Clonal Decomposition and DNA Replication States Defined by Scaled Single-Cell Genome Sequencing.

Accurate measurement of clonal genotypes, mutational processes, and replication states from individual tumor-cell genomes will facilitate improved understanding of tumor evolution. We have developed DLP+, a scalable single-cell whole-genome sequencing platform implemented using commodity instruments, image-based object recognition, and open source computational methods. Using DLP+, we have generated a resource of 51,926 single-cell genomes and matched cell images from diverse cell types including cell lines, xenografts, and diagnostic samples with limited material. From this resource we have defined variation in mitotic mis-segregation rates across tissue types and genotypes. Analysis of matched genomic and image measurements revealed correlations between cellular morphology and genome ploidy states. Aggregation of cells sharing copy number profiles allowed for calculation of single-nucleotide resolution clonal genotypes and inference of clonal phylogenies and avoided the limitations of bulk deconvolution. Finally, joint analysis over the above features defined clone-specific chromosomal aneuploidy in polyclonal populations.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

Structural basis of Cas3 activation in type I-C CRISPR-Cas system.

CRISPR-Cas systems function as adaptive immune mechanisms in bacteria and archaea and offer protection against phages and other mobile genetic elements. Among many types of CRISPR-Cas systems, Type I CRISPR-Cas systems are most abundant, with target interference depending on a multi-subunit, RNA-guided complex known as Cascade that recruits a transacting helicase nuclease, Cas3, to degrade the target. While structural studies on several other types of Cas3 have been conducted long ago, it was only recently that the structural study of Type I-C Cas3 in complex with Cascade was revealed, shedding light on how Cas3 achieve its activity in the Cascade complex. In the present study, we elucidated the first structure of standalone Type I-C Cas3 from Neisseria lactamica (NlaCas3). Structural analysis revealed that the histidine-aspartate (HD) nuclease active site of NlaCas3 was bound to two Fe2+ ions that inhibited its activity. Moreover, NlaCas3 could cleave both single-stranded and double-stranded DNA in the presence of Ni2+ or Co2+, showing the highest activity in the presence of both Ni2+ and Mg2+ ions. By comparing the structural studies of various Cas3 proteins, we determined that our NlaCas3 stays in an inactive conformation, allowing us to understand the structural changes associated with its activation and their implication.

Targeting ROS-sensing Nrf2 potentiates anti-tumor immunity of intratumoral CD8+ T and CAR-T cells.

Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8+ T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2-/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8+ T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors.

Clonal haematopoiesis of indeterminate potential and atrial fibrillation: an east Asian cohort study.

BACKGROUND AND AIMS: Both clonal haematopoiesis of indeterminate potential (CHIP) and atrial fibrillation (AF) are age-related conditions. This study investigated the potential role of CHIP in the development and progression of AF. METHODS: Deep-targeted sequencing of 24 CHIP mutations (a mean depth of coverage = 1000×) was performed in 1004 patients with AF and 3341 non-AF healthy subjects. Variant allele fraction ≥ 2.0% indicated the presence of CHIP mutations. The association between CHIP and AF was evaluated by the comparison of (i) the prevalence of CHIP mutations between AF and non-AF subjects and (ii) clinical characteristics discriminated by CHIP mutations within AF patients. Furthermore, the risk of clinical outcomes-the composite of heart failure, ischaemic stroke, or death-according to the presence of CHIP mutations in AF was investigated from the UK Biobank cohort. RESULTS: The mean age was 67.6 ± 6.9 vs. 58.5 ± 6.5 years in AF (paroxysmal, 39.0%; persistent, 61.0%) and non-AF cohorts, respectively. CHIP mutations with a variant allele fraction of ≥2.0% were found in 237 (23.6%) AF patients (DNMT3A, 13.5%; TET2, 6.6%; and ASXL1, 1.5%) and were more prevalent than non-AF subjects [356 (10.7%); P < .001] across the age. After multivariable adjustment (age, sex, smoking, body mass index, diabetes, and hypertension), CHIP mutations were 1.4-fold higher in AF [adjusted odds ratio (OR) 1.38; 95% confidence interval 1.10-1.74, P < .01]. The ORs of CHIP mutations were the highest in the long-standing persistent AF (adjusted OR 1.50; 95% confidence interval 1.14-1.99, P = .004) followed by persistent (adjusted OR 1.44) and paroxysmal (adjusted OR 1.33) AF. In gene-specific analyses, TET2 somatic mutation presented the highest association with AF (adjusted OR 1.65; 95% confidence interval 1.05-2.60, P = .030). AF patients with CHIP mutations were older and had a higher prevalence of diabetes, a longer AF duration, a higher E/E', and a more severely enlarged left atrium than those without CHIP mutations (all P < .05). In UK Biobank analysis of 21 286 AF subjects (1297 with CHIP and 19 989 without CHIP), the CHIP mutation in AF is associated with a 1.32-fold higher risk of a composite clinical event (heart failure, ischaemic stroke, or death). CONCLUSIONS: CHIP mutations, primarily DNMT3A or TET2, are more prevalent in patients with AF than non-AF subjects whilst their presence is associated with a more progressive nature of AF and unfavourable clinical outcomes.

Allergic rhinitis phenotypes with distinct transcriptome profiles in children: A birth cohort.

BACKGROUND: Allergic rhinitis (AR) phenotypes in childhood are unclear. OBJECTIVES: This study sought to determine AR phenotypes and investigate their natural course and clinical and transcriptomic characteristics. METHODS: Latent class trajectory analysis was used for phenotyping AR in 1050 children from birth through 12 years using a birth cohort study. Blood transcriptome analyses were performed to define the underlying mechanisms of each phenotype. RESULTS: Five AR phenotypes were identified: early onset (n = 88, 8.4%), intermediate transient (n = 110, 10.5%), late onset (n = 209, 19.9%), very late onset (n=187, 17.8%), and never/infrequent (n = 456, 43.4%). Children with early-onset AR were associated with higher AR severity and sensitizations to foods at age 1 year and inhalants at age 3 years and asthma symptoms, but not with bronchial hyperresponsiveness (BHR). Children with late-onset AR phenotype associated with sensitizations to various foods at age 1 year but not from age 3 years, and to inhalants from age 7 years and with asthma with BHR. Children with very late-onset AR phenotype associated with sensitizations to foods throughout preschool age and to inhalants at ages 7 and 9 years and with asthma with BHR. Transcriptome analysis showed that early-onset AR was associated with viral/bacterial infection-related defense response, whereas late-onset AR was associated with T cell-related immune response. CONCLUSIONS: Early-onset AR phenotype was associated with sensitization to foods and inhalants at an early age and asthma symptoms, but not with BHR, whereas very late- and late-onset AR phenotypes were positively associated with sensitization to inhalants and asthma with BHR. Transcriptomic analyses indicated that early- and late-onset AR phenotypes had distinct underlying mechanisms related to AR as well.

Tonic Activation of NR2D-Containing NMDARs Exacerbates Dopaminergic Neuronal Loss in MPTP-Injected Parkinsonian Mice.

NR2D subunit-containing NMDA receptors (NMDARs) gradually disappear during brain maturation but can be recruited by pathophysiological stimuli in the adult brain. Here, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication recruited NR2D subunit-containing NMDARs that generated an Mg2+-resistant tonic NMDA current (INMDA) in dopaminergic (DA) neurons in the midbrain of mature male mice. MPTP selectively generated an Mg2+-resistant tonic INMDA in DA neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Consistently, MPTP increased NR2D but not NR2B expression in the midbrain regions. Pharmacological or genetic NR2D interventions abolished the generation of Mg2+-resistant tonic INMDA in SNpc DA neurons, and thus attenuated subsequent DA neuronal loss and gait deficits in MPTP-treated mice. These results show that extrasynaptic NR2D recruitment generates Mg2+-resistant tonic INMDA and exacerbates DA neuronal loss, thus contributing to MPTP-induced Parkinsonism. The state-dependent NR2D recruitment could be a novel therapeutic target for mitigating cell type-specific neuronal death in neurodegenerative diseases.SIGNIFICANCE STATEMENT NR2D subunit-containing NMDA receptors (NMDARs) are widely expressed in the brain during late embryonic and early postnatal development, and then downregulated during brain maturation and preserved at low levels in a few regions of the adult brain. Certain stimuli can recruit NR2D subunits to generate tonic persistent NMDAR currents in nondepolarized neurons in the mature brain. Our results show that MPTP intoxication recruits NR2D subunits in midbrain dopaminergic (DA) neurons, which leads to tonic NMDAR current-promoting dopaminergic neuronal death and consequent abnormal gait behavior in the MPTP mouse model of Parkinson's disease (PD). This is the first study to indicate that extrasynaptic NR2D recruitment could be a target for preventing neuronal death in neurodegenerative diseases.

Selective Glycan Labeling of Mannose-Containing Glycolipids in Mycobacteria.

Mycobacterium tuberculosis (Mtb) is one of history's most successful human pathogens. By subverting typical immune responses, Mtb can persist within a host until conditions become favorable for growth and proliferation. Virulence factors that enable mycobacteria to modulate host immune systems include a suite of mannose-containing glycolipids: phosphatidylinositol mannosides, lipomannan, and lipoarabinomannan (LAM). Despite their importance, tools for their covalent capture, modification, and imaging are limited. Here, we describe a chemical biology strategy to detect and visualize these glycans. Our approach, biosynthetic incorporation, is to synthesize a lipid-glycan precursor that can be incorporated at a late-stage step in glycolipid biosynthesis. We previously demonstrated selective mycobacterial arabinan modification by biosynthetic incorporation using an exogenous donor. This report reveals that biosynthetic labeling is general and selective: it allows for cell surface mannose-containing glycolipid modification without nonspecific labeling of mannosylated glycoproteins. Specifically, we employed azido-(Z,Z)-farnesyl phosphoryl-ß-d-mannose probes and took advantage of the strain-promoted azide-alkyne cycloaddition to label and directly visualize the localization and dynamics of mycobacterial mannose-containing glycolipids. Our studies highlight the generality and utility of biosynthetic incorporation as the probe structure directs the selective labeling of distinct glycans. The disclosed agents allowed for direct tracking of the target immunomodulatory glycolipid dynamics in cellulo. We anticipate that these probes will facilitate investigating the diverse biological roles of these glycans.

Pogostemon cablin Extract Promotes Wound Healing through OR2AT4 Activation and Exhibits Anti-Inflammatory Activity.

Skin healing occurs through an intricate process called wound healing which comprises four phases: coagulation and hemostasis, inflammation, cellular proliferation, and remodeling. Chronic wounds often arise because of prolonged or excessive inflammation, which hinders the healing process and wound closure. Despite the recognized efficacy of Pogostemon cablin (patchouli) in wound healing, the precise mechanism of action of Pogostemon cablin extract (PCE) on inflammation and wound healing remains poorly understood. In this study, we investigated the effects of PCE on cell proliferation and wound healing, as well as its anti-inflammatory activity, using in vitro experiments. We found that PCE increased cell proliferation and expression of the cell proliferation marker Ki67 and accelerated wound healing in human keratinocytes through the activation of OR2AT4. Furthermore, PCE exhibited anti-inflammatory effects by decreasing the levels of pro-inflammatory cytokines interleukin-6 and -8 in lipopolysaccharide-treated and TNF-α-exposed THP-1 and HaCaT cells, respectively. Overall, these findings suggest that PCE holds therapeutic potential by promoting cell proliferation, facilitating wound healing, and exerting anti-inflammatory effects.

Reduction of Upper Gastrointestinal Bleeding Risk With Proton Pump Inhibitor Therapy in Asian Patients With Atrial Fibrillation Receiving Direct Oral Anticoagulant: A Nationwide Population-based Cohort Study.

BACKGROUND & AIMS: In patients with atrial fibrillation (AF) receiving direct oral anticoagulant (DOAC), upper gastrointestinal bleeding (UGIB) is a serious complication. There are limited data on the benefit of preventive proton pump inhibitor (PPI) use to reduce the risk of UGIB in DOAC users. METHODS: We included patients with AF receiving DOAC from 2015 to 2020 based on the Korean Health Insurance Review and Assessment database. The propensity score (PS) weighting method was used to compare patients with PPI use and those without PPI use. The primary outcome was hospitalization for UGIB. Weighted hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) were evaluated using the Cox proportional hazards regression model. RESULTS: A total of 165,624 patients were included (mean age: 72.2 ± 10.8 years; mean CHA2DS2-VASc score: 4.3 ± 1.8; mean HAS-BLED score: 3.3 ± 1.2). Among them, 99,868 and 65,756 were in the non-PPI group and PPI group, respectively. During a median follow-up of 1.5 years, the PPI group was associated with lower risks of hospitalization for UGIB and UGIB requiring red blood cell transfusion than non-PPI group (weighted HR, 0.825; 95% CI, 0.761-0.894 and 0.798; 95% CI, 0.717-0.887, respectively, both P < .001). The benefits of PPI on the risk of hospitalization for UGIB were greater in those with older age (≥75 years), higher HAS-BLED score (≥3), prior GIB history, and concomitant use of antiplatelet agent (all P-for-interaction < .1). Low-dose PPI was consistently associated with a lower risk of significant UGIB by 43.6-49.3% (P < .001). CONCLUSIONS: In this large Asian cohort of patients with AF on DOAC, PPI co-therapy is beneficial for reducing the risk of hospitalization for UGIB, particularly in high-risk patients.

Novel structure of secreted small molecular weight antigen Mtb12 from Mycobacterium tuberculosis.

Mtb12, a small protein secreted by Mycobacterium tuberculosis, is known to elicit immune responses in individuals infected with the pathogen. It serves as an antigen recognized by the host's immune system. Due to its immunogenic properties and pivotal role in tuberculosis (TB) pathogenesis, Mtb12 is considered a promising candidate for TB diagnosis and vaccine development. However, the structural and functional properties of Mtb12 are largely unexplored, representing a significant gap in our understanding of M. tuberculosis biology. In this study, we present the first structure of Mtb12, which features a unique tertiary configuration consisting of four beta strands and four alpha helices. Structural analysis reveals that Mtb12 has a surface adorned with a negatively charged pocket adjacent to a central cavity. The features of these structural elements and their potential effects on the function of Mtb12 warrant further exploration. These findings offer valuable insights for vaccine design and the development of diagnostic tools.

Novel structure of the anti-CRISPR protein AcrIE3 and its implication on the CRISPR-Cas inhibition.

As a response to viral infections, bacteria have evolved the CRISPR-Cas system as an adaptive immune mechanism, enabling them to target and eliminate viral genetic material introduced during infection. However, viruses have also evolved mechanisms to counteract this bacterial defense, including anti-CRISPR proteins, which can inactivate the CRISPR-Cas adaptive immune system, thus aiding the viruses in their survival and replication within bacterial hosts. In this study, we establish the high-resolution crystal structure of the Type IE anti-CRISPR protein, AcrIE3. Our structural examination showed that AcrIE3 adopts a helical bundle fold comprising four α-helices, with a notably extended loop at the N-terminus. Additionally, surface analysis of AcrIE3 revealed the presence of three acidic regions, which potentially play a crucial role in the inhibitory function of this protein. The structural information we have elucidated for AcrIE3 will provide crucial insights into fully understanding its inhibitory mechanism. Furthermore, this information is anticipated to be important for the application of the AcrIE family in genetic editing, paving the way for advancements in gene editing technologies.

Structural analysis of a bacterial ankyrin-like protein secreted by Acinetobacter baumannii.

In bacteria, the ankyrin-like protein AnkB helps overcome stress by regulating catalase activity when expressed under stressful conditions. As the structural properties of AnkB are largely unexplored, our understanding of various AnkB-mediated functions in bacteria remains limited. In the present study, we describe the structure of AnkB from Acinetobacter baumannii, hereafter referred to as "AbAnkB," which has a unique tertiary configuration compared with that of other ankyrin domain-containing proteins. Structural analysis revealed that AbAnkB has a relatively long loop between AKR3 and AKR4 and an oppositely positioned α8 helix. Based on amino acid conservation and protein surface analyses, we identified a hydrophobic patch that might be critical for the function of AbAnkB. To the best of our knowledge, our study is the first to report the structure of a bacterial AnkB protein; our findings will markedly enhance our understanding of its functions in bacteria.

Integrated Structural Modulation Inducing Fast Charge Transfer in Aqueous Zinc-Ion Batteries.

Aqueous Zn-ion batteries (AZIBs) are promising energy-storage devices owing to their exceptional safety, long cycle life, simple production, and high storage capacity. Manganese oxides are considered potential cathode materials for AZIBs, primarily because of their safety, low cost, simple synthesis, and high storage capacity. However, MnO2-based cathodes tend to deteriorate structurally during long-term cycling, which reduces their reversible capacity. In this study, an advanced α-MnO2@SnO2 nanocomposite via facile hydrothermal synthesis is developed. The synergistic effects of lattice disorder and increased electron conductivity in the α-MnO2@SnO2 nanocomposite mitigate structural degradation and enhance the overall electrochemical performance. The nanocomposite exhibits a high reversible capacity of 347 mAh g-1 at a current density of 100 mA g-1 after 50 cycles. Furthermore, it exhibits excellent rate performance and stable capacity even after 1000 cycles, maintaining a capacity of 78 mAh g-1 at a high current density of 5 A g-1. This excellent electrochemical performance is attributed to the reversible Zn intercalation in α-MnO2@SnO2 nanocomposites due to the increased structural stability and fast ion/electron exchange caused by the distortion of the tunnel structure, on the basis of various ex situ experiments, density functional theory calculations, and electrochemical characterizations.

Microstructure of the corneal endothelial transition zone in different laboratory animals.

Purpose: To compare the microstructure of the corneal endothelial transition zone in different laboratory animals. Methods: Flat-mount corneas of rabbits, rats, and mice were stained with Alizarin Red S (ARS) and observed using scanning electron microscopy (SEM). The progenitor cell markers p75 neurotrophin receptor (p75NTR), SRY-box transcription factor 9 (SOX9), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), telomerase reverse transcriptase (TERT), and proliferation marker Ki-67 were examined in the flat-mounted corneas of three laboratory animals using immunofluorescence microscopy. Results: On flat mounts, proximity to the trabecular meshwork correlated with weaker ARS staining and greater polymorphism of endothelial cells in the transition zone in all animals. On SEM, distinct and smooth structures of the transition zone were negligibly detected in all animals. The endothelial cells in the transition zone had irregular shapes, with less dense, less wavy intercellular junctions, especially in murine corneas, exhibiting unique intercellular cystic spaces. In the transition zone of the rabbit cornea, progenitor cell markers p75NTR, SOX9, Lgr5, TERT, and proliferation marker Ki-67 were expressed, in contrast to those in other murine corneas. Conclusions: Although the transition zone was not identified clearly, irregular cell morphology and loss of cell-cell contact were observed in all animal corneal endothelial cells. The proliferative capacity and the presence of progenitor cells were confirmed in the transition zone, especially in the rabbit cornea.

The impact of early cryoballoon ablation on clinical outcome in patients with atrial fibrillation: From the Korean cryoballoon ablation registry.

INTRODUCTION: Influence of early atrial fibrillation (AF) ablation, particularly cryoballoon ablation (CBA), on clinical outcome during long-term follow-up has not been clarified. The objective was to determine whether an early CBA (diagnosis-to-ablation of ≤6 months) strategy could affect freedom from AF recurrence after index CBA. METHODS: The study included 2605 patients from Korean CBA registry data with follow-up >12 months after de novo CBA. The primary outcome was recurrence of atrial tachyarrhythmias (ATs) of ≥30-s after a 3-month blanking period. RESULTS: Compared to patients in early CBA group, patients in late CBA group had higher prevalence of diabetes, congestive heart failure, and chronic kidney disease, and higher mean CHA2 DS2 -VAS score. During mean follow-up of >21 months, ATs recurrence was detected in 839 (32.2%) patients. The early CBA group showed a significantly lower 2-year recurrence rate of ATs than the late CBA group (26.1% vs. 31.7%, p = 0.043). In subgroup analysis, the early CBA group showed significantly higher 1-year and 2-year freedom from ATs recurrence than the late CBA group only in paroxysmal atrial fibrillation (PAF) patients in overall and propensity score matched cohorts. Multivariate analysis showed that early CBA was an independent factor for preventing ATs recurrence in PAF (hazard ratio: 0.637; 95% confidence intervals: 0.412-0.984). CONCLUSION: Early CBA strategy, resulting in significantly lower ATs recurrence during 2-year follow-up after index CBA, might be considered as an initial rhythm control therapy in patients with paroxysmal AF.

Long-term clinical impact of early recurrence of atrial tachyarrhythmia after cryoballoon ablation in patients with atrial fibrillation.

INTRODUCTION: The impact of early recurrence of atrial tachyarrhythmia (ERAT) within the 90-day blanking period on long-term outcomes in atrial fibrillation (AF) patients undergoing cryoballoon ablation (CBA) is controversial. This study aimed to assess the relationship between ERAT and late recurrence of atrial tachyarrhythmia (LRAT) post-CBA. METHODS: Utilizing data from a multicenter registry in Korea (May 2018 to June 2022), we analyzed the presence and timing of ERAT (<30, 30-60, and 60-90 days) and its association with LRAT risk after CBA. LRAT was defined as any recurrence of AF, atrial flutter, or atrial tachycardia lasting more than 30 s beyond the 90 days. RESULTS: Out of 2636 patients, 745 (28.2%) experienced ERAT post-CBA. Over an average follow-up period of 21.2 ± 10.3 months, LRAT was observed in 874 (33.1%) patients. Patients with ERAT had significantly lower 1-year LRAT freedom compared to those without ERAT (42.6% vs. 85.5%, p < .001). Multivariate analysis identified ERAT as a potential predictor of LRAT, with a hazard ratio (HR) of 3.98 (95% confidence interval [CI], 3.47-4.57). Significant associations were noted across all examined time frames (HR, 3.84; 95% CI, 3.32-4.45 in <30 days, HR, 5.53; 95% CI, 4.13-7.42 in 30-60 days, and HR, 4.29; 95% CI, 3.12-5.89 in 60-90 days). This finding was consistently observed across all types of AF. CONCLUSION: ERAT during the 90-day blanking period strongly predicts LRAT in AF patients undergoing CBA, indicating a need to reconsider the clinical significance of this period.

Effect of physical activity on incident atrial fibrillation in individuals with varying duration of diabetes: a nationwide population study.

BACKGROUND: Diabetes mellitus (DM) duration affects incident atrial fibrillation (AF) risk; the effect of physical activity on mitigating AF risk related to varying DM duration remains unknown. We assessed the effect of physical activity on incident AF in patients with DM with respect to known DM duration. METHODS: Patients with type 2 DM who underwent the Korean National Health Insurance Service health examination in 2015-2016 were grouped by DM duration: new onset and < 5, 5-9, and ≥ 10 years. Physical activity was classified into four levels: 0, < 500, 500-999, 1,000-1,499, and ≥ 1,500 metabolic equivalent task (MET)-min/week, with the primary outcome being new-onset AF. RESULTS: The study enrolled 2,392,486 patients (aged 59.3 ± 12.0 years, 39.8% female) with an average follow-up of 3.9 ± 0.8 years and mean DM duration of 5.3 ± 5.1 years. Greater physical activity was associated with a lower AF risk. Lowering of incident AF risk varied with different amounts of physical activity in relation to known DM duration. Among patients with new-onset DM, DM duration < 5 years and 5-9 years and 1,000-1,499 MET-min/week exhibited the lowest AF risk. Physical activity ≥ 1,500 MET-min/week was associated with the lowest incident AF risk in patients with DM duration ≥ 10 years (by 15%), followed DM duration of 5-9 years (12%) and < 5 years (9%) (p-for-interaction = 0.002). CONCLUSIONS: Longer DM duration was associated with a high risk of incident AF, while increased physical activity generally reduced AF risk. Engaging in > 1,500 MET-min/week was associated with the greatest AF risk reduction in patients with longer DM duration, highlighting the potential benefits of higher activity levels for AF prevention.

Polyoxometalate inhibition of SOX2-mediated tamoxifen resistance in breast cancer.

BACKGROUND: Increased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells. METHODS: Various POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis. RESULTS: POMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6- (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen. CONCLUSIONS: Together, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.