Pycallingcards: an integrated environment for visualizing, analyzing, and interpreting Calling Cards data.

MOTIVATION: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification. Despite these advantages, there has been a lack of dedicated bioinformatics tools for the detailed analysis of CC data. RESULTS: We introduce Pycallingcards, a comprehensive Python module specifically designed for the analysis of single-cell and bulk CC data across multiple species. Pycallingcards introduces two innovative peak callers, CCcaller and MACCs, enhancing the accuracy and speed of pinpointing TF binding sites from CC data. Pycallingcards offers a fully integrated environment for data visualization, motif finding, and comparative analysis with RNA-seq and ChIP-seq datasets. To illustrate its practical application, we have reanalyzed previously published mouse cortex and glioblastoma datasets. This analysis revealed novel cell-type-specific binding sites and potential sex-linked TF regulators, furthering our understanding of TF binding and gene expression relationships. Thus, Pycallingcards, with its user-friendly design and seamless interface with the Python data science ecosystem, stands as a critical tool for advancing the analysis of TF functions via CC data. AVAILABILITY AND IMPLEMENTATION: Pycallingcards can be accessed on the GitHub repository: https://github.com/The-Mitra-Lab/pycallingcards.

Joint association of sedentary behavior and vitamin D status with mortality among cancer survivors.

BACKGROUND: Sedentary behavior and vitamin D deficiency are independent risk factors for mortality in cancer survivors, but their joint association with mortality has not been investigated. METHODS: We analyzed data from 2914 cancer survivors who participated in the National Health and Nutrition Examination Survey (2007-2018) and followed up with them until December 31, 2019. Sedentary behavior was assessed by self-reported daily hours of sitting, and vitamin D status was measured by serum total 25-hydroxyvitamin D (25(OH)D) levels. RESULTS: Among 2914 cancer survivors, vitamin D deficiency was more prevalent in those with prolonged daily sitting time. During up to 13.2 years (median, 5.6 years) of follow-up, there were 676 deaths (cancer, 226; cardiovascular disease, 142; other causes, 308). The prolonged sitting time was associated with a higher risk of all-cause and noncancer mortality, and vitamin D deficiency was associated with a higher risk of all-cause and cancer mortality. Furthermore, cancer survivors with both prolonged sitting time (≥ 6 h/day) and vitamin D deficiency had a significantly higher risk of all-cause (HR, 2.05; 95% CI: 1.54-2.72), cancer (HR, 2.33; 95% CI, 1.47-3.70), and noncancer mortality (HR, 1.91; 95% CI, 1.33-2.74) than those with neither risk factor after adjustment for potential confounders. CONCLUSIONS: In a nationally representative sample of U.S. cancer survivors, the joint presence of sedentary behavior and vitamin D deficiency was significantly associated with an increased risk of all-cause and cancer-specific mortality.

Calling Cards: A Customizable Platform to Longitudinally Record Protein-DNA Interactions Over Time in Cells and Tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes. To achieve this, Calling Cards uses the piggyBac transposase to insert self-reporting transposon "Calling Cards" into the genome, leaving permanent marks at interaction sites. Calling Cards can be deployed in a variety of in vitro and in vivo biological systems to study gene regulatory networks involved in development, aging, and disease. Out of the box, it assesses enhancer usage but can be adapted to profile-specific transcription factor (TF) binding with custom TF-piggyBac fusion proteins. The Calling Cards workflow has five main stages: delivery of Calling Cards reagents, sample preparation, library preparation, sequencing, and data analysis. Here, we first present a comprehensive guide for experimental design, reagent selection, and optional customization of the platform to study additional TFs. Then, we provide an updated protocol for the five steps, using reagents that improve throughput and decrease costs, including an overview of a newly deployed computational pipeline. This protocol is designed for users with basic molecular biology experience to process samples into sequencing libraries in 2 days. Familiarity with bioinformatic analysis and command line tools is required to set up the pipeline in a high-performance computing environment and to conduct downstream analyses. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparation and delivery of Calling Cards reagents Support Protocol 1: Next-generation sequencing quantification of barcode distribution within self-reporting transposon plasmid pool and adeno-associated virus genome Basic Protocol 2: Sample collection and RNA purification Support Protocol 2: Library density quantitative PCR Basic Protocol 3: Sequencing library preparation Basic Protocol 4: Library pooling and sequencing Basic Protocol 5: Data analysis.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.

Contrast-enhanced image-guided lead deployment for left bundle branch pacing.

BACKGROUND: Left bundle branch pacing (LBBP) is a novel conduction system pacing modality, but pacing lead deployment remains challenging. OBJECTIVES: This study aimed to evaluate the feasibility of visualization-enhanced lead deployment for LBBP implantation and to assess LBBP characteristics on the basis of lead tip location. METHODS: Successful LBBP with a well-defined lead tip location by visualization of the tricuspid value annulus in 20 patients was retrospectively analyzed to develop an image-guided technique to identify the LBBP target site. This technique was then prospectively tested in 60 patients who were randomized into 2 groups, one using the standard approach (the standard group) and the other using the image-guided technique (the visualization group). The procedural details, electrophysiological characteristics, and short-term follow-up were compared between groups. RESULTS: LBBP was successfully achieved in 28 patients in the standard group and in 29 in the visualization group. The procedural and fluoroscopic durations in the visualization group (66.76 ± 14.62 and 7.83 ± 2.05 minutes) were significantly shorter than those in the standard group (85.46 ± 20.19 and 11.11 ± 3.51 minutes) (P < .01). The number of lead deployment attempts in the visualization group was lower than that in the standard group (2.03 ± 1.18 vs 2.96 ± 1.17; P < .01), and the proportion of left bundle branch potential recorded was higher (79.3% vs 46.4%; P = .01). CONCLUSION: Using a visualization technique, the procedural and fluoroscopic durations for LBBP implantation were significantly shortened with fewer lead repositioning attempts.

MicroRNA-144 Suppresses Prostate Cancer Growth and Metastasis by Targeting EZH2.

BACKGROUND: Prostate cancer is a common malignant tumor with a high incidence. MicroRNAs (miRNAs) have been shown to be important post-transcriptional regulators during tumorigenesis. This study aimed to explore the effect of miR-144 on PCa proliferation and apoptosis. MATERIAL AND METHODS: The expression of miR-144 and EZH2 were examined in clinical PCa tissues. PCa cell line LNCAP and DU-145 was employed and transfected with miR-144 mimics or inhibitors. The correlation between miR-144 and EZH2 was verified by luciferase reporter assay. Cell viability, apoptosis and migratory capacity were detected by CCK-8, flow cytometry assay and wound healing assay. The protein level of EZH2, E-Cadherin, N-Cadherin and vimentin were analyzed by western blotting. RESULTS: miR-144 was found to be negatively correlated to the expression of EZH2 in PCa tissues. Further studies identified EZH2 as a direct target of miR-144. Moreover, overexpression of miR-144 downregulated expression of EZH2, reduced cell viability and promoted cell apoptosis, while knockdown of miR-144 led to an inverse result. miR-144 also suppressed epithelial-mesenchymal transition level of PCa cells. CONCLUSION: Our study indicated that miR-144 negatively regulate the expression of EZH2 in clinical specimens and in vitro. miR-144 can inhibit cell proliferation and induce cell apoptosis in PCa cells. Therefore, miR-144 has the potential to be used as a biomarker for predicting the progression of PCa.

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells. The genomic locations of SRTs are recovered from mRNA, and SRTs deposited by exogenous, TF-transposase fusions can be used to map TFBS. We then present scCC, which map SRTs from scRNA-seq libraries, simultaneously identifying cell types and TFBS in those same cells. We benchmark multiple TFs with this technique. Next, we use scCC to discover BRD4-mediated cell-state transitions in K562 cells. Finally, we map BRD4 binding sites in the mouse cortex at single-cell resolution, establishing a new method for studying TF biology in situ.

A viral toolkit for recording transcription factor-DNA interactions in live mouse tissues.

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF-enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.

Transposase mapping identifies the genomic targets of BAP1 in uveal melanoma.

BACKGROUND: BAP1 is a histone deubiquitinase that acts as a tumor and metastasis suppressor associated with disease progression in human cancer. We have used the "Calling Card System" of transposase-directed transposon insertion mapping to identify the genomic targets of BAP1 in uveal melanoma (UM). This system was developed to identify the genomic loci visited by transcription factors that bind directly to DNA; our study is the first use of the system with a chromatin-remodeling factor that binds to histones but does not interact directly with DNA. METHODS: The transposase piggyBac (PBase) was fused to BAP1 and expressed in OCM-1A UM cells. The insertion of transposons near BAP1 binding sites in UM cells were identified by genomic sequencing. We also examined RNA expression in the same OCM-1A UM cells after BAP1 depletion to identify BAP1 binding sites associated with BAP1-responsive genes. Sets of significant genes were analyzed for common pathways, transcription factor binding sites, and ability to identify molecular tumor classes. RESULTS: We found a strong correlation between multiple calling-card transposon insertions targeted by BAP1-PBase and BAP1-responsive expression of adjacent genes. BAP1-bound genomic loci showed narrow distributions of insertions and were near transcription start sites, consistent with recruitment of BAP1 to these sites by specific DNA-binding proteins. Sequence consensus analysis of BAP1-bound sites showed enrichment of motifs specific for YY1, NRF1 and Ets transcription factors, which have been shown to interact with BAP1 in other cell types. Further, a subset of the BAP1 genomic target genes was able to discriminate aggressive tumors in published gene expression data from primary UM tumors. CONCLUSIONS: The calling card methodology works equally well for chromatin regulatory factors that do not interact directly with DNA as for transcription factors. This technique has generated a new and expanded list of BAP1 targets in UM that provides important insight into metastasis pathways and identifies novel potential therapeutic targets.

Effect of Obesity on Outcomes of Percutaneous Nephrolithotomy in Renal Stone Management: A Systematic Review and Meta-Analysis.

BACKGROUND: Percutaneous nephrolithotomy (PCNL) has been widely used to treat renal stones. The application of PCNL in obese patients results in the emergence of a number of challenges. This study compared the effect of obesity on the outcomes of PCNL in kidney stone treatment. METHODS: Eligible studies were searched in PubMed, Web of Science, and Cochrane Library databases. Data were analyzed using RevMan statistical software, weighted mean differences, ORs, and 95% CIs were calculated. RESULTS: Seven studies involving 2,720 normal-weight, 1,686 obese, and 286 super-obese individuals were included in this meta-analysis. A pooled analysis of safety revealed that no obvious differences in terms of complication rates after treatment existed between obese and normal-weight individuals (OR 0.97, 95% CI 0.80-1.16, p = 0.73), and between super-obese and normal-weight individuals (OR 0.88, 95% CI 0.61-1.27, p = 0.49). A pooled analysis of effectiveness revealed that no obvious difference in terms of stone-free rate after treatment existed between obese and normal-weight individuals (OR 0.98, 95% CI 0.84-1.15, p = 0.79), and between super-obese and normal-weight individuals (OR 1.20, 95% CI 0.88-1.63, p = 0.25). Moreover, no obvious differences in terms of length of hospital stay after treatment existed between super-obese and normal-weight individuals (95% CI -0.15 to 0.37, p = 0.39). Additionally, no obvious differences in terms of operation time existed between obese and normal-weight individuals (95% CI -3.36 to 1.17, p = 0.34). However, the operation time was longer among super-obese individuals than among normal-weight individuals (95% CI -22.64 to -1.40, p = 0.03), and the length of hospital stay was shorter among obese patients than among normal-weight patients (95% CI 0.04-0.34, p = 0.01). No publication bias was observed in this work. CONCLUSION: The PCNL performed in normal-weight, obese, and super-obese individuals for kidney stone treatment showed similar outcomes, except that operation time was longer among super-obese individuals and the hospital stay was shorter in obese individuals than in other groups. Thus, PCNL is a safe and efficacious treatment for renal stones in patients of all sizes.

An optimized, broadly applicable piggyBac transposon induction system.

The piggyBac (PB) transposon has been used in a number of biological applications. The insertion of PB transposons into the genome can disrupt genes or regulatory regions, impacting cellular function, so for many experiments it is important that PB transposition is tightly controlled. Here, we systematically characterize three methods for the post-translational control of the PB transposon in four cell lines. We investigated fusions of the PB transposase with ERT2 and two degradation domains (FKBP-DD, DHFR-DD), in multiple orientations, and determined (i) the fold-induction achieved, (ii) the absolute transposition efficiency of the activated construct and (iii) the effects of two inducer molecules on cellular transcription and function. We found that the FKBP-DD confers the PB transposase with a higher transposition activity and better dynamic range than can be achieved with the other systems. In addition, we found that the FKBP-DD regulates transposon activity in a reversible and dose-dependent manner. Finally, we showed that Shld1, the chemical inducer of FKBP-DD, does not interfere with stem cell differentiation, whereas tamoxifen has significant effects. We believe the FKBP-based PB transposon induction will be useful for transposon-mediated genome engineering, insertional mutagenesis and the genome-wide mapping of transcription factor binding.

Relation between N-terminal pro-brain natriuretic peptide and cardiac remodeling and function assessed by cardiovascular magnetic resonance imaging in patients with arrhythmogenic right ventricular cardiomyopathy.

Although N-terminal pro-brain natriuretic peptide (NT-proBNP) is a useful screening test of impaired right ventricular (RV) function in conditions affecting the right-sided cardiac muscle, the role of NT-proBNP remains unclear in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). This study was designed to clarify the relation between the plasma NT-proBNP level and the RV function evaluated by cardiovascular magnetic resonance (CMR) imaging. We selected 56 patients with confirmed ARVC only when their blood specimens for NT-proBNP measurements were collected within 48 hours of a CMR scan. The NT-proBNP level was significantly higher in patients with RV dysfunction than in patients without RV dysfunction (median of 655.3 [interquartile range 556.4 to 870.0] vs 347.0 [interquartile range 308.0 to 456.2] pmol/L, p <0.001). The NT-proBNP levels were positively correlated with RV end-diastolic and end-systolic volume indices (r = 0.49 and 0.70, respectively) and negatively correlated with RV ejection fraction (r = -0.76, all p <0.001), which remained significant after adjustment for age, gender, and body mass index. The area under the receiver-operating characteristic curve for NT-proBNP was 0.91 (95% confidence interval 0.80 to 0.97, p <0.001). The cut-off value of NT-proBNP (458 pmol/L) was associated with sensitivity, specificity, and positive and negative predictive values of 91%, 89%, 67%, and 98%, respectively. In conclusion, NT-proBNP is a useful marker for the detection of RV dysfunction and associated with extent of RV dilatation and dysfunction determined by CMR in patients with ARVC.

Comparative study of CMR characteristics between arrhythmogenic right ventricular cardiomyopathy patients with/without syncope.

To compare cardiovascular magnetic resonance (CMR) characteristics between arrhythmogenic right ventricular cardiomyopathy (ARVC) patients with syncope and without syncope and explore CMR parameters related with syncope. A consecutive series of 80 patients with ARVC were divided in two groups according to history of syncope prior to CMR examinations. The biventricular function and volumes were calculated and indexed by body surface area. Fatty infiltration and late-gadolinium enhancement (LGE) were self-quantitatively analyzed according to segmental model. Patients with syncope had statistically significant greater left ventricular end-diastolic volume index (LVEDVI) (79.6 ± 23.0 vs. 69.0 ± 17.9 mL/m(2), P = 0.030), right ventricular end-diastolic volume index (RVEDVI) (122.0 ± 30.0 vs. 107.4 ± 21.8 mL/m(2), P = 0.017), and LGE incidence (52.2 vs. 21.1 %, P = 0.006) than that of patients without syncope. Patients with syncope had a trend towards greater number of segments with LGE (8.6 ± 4.2 vs. 6.6 ± 3.1, P = 0.199) than that of patients without syncope in subgroup analyses of patients with LGE, but no statistical significance was reached. Multivariate regression analysis showed the presence of LGE was independently associated with syncope in patients with ARVC (odds ratios 8.827, 95 % confidence interval 1.945-40.068, P = 0.005). CMR is helpful in detection and management of the patients with ARVC. Patients with syncope had significantly higher LVEDVI, RVEDVI and LGE incidence, and larger studies with follow-up data are needed to elucidate the relationship between LGE and syncope in patients with ARVC.

[Association between abnormal electrocardiographic features and disease severity in patients with arrhythmogenic right ventricular cardiomyopathy].

OBJECTIVE: To analyze the relationship between electrocardiographic (ECG) features and disease severity in patients with the arrhythmogenic right ventricular cardiomyopathy (ARVC). METHOD: The study group consisted of 61 subjects with a definite diagnosis of ARVC on the basis of published guideline criteria and patients were divided into 3 subgroups according to the extent of diseased myocardium defined by cardiac magnetic resonance imaging (MRI): Group A: local involvement (n = 19, 31%), Group B: diffuse involvement of whole right ventricle (n = 28, 46%) and Group C: involvement of both right and left ventricles (n = 14, 23%). RESULTS: Normal electrocardiogram was shown in 1 patient in each group. Epsilon wave was detected in 24 (39%) patients, QRS duration was prolonged [≥ 110 ms (V(1)-V(3))] in 21 (34%) patients, S-wave upstroke was prolonged (≥ 55 ms) in 17 (28%) patients, complete right branch bundle block was evidenced in 10 (16%) patients and pathologic Q waves was found in 9 (15%) patients. The incidence of above abnormal ECG changes was increased in proportion to the degree of disease severity (group A < group B < group C). Incidence of Epsilon wave and prolonged QRS duration [ ≥ 110 ms (V(1)-V(3))] were significantly higher in Group C than in Group A. Incidence of prolonged S-wave upstroke (≥ 55 ms) was significantly higher in Group C than in Group A and Group B. T-wave inversion in V(1) leads was often found in Group A. T-wave inversion in inferior leads (V(1)-V(3) leads or beyond V(3)) was often presented in Group B and Group C. CONCLUSIONS: Normal ECG does not exclude the possibility of diagnosis of ARVC. The extent of T-wave inversion in the precordial leads and incidence of Epsilon wave, prolonged QRS duration [ ≥ 110 ms (V(1)-V(3))] and prolonged S-wave upstroke (≥ 55 ms) were related to degree of disease severity in patients with ARVC.

A neonatal encephalopathy with seizures in standard poodle dogs with a missense mutation in the canine ortholog of ATF2.

Neonatal encephalopathy with seizures (NEWS) is a previously undescribed autosomal recessive disease of standard poodle puppies. Affected puppies are small and weak at birth. Many die in their first week of life. Those surviving past 1 week develop ataxia, a whole-body tremor, and, by 4 to 6 weeks of age, severe generalized clonic-tonic seizures. None have survived to 7 weeks of age. Cerebella from affected puppies were reduced in size and often contained dysplastic foci consisting of clusters of intermixed granule and Purkinje neurons. We used deoxyribonucleic acid samples from related standard poodles to map the NEWS locus to a 2.87-Mb segment of CFA36, which contains the canine ortholog of ATF2. This gene encodes activating transcription factor 2 (ATF-2), which participates in the cellular responses to a wide variety of stimuli. We amplified and sequenced all coding regions of canine ATF2 from a NEWS-affected puppy and identified a T > G transversion that predicts a methionine-to-arginine missense mutation at amino acid position 51. Methionine-51 lies within a hydrophobic docking site for mitogen-activated protein kinases that activate ATF-2 so the arginine substitution is likely to interfere with ATF-2 activation. All 20 NEWS-affected puppies in the standard poodle family were homozygous for the mutant G allele. The 58 clinically normal family members were either G/T heterozygotes or homozygous for the ancestral T allele. There are no previous reports of spontaneous ATF2 mutations in people or animals; however, atf2-knockout mice have cerebellar lesions that are similar to those in puppies with NEWS.