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.

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.

Zinc cluster transcription factors frequently activate target genes using a non-canonical half-site binding mode.

Gene expression changes are orchestrated by transcription factors (TFs), which bind to DNA to regulate gene expression. It remains surprisingly difficult to predict basic features of the transcriptional process, including in vivo TF occupancy. Existing thermodynamic models of TF function are often not concordant with experimental measurements, suggesting undiscovered biology. Here, we analyzed one of the most well-studied TFs, the yeast zinc cluster Gal4, constructed a Shea-Ackers thermodynamic model to describe its binding, and compared the results of this model to experimentally measured Gal4p binding in vivo. We found that at many promoters, the model predicted no Gal4p binding, yet substantial binding was observed. These outlier promoters lacked canonical binding motifs, and subsequent investigation revealed Gal4p binds unexpectedly to DNA sequences with high densities of its half site (CGG). We confirmed this novel mode of binding through multiple experimental and computational paradigms; we also found most other zinc cluster TFs we tested frequently utilize this binding mode, at 27% of their targets on average. Together, these results demonstrate a novel mode of binding where zinc clusters, the largest class of TFs in yeast, bind DNA sequences with high densities of half sites.

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.

Dual threshold optimization and network inference reveal convergent evidence from TF binding locations and TF perturbation responses.

A high-confidence map of the direct, functional targets of each transcription factor (TF) requires convergent evidence from independent sources. Two significant sources of evidence are TF binding locations and the transcriptional responses to direct TF perturbations. Systematic data sets of both types exist for yeast and human, but they rarely converge on a common set of direct, functional targets for a TF. Even the few genes that are both bound and responsive may not be direct functional targets. Our analysis shows that when there are many nonfunctional binding sites and many indirect targets, nonfunctional sites are expected to occur in the cis-regulatory DNA of indirect targets by chance. To address this problem, we introduce dual threshold optimization (DTO), a new method for setting significance thresholds on binding and perturbation-response data, and show that it improves convergence. It also enables comparison of binding data to perturbation-response data that have been processed by network inference algorithms, which further improves convergence. The combination of dual threshold optimization and network inference greatly expands the high-confidence TF network map in both yeast and human. Next, we analyze a comprehensive new data set measuring the transcriptional response shortly after inducing overexpression of a yeast TF. We also present a new yeast binding location data set obtained by transposon calling cards and compare it to recent ChIP-exo data. These new data sets improve convergence and expand the high-confidence network synergistically.

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.

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.

Combined association of triglyceride-glucose index and systolic blood pressure with all-cause and cardiovascular mortality among the general population.

BACKGROUND: The combined association of triglyceride-glucose (TyG) index and different systolic blood pressure (SBP) levels with all-cause and cardiovascular mortality among the general population remains unclear. METHODS: In this study, 6245 individuals were from the National Health and Nutrition Examination Survey (1999-2002). The study endpoints were all-cause and cardiovascular mortality. Multivariate Cox proportional hazards regression models were used to explore the combined association of TyG index and different SBP levels with all-cause and cardiovascular mortality. RESULTS: During a mean follow-up period of 66.8 months, a total of 284 all-cause deaths (331/100000 person-years) and 61 cardiovascular deaths (66/100000 person-years) were recorded. Multivariate Cox regression analysis revealed that the combination of low TyG index and low SBP (< 120 mmHg and < 130 mmHg) was associated with a reduced risk of all-cause and cardiovascular mortality than others. However, survival benefit was not observed in the combined group with the low TyG index and SBP < 140 mmHg. Furthermore, the mortality rate in the combined group of low TyG index and low SBP gradually increased with the elevation of SBP level. CONCLUSION: The combination of low TyG index and low SBP (< 120 mmHg and < 130 mmHg) was associated with a lower risk of all-cause and cardiovascular mortality. However, no survival benefit was observed in the combined group of low TyG index and SBP < 140 mmHg.

Homotypic cooperativity and collective binding are determinants of bHLH specificity and function.

Eukaryotic cells express transcription factor (TF) paralogues that bind to nearly identical DNA sequences in vitro but bind at different genomic loci and perform different functions in vivo. Predicting how 2 paralogous TFs bind in vivo using DNA sequence alone is an important open problem. Here, we analyzed 2 yeast bHLH TFs, Cbf1p and Tye7p, which have highly similar binding preferences in vitro, yet bind at almost completely nonoverlapping target loci in vivo. We dissected the determinants of specificity for these 2 proteins by making a number of chimeric TFs in which we swapped different domains of Cbf1p and Tye7p and determined the effects on in vivo binding and cellular function. From these experiments, we learned that the Cbf1p dimer achieves its specificity by binding cooperatively with other Cbf1p dimers bound nearby. In contrast, we found that Tye7p achieves its specificity by binding cooperatively with 3 other DNA-binding proteins, Gcr1p, Gcr2p, and Rap1p. Remarkably, most promoters (63%) that are bound by Tye7p do not contain a consensus Tye7p binding site. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p. We then successfully reprogrammed the human bHLH NPAS2 to bind Cbf1p in vivo targets and a Tye7p target intergenic region to be bound by Cbf1p. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information, and provide lessons about TF specificity that can be applied across the phylogenetic tree.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Electrical characteristics of pacing different portions of the His bundle in bradycardia patients.

AIMS: His-bundle pacing (HBP) can be achieved in either atrial-side HBP (aHBP) or ventricular-side HBP (vHBP). The study compared the pacing parameters and electrophysiological characteristics between aHBP and vHBP in bradycardia patients. METHODS AND RESULTS: Fifty patients undergoing HBP implantation assisted by visualization of the tricuspid valvular annulus (TVA) were enrolled. The HBP lead position was identified by TVA angiography. Twenty-five patients were assigned to undergo aHBP and compared with 25 patients who underwent vHBP primarily in a prospective and randomized fashion. Pacing parameters and echocardiography were routinely assessed at implant and 3-month follow-up. His-bundle pacing was successfully performed in 45 patients (90% success rate with 44.4% aHBP and 55.6% vHBP). The capture threshold was lower in vHBP than aHBP at implant (vHBP: 1.1 ± 0.5 vs. aHBP: 1.4 ± 0.4 V/1.0 ms, P = 0.014) and 3-month follow-up (vHBP: 0.8 ± 0.4 vs. aHBP: 1.7 ± 0.8 V/0.4 ms, P < 0.001). The R-wave amplitude was higher in vHBP than in aHBP at implant (vHBP: 4.5 ± 1.4 vs. aHBP: 2.0 ± 0.8 mV, P < 0.001) and at 3-month follow-up (vHBP: 4.4 ± 1.5 vs. aHBP: 1.8 ± 0.7 mV, P < 0.001). No procedure-related complications and aggravation of tricuspid valve regurgitation were observed in most patients and echocardiographic assessment of cardiac function remained in the normal range in all patients during the follow-up. CONCLUSION: This study demonstrates that vHBP features a low and stable pacing capture threshold and high R-wave amplitude, suggesting better pacing mode management and battery longevity can be achieved by HBP in the ventricular side.

Identification of Host Adaptation Genes in Extraintestinal Pathogenic Escherichia coli during Infection in Different Hosts.

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important human and animal pathogen. Despite the apparent similarities in their known virulence attributes, some ExPEC strains can cross the host species barrier and present a zoonotic potential, whereas other strains exhibit host specificity, suggesting the existence of unknown mechanisms that remain to be identified. We applied a transposon-directed insertion site sequencing (TraDIS) strategy to investigate the ExPEC XM strain, which is capable of crossing the host species barrier, and to screen for virulence-essential genes in both mammalian (mouse) and avian (duck) models of E. coli-related septicemia. We identified 151 genes essential for systemic infection in both mammalian and avian models, 97 required only in the mammalian model, and 280 required only in the avian model. Ten genes/gene clusters were selected for further validation, and their contributions to ExPEC virulence in both mammalian and avian models or mammalian- or avian-only models were confirmed by animal tests. This represents the first comprehensive genome-wide analysis of virulence-essential genes required for systemic infections in two different host species and provides a further comprehensive understanding of ExPEC-related virulence, host specificity, and adaptation.

Visualization of tricuspid valve annulus for implantation of His bundle pacing in patients with symptomatic bradycardia.

BACKGROUND: His bundle pacing (HBP) is a physiological pacing modality, but HBP implantation remains a challenge. OBJECTIVE: This study explored the feasibility of using visualization of the tricuspid valve annulus (TVA) to locate the site for HBP. METHODS: During the lead placement in eight patients with symptomatic bradycardia, the TVA and tricuspid septal leaflet was revealed by contrast injection in the right ventricle under the fluoroscopic right anterior oblique view, and the target site for HBP was identified near the intersection of the tricuspid septal leaflet and the interventricular septum. On the basis of the imaging marker, the pacing lead was placed for HBP at either the atrial (aHBP) or ventricular side (vHBP). RESULTS: During the implantation, the pacing lead placement was attempted for aHBP in two patients, vHBP in five patients, and first for aHBP then vHBP in one patient. The aHBP was selective and had a capture threshold of 1.6 ± 0.5 V@ 1.0ms and R-wave amplitude of 1.2 ± 0.4 mV. Ventricular-side His bundle capture was selective in four patients and nonselective in two patients. The vHBP capture threshold was 0.8 ± 0.4 V@ 1.0ms (P < .05 vs aHBP) and R-wave amplitude was 4.1 ± 1.5 mV (P < .05 vs aHBP). At the final pacing programming of 3.0 V@ 1.0ms, vHBP was nonselective in all six patients and aHBP remained selective in two patients. Pacing parameters remained stable at 3 months. CONCLUSION: The location of the TVA and tricuspid septal leaflet revealed by right ventriculography can be used as a landmark to identify the HBP site.

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.

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.

Can "true bifurcation lesion" actually be regarded as an independent risk factor of acute side branch occlusion after main vessel stenting?: A retrospective analysis of 1,200 consecutive bifurcation lesions in a single center.

OBJECTIVES: True bifurcation lesion (TBL) is conventionally considered as a risk factor for acute side branch (SB) occlusion when using a single-stent strategy to treat bifurcation lesions. The impact of TBLs on acute SB occlusion after main vessel (MV) stenting was investigated. METHODS: A total of 1,170 consecutive patients with 1,200 bifurcation lesions undergoing one-stent or provisional two-stent techniques were studied. The TBLs were divided into two groups depending on their Medina classification. Multivariate logistic regression analysis was performed to identify independent predictors of acute SB occlusion. According to the median diameter of stenosis (DS) in the MV and the SB after pre-dilatation, the TBL group was divided into three subgroups: subgroup I (DS of both the MV and the SB

Comparison of cardiovascular magnetic resonance characteristics and clinical consequences in children and adolescents with isolated left ventricular non-compaction with and without late gadolinium enhancement.

BACKGROUND: Although cardiovascular magnetic resonance (CMR) is showing increasingly diagnostic potential in left ventricular non-compaction (LVNC), relatively little research relevant to CMR is conducted in children with LVNC. This study was performed to characterize and compare CMR features and clinical outcomes in children with LVNC with and without late gadolinium enhancement (LGE). METHODS: A cohort of 40 consecutive children (age, 13.7 ± 3.3 years; 29 boys and 11 girls) with isolated LVNC underwent a baseline CMR scan with subsequent clinical follow-up. Short-axis cine images were used to calculate left ventricular (LV) ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), myocardial mass, ratio of non-compacted-to-compacted myocardial thickness (NC/C ratio), and number of non-compacted segments. The LGE images were analyzed to assess visually presence and patterns of LGE. The primary end point was a composite of cardiac death and heart transplantation. RESULTS: The LGE was present in 10 (25%) children, and 46 (27%) segments were involved, including 23 non-compacted segments and 23 normal segments. Compared with LGE- cohort, LGE+ cohort had significantly lower LVEF (23.8 ± 10.7% vs. 42.9 ± 16.7%, p < 0.001) and greater LVEDV (169.2 ± 65.1 vs. 118.2 ± 48.9 mL/m2, p = 0.010), LVESV (131.3 ± 55.5 vs. 73.3 ± 46.7 mL/m2, p = 0.002), and sphericity indices (0.75 ± 0.19 vs. 0.60 ± 0.20, p = 0.045). There were no differences in terms of number and distribution of non-compacted segments, NC/C ratio, and myocardial mass index between LGE+ and LGE- cohort. In the LGE+ cohort, adverse events occurred in 6 patients compared to 2 events in the LGE- cohort. Kaplan-Meier analysis showed a significant difference in outcome between LGE+ and LGE- cohort for cardiac death and heart transplantation (p = 0.011). CONCLUSIONS: The LGE was present in up to one-fourth of children with LVNC, and the LGE+ children exhibited a more maladaptive LV remodeling and a higher incidence of cardiovascular death and heart transplantation.

Calling Cards enable multiplexed identification of the genomic targets of DNA-binding proteins.

Transcription factors direct gene expression, so there is much interest in mapping their genome-wide binding locations. Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a "Calling Card" marking the visit of the DNA-binding protein to that location. A unique sequence "barcode" in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively parallel DNA sequencing. To demonstrate the method's feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds.

Implication of geriatric nutritional risk index on treatment response and long-term prognosis in patients with cardiac resynchronization therapy.

PURPOSE: Geriatric Nutritional Risk Index (GNRI) is a simple tool for assessing the nutritional status of the aging population. This study aims to explore the clinical implication of GNRI on treatment response and long-term clinical outcomes in heart failure (HF) patients receiving cardiac resynchronization therapy (CRT). METHODS: Patients who underwent CRT implantation or upgrade at our hospital were retrospectively included. The association of GNRI and its tertiles with the echocardiographic response, all-cause mortality or heart transplantation, and the first hospitalization due to HF were investigated. RESULTS: Totally, 647 patients were enrolled, with a median age of 60 [Interquartile Range (IQR): 52-67] years and mean score of GNRI at 107.9 ± 23.7. Super-response rates increased significantly among the GNRI T1, T2, and T3 groups (25.1%, 29.8% vs. 41.1%, P = 0.002). Patients with higher GNRI were more likely to have better LVEF improvement after multiple adjustments (OR = 1.13, 95% CI: 1.04-1.23, P = 0.010). Higher GNRI was independently associated with a lower risk of all-cause mortality or heart implantation (HR = 0.95, 95% CI: 0.93-0.96, P < 0.001) and HF hospitalization (HR = 0.96, 95% CI: 0.95-0.98, P < 0.001). The inclusion of GNRI enhanced the predictability of all-cause mortality based on traditional model, including sex, New York Heart Association functional class, left bundle branch block, QRS reduction, and N-terminal pro-B-type natriuretic peptide level (C statistics improved from 0.785 to 0.813, P = 0.007). CONCLUSION: Higher GNRI was associated with better treatment response and long-term prognosis in HF patients with CRT. Evaluation of nutritional status among CRT population is necessary for individualized choice of potential responders.

MYT1L deficiency impairs excitatory neuron trajectory during cortical development.

Mutations that reduce the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. Furthermore, MYT1L is routinely used as a proneural factor in fibroblast-to-neuron transdifferentiation. MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss. In this study, we profiled 313,335 nuclei from the forebrains of wild-type and MYT1L-deficient mice at two developmental stages: E14 at the peak of neurogenesis and P21, when neurogenesis is complete, to examine the role of MYT1L levels in the trajectory of neuronal development. We found that MYT1L deficiency significantly disrupted the relative proportion of cortical excitatory neurons at E14 and P21. Significant changes in gene expression were largely concentrated in excitatory neurons, suggesting that transcriptional effects of MYT1L deficiency are largely due to disruption of neuronal maturation programs. Most effects on gene expression were cell autonomous and persistent through development. In addition, while MYT1L can both activate and repress gene expression, the repressive effects were most sensitive to haploinsufficiency, and thus more likely mediate MYT1L syndrome. These findings illuminate the intricate role of MYT1L in orchestrating gene expression dynamics during neuronal development, providing insights into the molecular underpinnings of MYT1L syndrome.