Metaphase Cells Research Articles

An efficient and simple method for enriching metaphase cells for dicentric chromosome assay.

As compared to peripheral blood mononuclear cell (PBMC) culture, a lower mitotic index (MI) is seen in whole blood (WB) culture, but WB can be directly used for culture in dicentric chromosome assay (DCA). The purpose of this study is to develop a simple protocol for metaphase enrichment to improve the metaphase frequency of WB culture. Fixed cells were obtained after performing WB and PBMC cultures for DCA after conventional fixation. An additional low-speed centrifugation of 200×g for 1min was performed, separating the fixed cells of WB culture into a pellet and a supernatant fraction. The additional low-speed centrifugation enriched metaphase frequency and provided an MI comparable to the PBMC culture in the pellet fraction. Our study suggests that it is possible to increase the number of metaphase cells on slides using the slow centrifugation method, which could contribute to the efficiency of chromosome aberration analysis in biodosimetry.

Chromosomal Localization and Diversity Analysis of 5S and 18S Ribosomal DNA in 13 Species from the Genus Ipomoea

Background: Sweet potato (Ipomoea batatas (L.) Lam.), a key global root crop, faces challenges due to its narrow genetic background. This issue can be addressed by utilizing the diverse genetic resources of sweet potato’s wild relatives, which are invaluable for its genetic improvement. Methods: The morphological differences in leaves, stems, and roots among 13 Ipomoea species were observed and compared. Chromosome numbers were determined by examining metaphase cells from root tips. Fluorescence in situ hybridization (FISH) was used to identify the number of 5S and 18S rDNA sites in these species. PCR amplification was performed for both 5S and 18S rDNA, and phylogenetic relationships among the species were analyzed based on the sequences of 18S rDNA. Results: Three species were found to have enlarged roots among the 13 Ipomoea species. Chromosome analysis revealed that I. batatas had 90 chromosomes, Ipomoea pes-tigridis had 28 chromosomes, while the remaining species possessed 30 chromosomes. Detection of rDNA sites in the 13 species showed two distinct 5S rDNA site patterns and six 18S rDNA site patterns in the 12 diploid species. These rDNA sites occurred in pairs, except for the seven 18S rDNA sites observed in Ipomoea digitata. PCR amplification of 5S rDNA identified four distinct patterns, while 18S rDNA showed only a single pattern across the species. Phylogenetic analysis divided the 13 species into two primary clades, with the closest relationships found between I. batatas and Ipomoea trifida, as well as between Ipomoea platensis and I. digitata. Conclusions: These results enhance our understanding of the diversity among Ipomoea species and provide valuable insights for breeders using these species to generate improved varieties.

Comparative karyological features of several populations of Populus euphratica Oliv., grown under different environmental conditions in Iran

Abstract Understanding genetic structure and chromosomal characteristics is essential for developing effective breeding programmes and improving plant species. This research compared karyotypic features of 10 plant populations of Populus euphratica from various regions of Iran. Fresh roots grown from cuttings of the populations were used to get metaphase cells. Then several chromosomal parameters were recorded and analysed using a nested statistical model. All the studied populations were diploid, with 2n = 38 chromosomes, consisting of medium and sub-medium chromosome types. Significant differences (P ⩽ 0.01) were observed between the plant populations in chromosomal dimensions and arm ratios, suggesting chromosomal rearrangements. Chromosome lengths in the studied populations ranged from 0.69 to 3.38 μm. Intra-chromosomal index (A1) showed clear asymmetrical differences between the plant populations. Furthermore, using Stebbins's standards, the studied populations classified as 1A and 1B classes, demonstrated more asymmetry than those categorized as 1B and 2B, respectively. Cytological differences between the plant populations, collected from different parts of the country, showed that chromosome structural rearrangements are responsible for the speciation and adaption of the species against the mentioned variable ecological conditions and play a key role in response to diverse climatic and geographical conditions.

Cytogenetic Analysis Associated with Hashimoto's Thyroiditis in Samples of Iraqi Patients: an in vitro study

Background: One of the most prevalent autoimmune diseases is Hashimoto's thyroiditis (HT), which is characterized by autoantibodies specific to the thyroid but whose precise cause is still unknown. Objectives: By comparing a sample of Iraqi patients with healthy controls using the comet assay, chromosomal abnormalities, micronucleus production, and mitotic index, the current study aims to clarify the cytogenetic impact of Hashimoto's thyroiditis. Methods: The study included ten Iraqi patients with HT (six males and four females), ages 20 to 75 years old, compared with ten healthy control groups (six males and four females). Results: The results showed that only cells in metaphase /1000 were being assessed, and the patient group (9.82 ± 0.0153) was more affected than the healthy control group (5.94 ± 0.170). The micronucleus formation result compared to the proportion of healthy controls was (0.004±0.0018 Mn/cell), while the frequency of Mn formation in HT was higher (0.0068±0.00132 Mn/cell). Three parameters were employed in the comet assay to indicate DNA damage in HT patients and healthy controls: tail length (9.2±6.016 and 25.5±10.607 px), tail moment (2.047±2.687 and 11.32±15.058), and DNA damage in the tail (20.892±11.225 and 35.153±44.429%). Conclusion: The percentage of mitotic index, micronucleus formation, and DNA damage detected by the comet assay in HT patients was higher than in healthy control.

Di(2-ethylexyl) phthalate and chromosomal damage: Insight on aneugenicity from the cytochalasin-block micronucleus assay

Bis(2-ethylhexyl) phthalate is the most abundant phthalate used as plasticizer to soften plastics and polymers included in medical devices. Human and environmental exposure may occur because DEHP is not chemically bound to plastics and can easily leach out of the materials. This phthalate is classified as reproductive toxicant and possible carcinogen to humans. The genotoxic potential has still to be clarified, but there are indications suggesting that DEHP may have aneugenic effects. To further investigate DEHP genotoxicity, the cytochalasin-block micronucleus assay was applied and combined with the CREST staining to characterise micronucleus content and gain insights on its genotoxic mode of action. Chromosomal damage was also analysed in metaphase and ana-telophase cells and the morphology of the mitotic spindle was investigated to evaluate the possible involvement of this cellular apparatus as a target of DEHP.Our findings indicated that DEHP induced a statistically significant increase in the frequency of micronuclei as well as in the frequency of CREST-positive micronuclei. Consistently, disturbance of chromosome segregation and induction of numerical chromosome changes were observed together with changes in spindle morphology, formation of multipolar spindles and alteration of the microtubule network. Experiments performed without metabolic activation demonstrated a direct action of DEHP on chromosome segregation not mediated by its metabolites.In conclusion, there is consistent evidence for an aneugenic activity of DEHP. A thresholded genotoxic activity was identified for DEHP, disclosing possible implications for risk assessment.

Abstract 410: ASXL3 is a potential biomarker of response of SCLC to targeted therapies

Abstract Small Cell Lung Cancer (SCLC) has been designated a recalcitrant cancer and a priority by National Cancer Institute. Despite recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential, these advances have yet to be fully translated to effective regimens for the treatment of SCLC. Previously, we identified Additional Sex Combs Like-3 (ASXL3) as a novel mediator of pluripotency in human respiratory epithelia and potential therapeutic target in SCLC. ASXL3 is one of three members of the ASXL family, whereas the functions of ASXL1 and ASXL2 as components of the PR-DUB complex have been extensively studied, the functions and significance of ASXL3 during malignant transformation remain elusive. To further investigate the relevance of ASXL3 activation in SCLC, we initially studied ASXL3 expression levels in various SCLC subtypes by qRT-PCR, digital RT-PCR, RNA-seq, and western blotting analysis. These studies demonstrated that ASCL (A) and ASCL and NeuroD1 (A+N) subtypes of SCLC cells contain thousand-folds more ASXL3 (ASXL3high) than Pou2f3 (P) and YAP1 (Y) SCLC subtypes (ASXL3low). ASXL3 levels were elevated when cancer cells were cultured under stemness-enriched conditions. Under normal culture conditions, ASXL3high SCLC cells showed higher intrinsic DNA damage, evidenced by a high percentage of γ-H2AX positive cells compared to ASXL3low SCLC cells. Additionally, ASXL3high SCLC cells displayed 38% γ-H2AX positive metaphase cells, and 42% of abnormal chromosome segregations during mitosis, including lagging chromosomes, DNA bridges, and unequal chromosome segregation indicative of profound genome instability. One reason for this extensive genomic instability is that ATM pathway activation in response to DNA replication stress is impaired in ASXL3high SCLC cells. Following exposures to 1mM hydroxyurea (HU) for 4, 8, and 24 hours, ATM autophosphorylation levels in ASXL3high SCLC cells were much lower than in ASXL3low SCLC cells. This insufficiency significantly affected the activation of downstream cascade targets including CHK2, KAP-1 and EZH2. Consistent with these findings, ASXL3high SCLC cells showed reduced sensitivities to ATM inhibitors. However, ASXL3high SCLC cells exhibited higher activity of PLK1 and were more sensitive to hydroxyurea. Importantly, low dose HU reduced the IC50 values of the PLK1 inhibitors, BI2536 or NMS-P937 by 80-100-fold in ASXL3high but not ASXL3low SCLC cells, suggesting a strong synergy between these two drugs in ASXL3high SCLC cells. Collectively, these findings suggest that ASXL3 functions to protect cells from lethality induced by genomic instability through monitoring ATM activation. Experiments are underway using SCLC PDX models to demonstrate the efficacy of this combined treatment in-vivo as a prelude to possible evaluation of this drug combination in SCLC patients. Citation Format: Ruihong Wang, Haitao Wang, Xinwei Wu, Keerti Mishra, Stephanie Shiffka, Frank Hernandez, Mary Zhang, Umay Tuana Tolunay, Sudheer Gara, Sichuan Xi, Shamus R. Carr, Choung D. Hoang, David S. Schrump. ASXL3 is a potential biomarker of response of SCLC to targeted therapies [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 410.

A Study of Hyaluronic Acid's Theoretical Reactivity and of Magnetic Nanoparticles Capped with Hyaluronic Acid.

Hyaluronic acid (HA) has attracted much attention in tumor-targeted drug delivery due to its ability to specifically bind to the CD44 cellular receptor, which is widely expressed on cancer cells. We present HA-capped magnetic nanoparticles (HA-MNPs) obtained via the co-precipitation method, followed by the electrostatic adsorption of HA onto the nanoparticles' surfaces. A theoretical study carried out with the PM3 method evidenced a dipole moment of 3.34 D and negatively charged atom groups able to participate in interactions with nanoparticle surface cations and surrounding water molecules. The ATR-FTIR spectrum evidenced the hyaluronic acid binding to the surface of the ferrophase, ensuring colloidal stability in the water dispersion. To verify the success of the synthesis and stabilization, HA-MNPs were also characterized using other investigation techniques: TEM, EDS, XRD, DSC, TG, NTA, and VSM. The results showed that the HA-MNPs had a mean physical size of 9.05 nm (TEM investigation), a crystallite dimension of about 8.35 nm (XRD investigation), and a magnetic core diameter of about 8.31 nm (VSM investigation). The HA-MNPs exhibited superparamagnetic behavior, with the magnetization curve showing saturation at a high magnetic field and a very small coercive field, corresponding to the net dominance of single-domain magnetic nanoparticles that were not aggregated with reversible magnetizability. These features satisfy the requirement for magnetic nanoparticles with a small size and good dispersibility for long-term stability. We performed some preliminary tests regarding the nanotoxicity in the environment, and some chromosomal aberrations were found to be induced in corn root meristems, especially in the anaphase and metaphase of mitotic cells. Due to their properties, HA-MNPs also seem to be suitable for use in the biomedical field.

Chromosomal characterization mediated by karyomorphological analysis and differential banding pattern in fenugreek (Trigonella foenum-graecum L.): a neglected legume

Fenugreek or Trigonella foenum-graecum L. is a commercially important yet neglected crop of the family Fabaceae, with potent medicinal applications, and can treat several diseases as well. Conventional breeding studies for higher yields of commercial crops largely depend on chromosomal information of the particular species. Despite a number of cytological research being conducted on T. foenum-graecum, a complete characterization of its chromosomes has not been achieved due to the limitations of traditional karyotype analysis methods. A range of chromosomal markers are advantageous to characterize at full extent and identify individual chromosomes rather than relying on only physical metrics. Thus, in this study, in addition to giemsa staining, other approaches like fluorochrome and silver staining were used for the precise karyomorphological analysis of this species. Enzyme maceration and air drying (EMA) based fluorochrome banding with GC-specific stain Chromomycin A3 (CMA), and AT-specific stain 4’,6-diamidino-2-phenylindole (DAPI) applied for the first time for chromosome characterization. The results showed 2n = 16 chromosomes in metaphase cells, with karyotype formula of 2m+6sm. The unique banding pattern observed in the CMA/DAPI and AgNOR staining highlights the AT and GC-rich regions as well as the nucleolar organizer regions (NORs). All this crucial information can further assist in conducting breeding studies of more precision with simultaneously encouraging similar studies that need to be done in other unexploited species of importance.

Erythroid variant evolving from chronic myeloid leukemia resistant to multiple tyrosine kinase inhibitors: a rare case report

BackgroundChronic myeloid leukemia (CML) is characterized by the presence of BCR::ABL1 fusion gene resulting from a reciprocal translocation, t(9;22)(q34;q11.2), leading to prominent granulocytic proliferation. The majority of patients initially present in chronic phase (CP), which may progress to advanced CML with predominantly granulocytic phenotypes in the absence of proper treatment or response to tyrosine kinase inhibitors (TKIs). We present an exceptionally rare case in which an erythroid variant emerged from a CML patient resistant to multiple TKIs. This variant is characterized by the detection of t(9;22) BCR::ABL1 fusion in erythroid precursors at various maturation stages and the absence of granulocytic progenitor hyperplasia typically seen in classical CML.Case presentationA 33-year-old female with CP-CML had received multiple TKI therapies since her initial diagnosis in 2015. Due to intolerable side effects and inconsistent adherence, she exhibited an inadequate response and developed new-onset pancytopenia. Bone marrow (BM) biopsy specimen revealed a hypercellular marrow with significant erythroid hyperplasia (90% of marrow cellularity) and a reversed myeloid-to-erythroid (M: E) ratio of 1:10. Both erythroid and myeloid cells displayed progressive maturation without dysplasia or excess blasts. Chromosomal analysis identified t(9;22) (q34;q11.2) in 19 out of 20 metaphase cells. BCR::ABL1 fusion transcript (p210 isoform) was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Notably, no additional pathogenic cytogenetic abnormalities or ABL1 kinase domain mutations were detected. Here, we report the first published case of an erythroid variant emerging in a CML patient resistant to multiple TKIs—a distinct entity from the erythroid blast crisis evolving from CML.ConclusionThe erythroid variant of CML is distinguished by the presence of t(9;22) (q34;q11.2) BCR::ABL1 in predominant erythroid precursors at different stages of maturation. In a myeloid neoplasm showing predominant erythroid hyperplasia without typical CML features, it is vital to correlate morphology and t(9;22) BCR::ABL1 cytogenetic testing for accurate diagnosis, and to prevent confusion with PEL transformation in CML.

Recognition of rare antinuclear antibody patterns based on a novel attention-based enhancement framework.

Rare antinuclear antibody (ANA) pattern recognition has been a widely applied technology for routine ANA screening in clinical laboratories. In recent years, the application of deep learning methods in recognizing ANA patterns has witnessed remarkable advancements. However, the majority of studies in this field have primarily focused on the classification of the most common ANA patterns, while another subset has concentrated on the detection of mitotic metaphase cells. To date, no prior research has been specifically dedicated to the identification of rare ANA patterns. In the present paper, we introduce a novel attention-based enhancement framework, which was designed for the recognition of rare ANA patterns in ANA-indirect immunofluorescence images. More specifically, we selected the algorithm with the best performance as our target detection network by conducting comparative experiments. We then further developed and enhanced the chosen algorithm through a series of optimizations. Then, attention mechanism was introduced to facilitate neural networks in expediting the learning process, extracting more essential and distinctive features for the target features that belong to the specific patterns. The proposed approach has helped to obtained high precision rate of 86.40%, 82.75% recall, 84.24% F1 score and 84.64% mean average precision for a 9-category rare ANA pattern detection task on our dataset. Finally, we evaluated the potential of the model as medical technologist assistant and observed that the technologist's performance improved after referring to the results of the model prediction. These promising results highlighted its potential as an efficient and reliable tool to assist medical technologists in their clinical practice.

M-FISH evaluation of chromosome aberrations to examine for historical exposure to ionising radiation due to participation at British nuclear test sites

Veterans of the British nuclear testing programme represent a population of ex-military personnel who had the potential to be exposed to ionising radiation through their participation at nuclear testing sites in the 1950s and 1960s. In the intervening years, members of this population have raised concerns about the status of their health and that of their descendants, as a consequence. Radiation dose estimates based on film badge measurements of external dose recorded at the time of the tests suggest any exposure to be limited for the majority of personnel, however, only ∼20% of personnel were monitored and no measurement for internalised exposure are on record. Here, to in-part address families concerns, we assay for chromosomal evidence of historical radiation exposure in a group of aged nuclear test (NT) veterans, using multiplex in situ hybridisation (M-FISH), for comparison with a matched group of veterans who were not present at NT sites. In total, we analysed 9379 and 7698 metaphase cells using M-FISH (24-colour karyotyping) from 48 NT and 38 control veteran samples, representing veteran servicemen from the army, Royal Airforce and Royal Navy. We observed stable and unstable simple- and complex-type chromosome aberrations in both NT and control veterans’ samples, however find no significant difference in yield of any chromosome aberration type between the two cohorts. We do observe higher average frequencies of complex chromosome aberrations in a very small subset of veterans previously identified as having a higher potential for radiation exposure, which may be indicative of internalised contamination to long-lived radionuclides from radiation fallout. By utilising recently published whole genome sequence analysis data of a sub-set of the same family groups, we examined for but found no relationship between paternal chromosome aberration burden, germline mutation frequency and self-reported concerns of adverse health in family members, suggesting that the previously reported health issues by participants in this study are unlikely to be associated with historical radiation exposure. We did observe a small number of families, representing both control and NT cohorts, showing a relationship between paternal chromosome aberrations and germline mutation sub-types which should be explored in future studies. In conclusion, we find no cytogenetic evidence of historical radiation exposure in the cohort of nuclear veterans sampled here, offering reassurance that attendance at NTs sites by the veterans sampled here, was not associated with significant levels of exposure to radiation.

DaCSeg: Divide and conquer for accurate overlapping chromosome instance segmentation in metaphase cell images

Chromosome segmentation is essential for karyotyping analysis, which provides a reliable basis for subsequent disease diagnosis. Recent deep segmentation techniques can significantly boost the efficiency of chromosome micrographs processing. However, existing classical Mask R-CNN-based methods cannot deal with chromosome overlapping issues highly susceptible to appearing in clinical data. In our opinion, it is difficult for current computational methods to achieve accurate segmentation without a specific design for complex overlapping. Therefore, we propose a divide-and-conquer automatic framework, which shows better robustness for complex chromosome instance segmentation. Exploiting a series of specially designed modules, our end-to-end model first performs an estimation of whether the chromosomes overlap and then generates precise attention masks for overlapping areas. With the assistance of the shape prior knowledge, it produces final instance segmentation predictions for the chromosomes. The experimental results show that the proposed method achieves mean Average Precision (mAP) of 84.10%, AP50 of 98.61%, AP75 of 97.62%, Precision of 89.32%, Recall of 88.87%, and F1-score of 89.09%, which are superior to other state-of-the-art methods. Code and models are available at https://github.com/labiip/DaCSeg.

Cytogenetic Bioindication in Root Meristems for Vitality Assessment of Trees.

Our method describes how to collect forest tree root tips in the field, to store them for transfer to the lab, to pretreat root tips in order to arrest cells in metaphase, fix root tips to preserve specific morphological organizations, to stain fixed root tips by Feulgen's Reaction in order to increase contrast, and to prepare the root meristem for analyzing mitotic stages and chromosomal aberrations via light microscopy. We further describe how to classify chromosomal abnormalities and quantify them via aberration indices.

Autopsy findings in a fetus with monosomy 20 mosaicism

Introduction. Mosaic monosomy 20 is a rare chromosomal aberration, without characteristic clinical features. We present a case of a fetus with monosomy 20 mosaicism revealed after prenatal ultrasound detection of anhydramnios and multiple anomalies. Case outline. The second pregnancy of a 33-year-old woman, was terminated at 23rd gestational week, because of the multiple fetal anomalies and anhydramnios, detected by ultrasound. The autopsy of a female fetus revealed multiple congenital anomalies: ventriculomegaly, bilateral choroid plexus cysts, perivascular gliosis in periventricular region of cerebri, hydropericardium, severe cardiomegaly, severe myocardial hypertrophy, hydrothorax, glandular/canalicular stage of fetal lung development, bilateral renal and ureter agenesis (Potter syndrome), bladder aplasia, agenesis of the uterus, fallopian tubes and proximal vagina and valgus deformity of left foot (pes valgus). Fetal growth was adequate for gestational age with no craniofacial dysmorphia or radiographically visible anomalies of the skeleton, without signs of infection. The umbilical cord was too long for gestational age ? 48 cm. Analysis of fetal karyotype from fetal blood sampling revealed monosomy of chromosome 20 in 10% of analyzed cells in metaphase. Conclusion. Revealing the genetic basis of fetal anomalies is at outmost importance not only for further evaluation of pregnancy, but also for proper genetic informing of patients.

Chromosomal abnormalities in children with acute lymphoblastic leukemia

Acute lymphoblastic leukemia is the most conventional cancer in children and teenagers. In adults, this pathology is much less common. The paper presents the results of a retrospective analysis of chromosomal aberrations in clinically identified 1269 feeble children with acute lymphoblastic leukemia for the period from January 2013 to January 2023. The average age of the patients was 10.2±1.69 years. The material for the study was bone marrow and blood cells obtained from a puncture biopsy of the sternum and peripheral vein. Cytogenetic studies were carried out using the generally accepted standard method using GTG banding. The results of the analysis showed the absence of genomic and chromosomal mutations in 215 (17%) children; translocation t(1;19)(q23;p13) was detected in 75 (6%) children; in 221 (17.5%) children - t(4;11)(q21;q23); in 234 (18.5%) children - t(9;22)(q34;q11); 88 (7%) - t(12;21)(p33;q22) and 139 (11%) patients had translocations between other chromosomes. Hyperdiploid translocations were detected in 139 (11%) children. No sick children with hypodiploid cells were found. No metaphase cells were obtained from 152 patients (12%). In sick children, a relatively low frequency of optimistic predicting chromosomal abnormalities, namely t(12;21)(p13;q22) and hyperdiploid, has been established. The pervasiveness of chromosomal aberrations with a negative treatment prediction, such as t(9;22)(q34;q11.2), is consistent with the accessible international scientific literature data. The results obtained indicate the importance of cytogenetic studies in the diagnosis and predictions of children with ALL.

Cytogenetics of Diphysa americana (Mill.) M.Sousa (Leguminosae-Papilionoideae-dalbergioid clade), a rare species from the coast of Oaxaca, Mexico

Diphysa Jacq. is an essentially Mexican and Central American genus that includes 21 species and only one cytogenetic report. In this work, a surface spread and air-drying method was used to obtain the karyotype of Diphysa americana (Mill) M.Sousa, a rare native tree that grows in a coastal town in the State of Oaxaca, Mexico. Metaphase cells showed a 2n = 20, consistent with the predominant diploid number in the dalbergioid clade. This number contrasts with a previously reported 2n = 16. The karyotypic formula 5m + 5sm, first proposed for a species of the genus, denotes a slightly asymmetric karyotype. The presence of secondary constrictions associated with satellites on the short arms of a pair of sm chromosomes and other cytogenetic parameters require studies in other species of the genus to verify their taxonomic utility. In addition, cells in prometaphase exhibited a circular fragment of unknown origin like that observed in a species of the genus Aeschynomene, also dalbergioid. This fragment could be related to extrachromosomal circular DNA (eccDNA) observed in other plants. Diphysa is a small, cytogenetically favorable genus, and further studies will exhibit the karyotypic diversity that underlies its diversification.

Mechanisms underlying Myosin 10's contribution to the maintenance of mitotic spindle bipolarity.

Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.

Diagnostic Utility of Comprehensive RNA-Seq Analysis in Adult B-ALL

Introduction Adult B-cell acute lymphoblastic leukemia (B-ALL) comprises 75% of adult ALL cases. The classification and risk stratification of patients have conventionally relied on cytogenetics analysis. However, cytogenetics has limitations due to its reliance on metaphase cells, which can mitigate classification accuracy. In recent years, transcriptome sequencing (RNA-seq) of large pediatrics ALL cohorts has expanded the number of recurrent gene fusions recognized as disease defining events and informed risk stratification [Brady et al. 2022, Mullighan et al. 2017]. This study aims to evaluate the diagnostic utility of RNA-seq for clinical subtype classification in adult B-ALL in a large representative cohort. Methods The study cohort comprised 338 adult patients (25-65 years) with newly diagnosed ALL (UKALL14, ISRCTN66541317, NCT01085617). Paired-end RNA-seq data at 60 Million reads per sample were generated. Analysis focused on the detection of disease-defining gene fusions and gene expression signatures. Three RNA fusion callers (FusionCatcher, FuSeq, and STAR-Fusion) and three expression-based classifiers (ALLSorts, ALLspice, and ALLCatchR) were used for consensus fusion calling and expression-based classification, respectively [Nicorici et al. 2014, Vu et al. 2018, Haas et al. 2019, Schmidt et al., Mäkinen et al., Bader et al. 2022]. Integration of classification results from expression-based classifiers and the consensus gene fusions provides 4 levels of RNA-derived subtype information, denoted as “RNA evidence” in this study. These RNA-seq based classification results were compared with clinical cytogenetics findings. Results Out of 338 samples in this cohort, cytogenetics unequivocally classified 73% (246/338) of the samples. By integrating RNA expression-based classification with fusion subtypes, we confidently classified 87% (295/338) of the cohort with at least 2 levels of RNA evidence. In total, consensus gene fusions were identified in 74% (249/338), while expression-based classifiers assigned a confident subtype to 82% (319/338). Subsequently, we compared cytogenetics with RNA-seq derived classification and found a high concordance of 98% (240/246) between the two approaches. Only 6 (<2%) samples with cytogenetics subtypes were not classified by RNA-seq, attributed to specific subtype characteristics (near-haploid) or lower RNA purity/quality. 27% (92/338) of subjects were not classified by cytogenetics (31 missing/failed and 61 indeterminate cytogenetics). Of the 31 samples with failed/missing cytogenetics, RNA-seq confidently assigned a class in 74% (23/31). For the remaining 61 cases with indeterminate cytogenetic (complex/other), RNA-seq resolved classification in 74% (45/61). Taken together, this improved the classification for 20% (68/338) of the entire cohort that was previously unclassified through cytogenetics. Notably, RNA-seq-exclusive findings were enriched in subtypes like DUX4, PAX5alt, and ZNF384, which are challenging for cytogenetics to discern. Importantly, integration of RNA-seq with cytogenetics led to a confident subtype classification for 93% of the cohort (314/338), a significant improvement over the initial 73% based on cytogenetics alone. Conclusions In conclusion, our study highlights the relevance of integrated RNA-seq analysis for reliable and comprehensive subtype classification in adult B-ALL. The high concordance between cytogenetics and RNA-derived subtypes underscores their complementary roles, providing valuable insights into disease mechanisms and guiding personalized treatment strategies.

Modified YOLO network model for metaphase cell detection in antinuclear antibody images

The antinuclear antibody (ANA) test plays the role of screening, diagnosis, treatment and prognosis for autoimmune diseases. However, the ANA test is time-consuming, and strongly depends on the interpretation of experienced medical examiners. With the rise of artificial intelligence, a lot of convolutional neural networks (CNN) have been used to recognize the features of ANA images, such as GoogLeNet, ResNet, and MobileNet. By extracting the disease-related features from the entire ANA image, these models achieve acceptable accuracy, but none of them can replace the manual ANA test completely. This is because they ignore the key features of ANA images. Accordingly, this paper aims to design an object detection model that can directly identify the key features of ANA images, i.e., the Metaphase cells. This process is critical in the manual ANA test performed by the medical examiners. Based on the YOLO model, our design first applies various image preprocessing and data augmentation techniques to enhance the critical features in the training data. We modify the original YOLO model, in which the residual block is replaced with SENet module to improve the classification performance by emphasizing critical features and reducing the influence of low-level features. Moreover, we use the different activation function to prevent the modified model from partial judgment. The experimental results show that our design can effectively improve the performance of ANA metaphase identification. Compared to the related CNN models, our design can improve F1-score and mAP by range 5%–14% and 11%–13%, respectively. In particular, the Kappa value of our design is 0.82 that is almost perfect agreement with the ANA experts.

7. AI-Based Algorithms for neoplastic metaphase cells boost efficiencies in the cytogenetics laboratory

7. AI-Based Algorithms for neoplastic metaphase cells boost efficiencies in the cytogenetics laboratory