Miconazole induces aneuploidy-mediated tolerance in Candida albicans that is dependent on Hsp90 and calcineurin.

Antifungal resistance and antifungal tolerance are two distinct terms that describe different cellular responses to drugs. Antifungal resistance describes the ability of a fungus to grow above the minimal inhibitory concentration (MIC) of a drug. Antifungal tolerance describes the ability of drug susceptible strains to grow slowly at inhibitory drug concentrations. Recent studies indicate antifungal resistance and tolerance have distinct evolutionary trajectories. Superficial candidiasis bothers millions of people yearly. Miconazole has been used for topical treatment of yeast infections for over 40 years. Yet, fungal resistance to miconazole remains relatively low. Here we found different clinical isolates of Candida albicans had different profile of tolerance to miconazole, and the tolerance was modulated by physiological factors including temperature and medium composition. Exposure of non-tolerant strains with different genetic backgrounds to miconazole mainly induced development of tolerance, not resistance, and the tolerance was mainly due to whole chromosomal or segmental amplification of chromosome R. The efflux gene CDR1 was required for maintenance of tolerance in wild type strains but not required for gain of aneuploidy-mediated tolerance. Heat shock protein Hsp90 and calcineurin were essential for maintenance as well as gain of tolerance. Our study indicates development of aneuploidy-mediated tolerance, not resistance, is the predominant mechanism of rapid adaptation to miconazole in C. albicans, and the clinical relevance of tolerance deserves further investigations.

Fbxo28 is essential for spindle migration and morphology during mouse oocyte meiosis I.

Spindle migration and assembly regulates asymmetric oocyte division, which is essential for fertility. Fbxo28, as a member of SCF (Skp1-Cul1-F-box) ubiquitin E3 ligases complex, is specifically expressed in oocytes. However, little is known about the functions of Fbxo28 in spindle assembly and migration during oocyte meiosis I. In present study, microinjection with morpholino oligonucleotides and exogenous mRNA for knockdown and rescue experiments, and immunofluorescence staining, western blot, timelapse confocal microscopy and chromosome spreading were utilized to explore the roles of Fbxo28 in asymmetric division during meiotic maturation. Our data suggested that Fbxo28 mainly localized at chromosomes and acentriolar microtubule-organizing centers (aMTOCs). Depletion of Fbxo28 did not affect polar body extrusion but caused defects in spindle morphology and migration, indicative of the failure of asymmetric division. Moreover, absence of Fbxo28 disrupted both cortical and cytoplasmic actin assembly and decreased the expression of ARPC2 and ARP3. These defects could be rescued by exogenous Fbxo28-myc mRNA supplement. Collectively, this study demonstrated that Fbxo28 affects spindle morphology and actin-based spindle migration during mouse oocyte meiotic maturation.

A 12-year Life History of a Girl with Profound Intellectual Disability and Leukoencephalopathy: A Rare Clinical Presentation of X Chromosome Pentasomy.

This paper presents a unique 12-year case analysis of a girl with Penta-X syndrome, a chromosomal abnormality characterized by five X chromosomes instead of the normal two in healthy women. Pentasomy of X is a genetic, but not a hereditary disease affecting only women. Our patient demonstrated delayed mental, speech, and motor development along with physical anomalies such as craniofacial deformities, and eye pathology and was diagnosed with pentasomy of the X chromosome at the age of 3 after a cytogenetic examination. She developed epileptic seizures at the age of nine. Magnetic resonance imaging(MRI) revealed leukoencephalopathy with ventriculomegaly. The peculiarity of this observation is that the polysomy 49, XXXXX detected in the patient is characterized by a typical phenotypic presentation combined with demyelinating leukoencephalopathy, which has not been a typical feature of the disorder.

Basal FSH values are positively associated with aneuploidy incidence in pre-advanced maternal age (AMA) but not in AMA patients.

PURPOSE: To assess the impact of maternal age on the association between maternal basal FSH and aneuploidy. METHODS: A retrospective study including data from 1749 blastocysts diagnosed as euploid or aneuploid by PGT-A (preimplantation genetic testing for aneuploidy). Aneuploidy incidence was compared between embryos from mothers with high vs. low basal FSH levels (above and below the group median, respectively) in total, pre-AMA (advanced maternal age; < 35 years, 198 embryos) and AMA (≥ 35 years, 1551 embryos) patient groups, separately. To control for the interference of potentially confounding variables, the association between aneuploidy and high basal FSH levels was assessed by multivariate logistic analysis in overall, pre-AMA and AMA patient groups. RESULTS: Overall, aneuploidy rate was 9% higher (p = 0.02) in embryos from patients with high basal FSH (63.7%) compared to those with low basal FSH (58.4%). In the pre-AMA subgroup, aneuploidy incidence was 35% higher (p = 0.04) in embryos from patients with high basal FSH (53.5%) compared to those with low basal FSH (39.4%). Differently, aneuploidy occurrence did not vary between embryos from AMA patients with low (61.0%) and high (64.8%) basal FSH (p = 0.12). The multivariate analysis revealed that, in pre-AMA embryos, the association between aneuploidy occurrence and high basal FSH is independent of potential confounding variables (p = 0.04). CONCLUSION: Maternal basal FSH values are associated with embryo aneuploidy in pre-AMA but not in AMA patients. The present findings suggest that basal FSH is a useful parameter to assess aneuploidy risk in pre-AMA patients and reinforce the hypothesis that excessive FSH signalling can predispose to oocyte meiotic errors.

Mechanical stress during confined migration causes aberrant mitoses and c-MYC amplification.

Confined cell migration hampers genome integrity and activates the ATR and ATM mechano-transduction pathways. We investigated whether the mechanical stress generated by metastatic interstitial migration contributes to the enhanced chromosomal instability observed in metastatic tumor cells. We employed live cell imaging, micro-fluidic approaches, and scRNA-seq to follow the fate of tumor cells experiencing confined migration. We found that, despite functional ATR, ATM, and spindle assembly checkpoint (SAC) pathways, tumor cells dividing across constriction frequently exhibited altered spindle pole organization, chromosome mis-segregations, micronuclei formation, chromosome fragility, high gene copy number variation, and transcriptional de-regulation and up-regulation of c-MYC oncogenic transcriptional signature via c-MYC locus amplifications. In vivo tumor settings showed that malignant cells populating metastatic foci or infiltrating the interstitial stroma gave rise to cells expressing high levels of c-MYC. Altogether, our data suggest that mechanical stress during metastatic migration contributes to override the checkpoint controls and boosts genotoxic and oncogenic events. Our findings may explain why cancer aneuploidy often does not correlate with mutations in SAC genes and why c-MYC amplification is strongly linked to metastatic tumors.

Mapping of long interspersed element-1 (L1) insertions by TIPseq provides information about sub chromosomal genetic variation in human embryos.

PURPOSE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos. METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls. RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos. CONCLUSION: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans.

Cryptococcus neoformans is at the top of the list of "most wanted" human pathogens. Only three classes of antifungal drugs are available for the treatment of cryptococcosis. Studies on antifungal resistance mechanisms are limited to the investigation of how a particular antifungal drug induces resistance to a particular drug, and the impact of stresses other than antifungals on the development of antifungal resistance and even cross-resistance is largely unexplored. The endoplasmic reticulum (ER) is a ubiquitous subcellular organelle of eukaryotic cells. Brefeldin A (BFA) is a widely used chemical inducer of ER stress. Here, we found that both weak and strong selection by BFA caused aneuploidy formation in C. neoformans, mainly disomy of chromosome 1, chromosome 3, and chromosome 7. Disomy of chromosome 1 conferred cross-resistance to two classes of antifungal drugs: fluconazole and 5-flucytosine, as well as hypersensitivity to amphotericin B. However, drug resistance was unstable, due to the intrinsic instability of aneuploidy. We found overexpression of AFR1 on Chr1 and GEA2 on Chr3 phenocopied BFA resistance conferred by chromosome disomy. Overexpression of AFR1 also caused resistance to fluconazole and hypersensitivity to amphotericin B. Furthermore, a strain with a deletion of AFR1 failed to form chromosome 1 disomy upon BFA treatment. Transcriptome analysis indicated that chromosome 1 disomy simultaneously upregulated AFR1, ERG11, and other efflux and ERG genes. Thus, we posit that BFA has the potential to drive the rapid development of drug resistance and even cross-resistance in C. neoformans, with genome plasticity as the accomplice.

Formation of the first plane of division relative to the pronuclear axis predicts embryonic ploidy.

RESEARCH QUESTION: Is there a relationship between the pronuclear axis and the first cleavage plane formation in human pronuclear-stage embryos, and what are the effects on ploidy and clinical pregnancy rates? DESIGN: Transferred embryos were followed up until their prognoses. A total of 762 embryos formed two cells and reached the blastocyst stage after normal fertilization in a time-lapse incubator. Embryos were classified into three groups: group A: embryos in which the first plane of division was formed parallel to the axis of the pronucleus; group B: embryos in which cases of oblique formation were observed; and group C: embryos in which cases of perpendicular formation were observed. RESULTS: The euploidy rate was significantly higher in groups A and B than those in group C (P < 0.01), whereas the aneuploidy rate was significantly higher in group C (P < 0.01) than in groups A and B. No differences were found between the three groups in frequency of positive HCG-based pregnancy tests, frequency of clinical pregnancies, miscarriage rates or delivery rates. CONCLUSIONS: The formation pattern of the first plane of division relative to the pronuclear axis was a predictor of embryonic ploidy, with a reduced rate of euploidy and a high probability of aneuploidy observed when the first plane of division was perpendicular to the pronuclear axis.

Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.

Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.

Non-invasive preimplantation genetic testing for aneuploidy: is the promise real?

Recent advances in preimplantation genetic testing for aneuploidy (PGT-A) have significantly enhanced its application in ART, providing critical insights into embryo viability, and potentially reducing both the time spent in fertility treatments and the risk of pregnancy loss. With the integration of next-generation sequencing, PGT-A now offers greater diagnostic precision, although challenges related to segmental aneuploidies and mosaicism remain. The emergence of non-invasive PGT-A (niPGT-A), which analyzes DNA in spent embryo culture media, promises a simpler aneuploidy screening method. This mini review assesses the methodological criteria for test validation, the current landscape of PGT-A, and the potential of niPGT-A, while evaluating its advantages and potential pitfalls. It underscores the importance of a robust three-phase validation process to ensure the clinical reliability of PGT-A. Despite initial encouraging data, niPGT-A not only confronts issues of DNA amplification failure and diagnostic inaccuracies but also has yet to meet the three-prong criteria required for appropriate test validation, necessitating further research for its clinical adoption. The review underscores that niPGT-A, like traditional PGT-A, must attain the high standards of precision and reliability expected of any genetic testing platform used in clinical settings before it can be adopted into routine ART protocols.

Maternal Reassurance, Satisfaction, and Anxiety after First-Trimester Screening for Aneuploidies: Comparison between Contingent Screening and Universal Cell-Free DNA Testing.

BACKGROUND: This study aims to evaluate maternal reassurance, satisfaction, and anxiety after two different strategies for the first-trimester screening for aneuploidies. METHODS: Patients between 11 + 3 and 13 + 6 weeks of gestation attending the first-trimester screening at Department of Mother and Child, University Hospital Federico II, Naples, Italy have been recruited and randomly allocated to contingent screening or universal cell-free fetal DNA testing (cffDNA). Questionnaires to measure reassurance, satisfaction, and anxiety have been filled twice: (Q1) after randomization and (Q2) after receiving results. Anxiety was measured by an Italian-version short form of the state scale of the Spielberger State-Trait Anxiety Inventory (STAI); child-related anxiety was measured by the 11-item Pregnancy-Related Anxiety Questionnaire-Revised Regardless of Parity (PRAQ-R2 scale); fear of bearing a physically or mentally handicapped child was measured considering only four items (item 4, 9, 10, and 11) of the PRAQ-R2 scale. RESULTS: 431 patients were recruited: 205 (49%) were randomized in the contingent screening arm, 226 (51%) in the cfDNA arm. Maternal reassurance, satisfaction, and anxiety were not different in the two groups. CONCLUSION: A contingent screening for aneuploidies in the first trimester seems able to ensure the same maternal reassurance and satisfaction as a cfDNA analysis in the low-risk population and to not affect maternal anxiety.

Re-Examination of PGT-A Detected Genetic Pathology in Compartments of Human Blastocysts: A Series of 23 Cases.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments-the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

Implementation of an Enhanced Prenatal Checklist to Increase Rates of Counseling of Prenatal Fetal Aneuploidy Testing.

Aim This study aims to assess the effect of implementing an enhanced prenatal genetic checklist to guide the provider's discussion on both screening and diagnostic options for fetal aneuploidy testing at the initial prenatal visit. Methods A retrospective quality improvement (QI) project was performed at a single, large, urban academic medical center. The implementation of this project was prospective; however, data was examined retrospectively after the QI initiative was implemented for three months. Patients were included if they were less than 24 weeks gestational age with a live intrauterine gestation at their initial obstetric (OB) visit. Patients less than 18 years old at the initial OB visit were excluded. The results were analyzed using the statistical software R. Chi-squared tests were used to examine proportional differences between the pre- and post-intervention groups with respect to demographic and clinical characteristics and documented genetic counseling discussions. Results A total of 416 patients were included in the final cohort. As measured by documentation, the rate of discussion of diagnostic prenatal genetic testing increased significantly from the pre-intervention proportion of 54% to the post-intervention proportion of 72% (p < 0.001). In the subgroup analysis of patients with advanced maternal age, the rate of discussion of diagnostic prenatal genetic testing increased significantly from the pre-intervention proportion of 53% to the post-intervention proportion of 83% (p = 0.003), and the rate of genetics counseling referrals made at the initial prenatal visit increased significantly from 4% pre-intervention to 38% post-intervention (p < 0.001). Conclusions The use of an enhanced prenatal genetic checklist led to increased discussion of diagnostic fetal aneuploidy testing and increased rates of referral to genetics counseling.

Selection forces underlying aneuploidy patterns in cancer.

Aneuploidy, the presence of an aberrant number of chromosomes, has been associated with tumorigenesis for over a century. More recently, advances in karyotyping techniques have revealed its high prevalence in cancer: About 90% of solid tumors and 50-70% of hematopoietic cancers exhibit chromosome gains or losses. When analyzed at the level of specific chromosomes, there are strong patterns that are observed in cancer karyotypes both pan-cancer and for specific cancer types. These specific aneuploidy patterns correlate strongly with outcomes for tumor initiation, progression, metastasis formation, immune evasion and resistance to therapeutic treatment. Despite their prominence, understanding the basis underlying aneuploidy patterns in cancer has been challenging. Advances in genetic engineering and bioinformatic analyses now offer insights into the genetic determinants of aneuploidy pattern selection. Overall, there is substantial evidence that expression changes of particular genes can act as the positive selective forces for adaptation through aneuploidy. Recent findings suggest that multiple genes contribute to the selection of specific aneuploid chromosomes in cancer; however, further research is necessary to identify the most impactful driver genes. Determining the genetic basis and accompanying vulnerabilities of specific aneuploidy patterns is an essential step in selectively targeting these hallmarks of tumors.

An egg sabotaging mechanism drives non-Mendelian transmission in mice.

During meiosis, homologous chromosomes segregate so that alleles are transmitted equally to haploid gametes, following Mendel's Law of Segregation. However, some selfish genetic elements drive in meiosis to distort the transmission ratio and increase their representation in gametes. The established paradigms for drive are fundamentally different for female vs male meiosis. In male meiosis, selfish elements typically kill gametes that do not contain them. In female meiosis, killing is predetermined, and selfish elements bias their segregation to the single surviving gamete (i.e., the egg in animal meiosis). Here we show that a selfish element on mouse chromosome 2, R2d2, drives using a hybrid mechanism in female meiosis, incorporating elements of both male and female drivers. If R2d2 is destined for the polar body, it manipulates segregation to sabotage the egg by causing aneuploidy that is subsequently lethal in the embryo, so that surviving progeny preferentially contain R2d2. In heterozygous females, R2d2 orients randomly on the metaphase spindle but lags during anaphase and preferentially remains in the egg, regardless of its initial orientation. Thus, the egg genotype is either euploid with R2d2 or aneuploid with both homologs of chromosome 2, with only the former generating viable embryos. Consistent with this model, R2d2 heterozygous females produce eggs with increased aneuploidy for chromosome 2, increased embryonic lethality, and increased transmission of R2d2. In contrast to a male meiotic driver, which kills its sister gametes produced as daughter cells in the same meiosis, R2d2 eliminates "cousins" produced from meioses in which it should have been excluded from the egg.

The Blastulation Rate Is Negatively Associated With Euploid Rate.

OBJECTIVES: To study the association between the blastulation rate, the presence of 1 pronucleus (1PN) zygotes, and the ploidy of the cohort of blastocysts. METHODS: A cross-sectional study using the existing databases of 2 university fertility centres in Canada. We included 345 cycles from 235 couples who underwent next-generation sequencing preimplantation genetic testing for the detection of aneuploidy in the study. RESULTS: A total of 1456 blastocysts were biopsied. In multivariate analysis, only female age and the number of 1PN/2PN embryos showed a negative association with euploid ratio. Surprisingly, when the analysis was limited to cycles with no delayed blastulation, the blastulation rate was also negatively associated with the euploid ratio. CONCLUSIONS: This study sheds some light on the stages of early embryo development. Further study on the mechanisms governing embryo development and the different cell cycle checkpoints in embryo development is warranted.

Assessing the necessity of screening ≤5 Mb segmental aneuploidy in routine PGT for aneuploidies.

RESEARCH QUESTION: Does routine clinical practice require an increase in the resolution of preimplantation genetic testing for aneuploidies (PGT-A) to detect segmental aneuploidies ≤5 Mb? DESIGN: This retrospective study analysed 963 trophectoderm biopsies from 346 couples undergoing PGT between 2019 and 2023. Segmental aneuploidies ≥1 Mb were reported. The characteristics, clinical interpretation and concordance of segmental aneuploidies ≤5 Mb were analysed. RESULTS: The incidence of segmental aneuploidies was 15.1% (145/963) in blastocysts, with segmental aneuploidies of ≤5 Mb accounting for 2.3% (22/963). The size of the segmental aneuploidies showed a skewed distribution. Segmental aneuploidies ≤5 Mb were found to occur more frequently on the q arm of the chromosome, compared with the p arm. Losses of ≤5 Mb segmental aneuploidies were more prevalent than gains, with 17 deletions compared with 5 duplications. Of the segmental aneuploidies, 63.6% (14/22) ≤5 Mb were de novo, and 50.0% (7/14) of de-novo segmental aneuploidies were pathogenic/likely pathogenic (P/LP) copy number variations, accounting for 0.7% of 963 blastocysts. For blastocysts carrying ≤5 Mb segmental aneuploidies, a re-analysis of back-up biopsy samples showed that 35.7% of de-novo segmental aneuploidies (5/14) were not detected in the back-up samples. Cases were reported in which prenatal diagnosis (amniocentesis) revealed the absence of embryonic ≤5 Mb segmental aneuploidies detected at the blastocyst stage. CONCLUSIONS: The incidence of P/LP de-novo ≤5 Mb segmental aneuploidies in human blastocysts is extremely low. There is no compelling need to increase the resolution of PGT-A to 5 Mb in routine clinical practice.

Integrated bulk and single-cell RNA sequencing identifies an aneuploidy-based gene signature to predict sensitivity of lung adenocarcinoma to traditional chemotherapy drugs and patients' prognosis.

Background: Patients with lung adenocarcinoma (LUAD) often develop a poor prognosis. Currently, researches on prognostic and immunotherapeutic capacity of aneuploidy-related genes in LUAD are limited. Methods: Genes related to aneuploidy were screened based on bulk RNA sequencing data from public databases using Spearman method. Next, univariate Cox and Lasso regression analyses were performed to establish an aneuploidy-related riskscore (ARS) model. Results derived from bioinformatics analysis were further validated using cellular experiments. In addition, typical LUAD cells were identified by subtype clustering, followed by SCENIC and intercellular communication analyses. Finally, ESTIMATE, ssGSEA and CIBERSORT algorithms were employed to analyze the potential relationship between ARS and tumor immune environment. Results: A five-gene ARS signature was developed. These genes were abnormally high-expressed in LUAD cell lines, and in particular the high expression of CKS1B promoted the proliferative, migratory and invasive phenotypes of LUAD cell lines. Low ARS group had longer overall survival time, higher degrees of inflammatory infiltration, and could benefit more from receiving immunotherapy. Patients in low ASR group responded more actively to traditional chemotherapy drugs (Erlotinib and Roscovitine). The scRNA-seq analysis annotated 17 cell subpopulations into seven cell clusters. Core transcription factors (TFs) such as CREB3L1 and CEBPD were enriched in high ARS cell group, while TFs such as BCLAF1 and UQCRB were enriched in low ARS cell group. CellChat analysis revealed that high ARS cell groups communicated with immune cells via SPP1 (ITGA4-ITGB1) and MK (MDK-NCl) signaling pathways. Conclusion: In this research, integrative analysis based on the ARS model provided a potential direction for improving the diagnosis and treatment of LUAD.

Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma.

Chemotherapy induced polyploidy is a mechanism of inherited drug resistance resulting in an aggressive disease course in cancer patients. Alisertib, an Aurora Kinase A (AK-A) ATP site inhibitor, induces cell cycle disruption resulting in polyaneuploidy in Diffuse Large B Cell Lymphoma (DLBCL). Propidium iodide flow cytometry was utilized to quantify alisertib induced polyploidy in U2932 and VAL cell lines. In U2932 cells, 1µM alisertib generated 8n+ polyploidy in 48% of the total cell population after 5 days of treatment. Combination of Aurkin A an AK-A/TPX2 site inhibitor, plus alisertib disrupted alisertib induced polyploidy in a dose-dependent manner with associated increased apoptosis. We generated a stable FUCCI U2932 cell line expressing Geminin-clover (S/G2/M) and cdt1-mKO (G1), to monitor cell cycle progression. Using this system, we identified alisertib induces polyploidy through endomitosis, which was eliminated with Aurkin A treatment. In a VAL mouse xenograft model, we show polyploidy generation in alisertib treated mice versus vehicle control or Aurkin A. Aurkin A plus alisertib significantly reduced polyploidy to vehicle control levels. Our in vitro and in vivo studies show that Aurkin A synergizes with alisertib and significantly decreases the alisertib dose needed to disrupt polyploidy while increasing apoptosis in DLBCL cells.

Oral cancer detection and progression prediction using noninvasive cytology-based DNA ploidy approach.

BACKGROUND: Despite the oral cavity being readily accessible, oral cancer (OC) remains a significant burden. The objective of this study is to develop a DNA ploidy-based cytology test for early detection of high-risk oral lesions. METHODS: This retrospective study was conducted using 569 oral brushing samples collected from 95 normal and 474 clinically abnormal mucosa with biopsy diagnosis of reactive, low-grade or high-grade precancer or cancers. Brushing cells were processed to characterize DNA ploidy. A two-step DNA ploidy-based algorithm, the DNA ploidy oral cytology (DOC) test, was developed using a training set, and verified in test and validation sets to differentiate high-grade lesions (HGLs) from normal. The prognostic value of the test was evaluated by an independent outcome cohort, including progressed and non-progressing normal, reactive and low-grade lesions. Classification performance was assessed by accuracy, sensitivity, and specificity, while the prognostic value was evaluated by using the Cox proportional hazards analysis on 3-year progression-free survival (PFS). RESULTS: The developed DOC test exhibited high accuracy for detecting HGLs in the test and validation sets, with a sensitivity of 0.97 and 0.96, respectively. Its application to the Outcome cohort demonstrated significant prognostic value for 3-year PFS (log rank, p < 0.001). Multivariate analysis showed that high-grade pathology was the only variable explaining positive DOC test, not age, smoking, or lesional site. CONCLUSION: Clinical implementation of the DOC test could provide an effective screening method for detecting HGLs for biopsy and lesions at risk of progression.