results showed that there were significant changes (P<0.001) in the mean weight, length, and width of the placentomes in all three stages. Three types of placenta appearances were identified in the Yankasa ewe: concave, convex, and flat, while two types of appearances in the Balami ewe were concave and convex. In the light microscopy evaluation, the trophoblastic epithelia of both ewes were composed of mononucleated, binucleated cells and syncytia. Endometrial glands were also observed in the inter-placental space. In conclusion, this study has shown that in both breeds, the cotyledonary surfaces of the placentae were mostly concave, followed by convex, and a mixture of the two (concave/convex). The trophoblast epithelia are positioned to control the movement of substances across foetal and maternal tissues. Even though little is still known about the regulation of these cells in the trophoblast epithelia, they are believed to be responsible for the synthesis of hormones such as lactogen for the maintenance of the pregnancy before parturition.

Genotoxicity evaluation of two derived products from Allium extracts: s-propylmercaptocysteine and s-propyl mercaptoglutathione

Propyl-propane-thiosulfonate (PTSO) is one of the main organosulfur compounds present in Allium essential oils with a widely documented biological activity. For this reason, it could be used as a food and feed additive in the agri-food industry. A genotoxicity evaluation of substances and their metabolites present in food is necessary to guarantee the consumer's health following the recommendations of the European Food Safety Authority (EFSA). To evaluate the toxicological profile of derivatives of PTSO, the cytotoxicity, an Ames test, a micronucleus test and the comet assay were performed. Results showed that non-cytotoxic effects were observed in Caco-2 exposed to s-propyl mercaptocysteine (CSSP) and s-propyl mercaptoglutathione (GSSP) (0–450 μM). The mutagenicity index remained in the range of 0.6–1.4 for both compounds, showing no mutagenic effects for the concentrations of 5000–312.5 μg GSSP/plate and 250–15.63 μg CSSP/plate. Moreover, the % binucleated cells with micronuclei were 1.3–2.2 and 1.6–2.7 for GSSP and GSSP, respectively. For comet assays there was no DNA-genotoxic or oxidative damage in a concentration range of 112.5–450 μM. Therefore, we can conclude that these compounds are not genotoxic at the conditions tested. These results support that the presence of CSSP and GSSP in the food/feed is not of concern, although further studies are needed to complete their safety profile.

Corticotropin releasing hormone is present in the feline placenta and maternal serum.

In women, placental corticotropin releasing hormone (CRH) can be detected in maternal blood throughout pregnancy and is important in the regulation of the timing of parturition. However, its role in other mammalian species is unclear. In fact, very little is known about the presence and localization of CRH in placentas other than human. In this study we report for the first time the presence of CRH in feline placenta and maternal serum. Presence of CRH mRNA and protein was assessed using RT-PCR and Western blot, respectively, in at term domestic cat placentas opportunistically obtained at a local animal shelter and spay clinic. In addition, CRH localization within the placenta was demonstrated via immunohistochemistry. Finally, presence of CRH in maternal blood from early (¾21 days) and mid (25-35 days) stages of pregnancy was investigated by ELISA. CRH mRNA and protein were detected in feline placentas, and localized to larger decidual cells and fetal trophoblast cells, including the binucleate cells. CRH was detectable in maternal blood collected from early-stage pregnancies, and amounts significantly increased in mid-gestation samples. This is the first report on the presence and localization of CRH in the feline placenta, and its increase in maternal serum during the first half of pregnancy. These data lay the foundation for future studies to determine if CRH can be used as potential novel marker for early pregnancy diagnosis, determination, and monitoring in felids, and could greatly increase efficiency and success in zoo breeding programs utilizing artificial reproductive technologies for endangered feline species.

Abstract LB237: Functional characterisation of TRACERx reveals mechanisms of NSCLC evolution

Abstract Chromosomal instability (CIN), the increase in the rate of whole/partial chromosome gains and losses, is a driving feature of cancer identified in ~90% of solid tumours. CIN generates intratumor heterogeneity (ITH), drives evolutionary adaptation, and is associated with highly aggressive, drug-resistant tumours and poor prognosis. Aberrations in numerous pathways have been suggested to increase CIN. However, how CIN is initiated and maintained in non-small cell lung cancer (NSCLC) is largely unknown. Here we have analysed multi-region WES sequencing data from patients of the TRACERx study, and identified novel genes involved in the DNA damage response (DDR) that may contribute to chromosomal instability. Using orthogonal methods, we have identified that genetic alteration in 6 genes, particularly FAT1, culminates in deficiencies in homologous recombination repair (HRR). This is manifested by a reduction of RAD51 and BRCA1 foci formation, reduced end-resection rate and increased 53BP1 bodies. We find that these deficiencies in the HRR pathway can lead to an increase in structural CIN and deviation from the modal cjromosome number. Furthermore, we observed an early selection of FAT1 mutations in the TRACERx421 LUSC cohort, which resulted in more genome-doubling. We reprot mirrored sub-clonal allelic imbalance (MSAI) events at the FAT1 gene locus, indicating parallel evolution. We have proceeded to validate these observations using several cell lines, showing that FAT1 loss promotes whole genome doubling (WGD) through the generation of actively replicating binucleated cells through an elevated mitotic error rate. Importantly, the elevated CIN induced by FAT1 loss could be partially ameliorated by co-depletion of the oncogene YAP1. We hypothesize that elevated CIN could synergize with WGD to generate increased intratumor heterogeneity. To investigate this, we modelled the relationship between FAT1 loss and genome doubling in an isogenic cell culture system using PC9 cells which are sensitive to the EGFR inhibitor, Osimertinib. Indeed, we observed an elevated rate of acquired resistance in FAT1 KO genome-doubled PC9 cells, which we attribute to the high CIN level generated by FAT1 loss. In line with our hypothesis, we also observed a significant increase in genome content in FAT1 KO cells, suggesting FAT1 loss induces a second WGD event to escape targeted therapy. In conclusion, FAT1 is one of the most frequently mutated genes both in the TRACERx421 cohort and in somatic tissues (Martincorena et al, Science 2015). We postulate that FAT1 loss attenuates DDR and exacerbates CIN to enhance tumour heterogeneity, early in lung cancer evolution. Our observation that FAT1 loss leads to elevated EGFRi resistance also provides a unique opportunity to understand how EGFRi resistance arises through elevated CIN. Citation Format: Wei-Ting Lu, Lykourgos-Panagiotis Zalmas, Chris Bailey, Oriol Pich, Carlos M. Ruiz, James Black, Georgia Stavrou, Dhruva Biswas, Francisco Gimeno-Valiente, Kevin Litchfield, Jiri Bartek, Nicholas McGranahan, Nnennaya Kanu, Charles Swanton. Functional characterisation of TRACERx reveals mechanisms of NSCLC evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB237.

RENEB Inter-Laboratory Comparison 2021: The Cytokinesis-Block Micronucleus Assay.

The goal of the RENEB inter-laboratory comparison 2021 exercise was to simulate a large-scale radiation accident involving a network of biodosimetry labs. Labs were required to perform their analyses using different biodosimetric assays in triage mode scoring and to rapidly report estimated radiation doses to the organizing institution. This article reports the results obtained with the cytokinesis-block micronucleus assay. Three test samples were exposed to blinded doses of 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 13 mA, ∼75 keV, 1 Gy/min). These doses belong to 3 triage categories of clinical relevance: a low dose category, for no exposure or exposures inferior to 1 Gy, requiring no direct treatment of subjects; a medium dose category, with doses ranging from 1 to 2 Gy, and a high dose category, after exposure to doses higher than 2 Gy, with the two latter requiring increasing medical attention. After irradiation the test samples (no. 1, no. 2 and no. 3) were sent by the organizing laboratory to 14 centers participating in the micronucleus assay exercise. Laboratories were asked to setup micronucleus cultures and to perform the micronucleus assay in triage mode, scoring 500 binucleated cells manually, or 1,000 binucleated cells in automated/semi-automated mode. One laboratory received no blood samples, but scored pictures from another lab. Based on their calibration curves, laboratories had to provide estimates of the administered doses. The accuracy of the reported dose estimates was further analyzed by the micronucleus assay lead. The micronucleus assay allowed classification of samples in the corresponding clinical triage categories (low, medium, high dose category) in 88% of cases (manual scoring, 88%; semi-automated scoring, 100%; automated scoring, 73%). Agreement between scoring laboratories, assessed by calculating the Fleiss' kappa, was excellent (100%) for semi-automated scoring, good (83%) for manual scoring and poor (53%) for fully automated scoring. Correct classification into triage scoring dose intervals (reference dose ±0.5 Gy for doses ≤2.5 Gy, or reference dose ±1 Gy for doses >2.5 Gy), recommended for triage biodosimetry, was obtained in 79% of cases (manual scoring, 73%; semi-automated scoring, 100%; automated scoring, 67%). The percentage of dose estimates whose 95% confidence intervals included the reference dose was 58% (manual scoring, 48%; semiautomated scoring, 72%; automated scoring, 60%). For the irradiated samples no. 2 and no. 3, a systematic shift towards higher dose estimations was observed. This was also noticed with the other cytogenetic assays in this intercomparison exercise. Accuracy of the rapid triage modality could be maintained when the number of manually scored cells was scaled down to 200 binucleated cells. In conclusion, the micronucleus assay, preferably performed in a semi-automated or manual scoring mode, is a reliable technique to perform rapid biodosimetry analysis in large-scale radiation emergencies.

Development of a micronucleus test using the EpiAirway™ organotypic human airway model

BackgroundThe use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al., Mutat. Res. 850–851 (2020) 503135]. Organotypic human airway model is composed of fully differentiated and functional respiratory epithelium. However, because cell proliferation in organotypic airway models is thought to be less active, assessing their MN-inducing potential is an issue, even in the cytokinesis-blocking approach using cytochalasin B (CB) [Wang et al., Environ. Mol. Mutagen. 62 (2021) 306–318]. Here, we developed a MN test using EpiAirway™ in which epidermal growth factor (EGF) was included as a stimulant of cell division.ResultsBy incubating EpiAirway™ tissue with medium containing various concentrations of CB, we found that the percentage of binucleated cells (%BNCs) almost plateaued at 3 μg/mL CB for 72 h incubation. Additionally, we confirmed that EGF stimulation with CB incubation produced an additional increase in %BNCs with a peak at 5 ng/mL EGF. Transepithelial electrical resistance measurement and tissue histology revealed that CB incubation caused the reduced barrier integrity and cyst formation in EpiAirway™. Adenylate kinase assay confirmed that the cytotoxicity increased with each day of culture in the CB incubation period with EGF stimulation. These results indicated that chemical treatment should be conducted prior to CB incubation. Under these experimental conditions, it was confirmed that the frequency of micronucleated cells was dose-dependently increased by apical applications of two clastogens, mitomycin C and methyl methanesulfonate, and an aneugen, colchicine, at the subcytotoxic concentrations assessed in %BNCs.ConclusionsWell-studied genotoxicants demonstrated capability in an organotypic human airway model as a MN test system. For further utilization, investigations of aerosol exposure, repeating exposure protocol, and metabolic activation are required.

Description and quantification of micronucleus and nuclear abnormalities in erythrocytes of the sentinel green turtle (Chelonia mydas) with fluorescence microscopy.

Contamination in marine ecosystems is of the most critical threats to marine turtles. The identification of useful biomarkers to detect and monitor the physiological and clinical effects of pollutants on these populations will allow early detection of alterations (e.g., mutagenic damages) that could risk their viability or favor the development of diseases, thus threatening the biodiversity of these ecosystems and human population. This study is aimed at describing and quantifying nuclear anomalies in peripheral blood erythrocytes of green turtles (Chelonia mydas) from three distinct foraging areas in Mexico (Akumal, Xcalak, and Punta Herrero). We developed a novel morphological index that could be used as a biomarker to identify abnormal nuclei in peripheral blood erythrocytes. Here we describe for the first time in C. mydas, with a species-specific staining protocol, distinct nuclear abnormalities such as blebbed, lobed, notched, eight shape nuclei, and binucleated cells. These nuclear abnormalities were present in >90 % of the subjects (n=30). Moreover, 50 % of the organisms presented erythrocytes with micronuclei. The number of nuclear abnormalities did not correlate with size of the green turtles or differ between sites, or health status. We found a higher frequency of green turtles with nuclear abnormalities in the southern region (Punta Herrero and Xcalak) with the highest frequency of micronucleus and buds. The former could be associated to the constant exposure to chemical pollutants of oceanographic origin in the southern coast of Quintana Roo. Furthermore, the increasing anthropogenic pollution in Akumal could also explain the highest variability in the number of nuclear abnormalities presented in resident individuals. We propose that a long-term monitoring programs of green turtle populations in the Mexican Caribbean that include a micronucleus test could be a useful to determine possible mutagenic damage in these animals.

Micronucleus assay of DNA damage among welders: Effects of welding processes

Metal fumes, gases, noise, and radiation are hazardous occupational exposures that may be encountered by welders. We have evaluated DNA damage among welders; the buccal micronucleus cytome (BMCyt) assay was used. Thirty-four exposed welders (cases) and an equal number of non-welders (controls) participated in this study. Cell types including basal, early and late differentiated cells with micronucleus (MN), dense chromatin, karyorrhectic, pyknotic, karyolitic, and binucleated cells (NBUD) were measured. Damage levels among, arc, argon, and CO2 welders were statistically significantly higher, compared to the control group. Results showed that mean of MN and NBUDs as indicators of DNA damages among arc, argon and CO2 welding's were significantly higher compared to control group. Also, the mean of DNA damage levels were statistically higher among the arc welders than among the argon or CO2 welders; and levels were higher among the argon welders than the CO2 welders. Preventative measures need to be implemented to reduce exposure to harmful agents during welding.

Evaluation of Cyto-Genotoxicity of Pharmaceutical Industrial Effluent in Kano Metropolis, Kano State, Nigeria, Using Allium Cepa L. Assay

ABSTRACT
 An Allium cepa root cells assay was used to assess cytotoxic and genotoxic impacts on Pharmaceutical industrial effluent in Kano Metropolis. An industrial effluent's physicochemical characteristics and heavy metal composition were assessed, and the readings were found to be higher than the required levels, demonstrating that it had not been treated before disposal. A set of 45 onion bulbs were grown for 96 hours in pharmaceutical effluent that included 2.5, 5.0, 7.5, and 10.0% (v/v), with distilled water serving as the control. All three root tips from each replication's treated bulbs were plucked at 96 hours and prepared for cytogenetic analysis using the aceto-carmine squashed procedure. At higher doses of industrial effluents, the root tips were highly cytotoxic, and their growth was strongly retarded. Exposure to the effluents inhibited root growth with an EC50 value of 6.3%. An analysis of variance (ANOVA) revealed a significant difference (P 0.05) in the average root growth of Allium cepa subjected to various pharmaceutical effluent concentrations. Mitosis Index (MI) rapidly reduced when effluent concentrations rose compared to control, whereas mitotic inhibition rose with rising effluent concentrations compared to controls. The pharmaceutical effluent triggered chromosomal abnormalities in Allium cepa root tip cells, particularly sticky chromosomes, Binucleated cells, and Bridge chromosomes being most commonly seen at lower doses of 2.5%. It was discovered that the compounds present in effluent might harm living things and, if left untreated, could poison the environment. Industrialists need to be legally required to switch their operations to environmentally friendly technology after it was determined that industrial effluents pose an environmental danger and can result in a number of human illnesses.

Longitudinal multi-omic changes in the transcriptome and proteome of peripheral blood cells after a 4 Gy total body radiation dose to Rhesus macaques

BackgroundNon-human primates, such as Rhesus macaques, are a powerful model for studies of the cellular and physiological effects of radiation, development of radiation biodosimetry, and for understanding the impact of radiation on human health. Here, we study the effects of 4 Gy total body irradiation (TBI) at the molecular level out to 28 days and at the cytogenetic level out to 56 days after exposure. We combine the global transcriptomic and proteomic responses in peripheral whole blood to assess the impact of acute TBI exposure at extended times post irradiation.ResultsThe overall mRNA response in the first week reflects a strong inflammatory reaction, infection response with neutrophil and platelet activation. At 1 week, cell cycle arrest and re-entry processes were enriched among mRNA changes, oncogene-induced senescence and MAPK signaling among the proteome changes. Influenza life cycle and infection pathways initiated earlier in mRNA and are reflected among the proteomic changes during the first week. Transcription factor proteins SRC, TGFβ and NFATC2 were immediately induced at 1 day after irradiation with increased transcriptional activity as predicted by mRNA changes persisting up to 1 week. Cell counts revealed a mild / moderate hematopoietic acute radiation syndrome (H-ARS) reaction to irradiation with expected lymphopenia, neutropenia and thrombocytopenia that resolved within 30 days. Measurements of micronuclei per binucleated cell levels in cytokinesis-blocked T-lymphocytes remained high in the range 0.27–0.33 up to 28 days and declined to 0.1 by day 56.ConclusionsOverall, we show that the TBI 4 Gy dose in NHPs induces many cellular changes that persist up to 1 month after exposure, consistent with damage, death, and repopulation of blood cells.

Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

Analysis of the cytotoxic and genotoxic effects in a population chronically exposed to coal mining residues

During coal mining activities, many compounds are released into the environment that can negatively impact human health. Particulate matter, polycyclic aromatic hydrocarbons (PAHs), metals, and oxides are part of the complex mixture that can affect nearby populations. Therefore, we designed this study to evaluate the potential cytotoxic and genotoxic effects in individuals chronically exposed to coal residues from peripheral blood lymphocytes and buccal cells. We recruited 150 individuals who lived more than 20 years in La Loma-Colombia and 120 control individuals from the city of Barranquilla without a history of exposure to coal mining. In the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay, significant differences in the frequency of micronucleus (MN), nucleoplasmic bridge (NPB), nuclear bud (NBUD), and apoptotic cells (APOP) were observed between the two groups. In the buccal micronucleus cytome (BM-Cyt) assay, a significant formation of NBUD, karyorrhexis (KRX), karyolysis (KRL), condensed chromatin (CC), and binucleated (BN) cells was observed in the exposed group. Considering the characteristics of the study group, a significant correlation for CBMN-Cyt was found between NBUD and vitamin consumption, between MN or APOP and meat consumption, and between MN and age. Moreover, a significant correlation for BM-Cyt was found between KRL and vitamin consumption or age, and BN versus alcohol consumption. Using Raman spectroscopy, a significant increase in the concentration of DNA/RNA bases, creatinine, polysaccharides, and fatty acids was detected in the urine of individuals exposed to coal mining compared to the control group. These results contribute to the discussion on the effects of coal mining on nearby populations and the development of diseases due to chronic exposure to these residues.

Machine learning identification of thresholds to discriminate osteoarthritis and rheumatoid arthritis synovial inflammation

BackgroundWe sought to identify features that distinguish osteoarthritis (OA) and rheumatoid arthritis (RA) hematoxylin and eosin (H&E)-stained synovial tissue samples.MethodsWe compared fourteen pathologist-scored histology features and computer vision-quantified cell density (147 OA and 60 RA patients) in H&E-stained synovial tissue samples from total knee replacement (TKR) explants. A random forest model was trained using disease state (OA vs RA) as a classifier and histology features and/or computer vision-quantified cell density as inputs.ResultsSynovium from OA patients had increased mast cells and fibrosis (p < 0.001), while synovium from RA patients exhibited increased lymphocytic inflammation, lining hyperplasia, neutrophils, detritus, plasma cells, binucleate plasma cells, sub-lining giant cells, fibrin (all p < 0.001), Russell bodies (p = 0.019), and synovial lining giant cells (p = 0.003). Fourteen pathologist-scored features allowed for discrimination between OA and RA, producing a micro-averaged area under the receiver operating curve (micro-AUC) of 0.85±0.06. This discriminatory ability was comparable to that of computer vision cell density alone (micro-AUC = 0.87±0.04). Combining the pathologist scores with the cell density metric improved the discriminatory power of the model (micro-AUC = 0.92±0.06). The optimal cell density threshold to distinguish OA from RA synovium was 3400 cells/mm2, which yielded a sensitivity of 0.82 and specificity of 0.82.ConclusionsH&E-stained images of TKR explant synovium can be correctly classified as OA or RA in 82% of samples. Cell density greater than 3400 cells/mm2 and the presence of mast cells and fibrosis are the most important features for making this distinction.

MACRO- AND MICROSTRUCTURAL HEART ARRANGEMENT IN WHITE RATS IN HEALTH

The аim of research – to study the histological structure of the heart of a white rat in healthfor a comparative analysis of possible changes during further experimental interventions.Material and methods. The material for the study was heart biopsies of male rats witha body weight of 200±30 g. For the production of histological preparations, heart sampleswere taken randomly from different parts at least of 5 fragments. The thickness of samplesfor histological preparations was no more than 4 mm. For morphometry, the sectionswere stained with hematoxylin- eosin, azan and fuxilin and picrofuxin.The results. Myocardial muscle fibers are formed by mono- and binucleate cells ofrectangular shape in cross- section, which contact with each other in the form of chainsand provide a contractile function. The myocardium of the ventricles is three- layered:it has superficial, middle and deep layers. The fibers of the superficial layer are locatedlongitudinally. Bundles of the middle layer envelop each ventricle separately. Themuscle bundles of the inner layer form the trabeculae of the ventricles of the heart. Theatrial myocardium consists of two muscle layers – superficial and deep. The superficiallayer is made of oblique- circular muscle bundles that continuously cover the two atria.The deep layer is made of longitudinal muscle bundles, and is separate for each atrium.Conclusions. The histological structure of the white rat’s heart in health has beenspecified, as a basis for a comparative analysis of possible changes during experimentalinterventions, accompanied by morphofunctional changes of the myocardium.

Toxic metal(loids) levels in the aquatic environment and nuclear alterations in fish in a tropical river impacted by gold mining

The Atrato River basin was protected by Colombian law due to anthropogenic impacts, mainly from illegal gold mining, which triggered a critical environmental health problem. In this study we quantified mercury (Hg), methylmercury (MeHg) and arsenic (As) concentrations in aquatic environmental matrices, and explored for the first-time nuclear degenerations in fish from the Atrato River. The median concentrations (μg/kg) for T-Hg, MeHg and As in fish were 195.0, 175.5, and 30.0; in sediments (μg/kg) 165.5, 13.8 and 3.1; and in water (ng/L), 154.7 for T-Hg and 2.1 for As. A 38% and 10% of the fish exceeded the WHO limit for the protection of populations at risk (200 μg Hg/kg) and for human consumption (500 μg Hg/kg); while As concentrations were below the international standard (1000 μg/kg) in all fish. The percentage of MeHg was 89.7% and the highest accumulation was observed in carnivorous fish (336.3 ± 245.6 μg/kg, p < 0.05) of high consumption, indicating risk to human health. In water, T-Hg concentrations exceeded the threshold effect value of 12 ng/L, whereas As concentrations were below the threshold of 10,000 ng/L, established by USEPA. On the contrary, 33% of the sediments exceeded the quality standard of 200 μg/kg for Hg. We found that Prochilodus magdalenae was the species with the highest susceptibility to nuclear alterations in its order, nuclear bud (CNB, 3.7 ± 5.4%), micronuclei (MN, 1.6 ± 2.5%) and binucleated cells (BC, 1.6 ± 2.3%). These results indicate that the species appears to be a good predictor of genotoxicity in the Atrato River. Fulton's condition factor (K) indicated that 31.7% of the fishes had poor growth condition, suggesting that the Atrato River basin needs to be monitored and restored in accordance with the agreements reached in the Minamata Convention on Mercury.

Bone Marrow Mesenchymal Stem Cells Combined with Bone Transport Improves Bone Defect in Rats

This study assessed the mechanism of BMSC combined with bone transport in improving bone defect. Fifty rats were divided into five sets randomly including NC set, DEX set, BMSC set and BT set. There were ten rats in each set. The BMSC was isolated using whole bone marrow adherent method and then cultivated. The general condition of rats in each set was observed and morphological parameter, pathological change in bone defect tissue was detected along with analysis of the expression of MCP-1, p53, TNF-αand STAT1 in bone tissue. The primary BMSC was cultivated for seven days and the fusiform BMSC was enlarged and the quantity of binucleate or multinucleate cells was increased after passage. The bone defect model was prepared successfully when the degree of fusion reached 100%. The mental condition was good. DEX set showed significantly reduced TBV and increased TRS compared with NC set. However, TBV was increased and TRS was reduced in BMSC set, BT set and BMSC+BT set significantly compared with DEX set. MCP-1 mRNA level in DEX set was lower and increased in the treatment group. In addition, p53, TNF-αand STAT1 was increased in DEX set but reduced in BMSC set, BT set and BMSC+BT set. In conclusion, MCP-1 in rats’ bone defect tissue is upregulated and the p53/TNF-α/STAT1 signal activity is restrained by BMSC combined with bone transport so as to treat the bone defect.