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Investigations of 2 cases of high mortality in cull sows and feeder pigs from a buying station in Ohio and cull sows at an abattoir in Tennessee were conducted at the Iowa State University Veterinary Diagnostic Laboratory. The animals were presented as weak, lethargic, and some with high fever. Rapidly escalating mortality was reported to be as high as 30–50% within groups at the buying station over 8–10 d, and 30–40% over 5–7 d at the abattoir. Splenomegaly and red lymph nodes were the most consistent macroscopic findings, with scant fibrinous polyserositis observed in one sow. The microscopic lesions of vasculitis, fibrin thrombi, fibrinosuppurative polyserositis, and intralesional bacteria were consistent with acute bacterial septicemia. Bacterial culture isolated Streptococcus equi subsp. zooepidemicus ( S. zooepidemicus) from multiple organs, including spleen, lung, and kidney. PCR tests were negative for African swine fever virus, classical swine fever virus, Erysipelothrix rhusiopathiae, porcine reproductive and respiratory syndrome virus, porcine circovirus 2, and Salmonella spp. Porcine circovirus 3 was inconsistently detected at low levels by PCR, with a lack of associated lesions. Next-generation sequencing identified S. zooepidemicus and porcine partetravirus in the serum sample of the feeder pig from the buying station. Phylogenetic analysis of the szP gene indicated that the S. zooepidemicus isolates from Ohio and Tennessee are in genotype VI. We conclude that the cause of these high mortality events in swine was S. zooepidemicus septicemia.

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 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.

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 three 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.

Pacing the cardiac conduction system has been explored in patients with conduction system disease, but comprehensive comparisons between different pacing modalities are not well investigated.To compare pacing characteristics and ventricular synchrony between His-bundle pacing (HBP) and left bundle branch pacing (LBBP) in patients with atrioventricular block (AVB).Fifty pacemaker-indicated patients with AVB were enrolled. Twenty-five patients underwent HBP, and another 25 patients underwent LBBP. Success rate, procedural and fluoroscopy duration, pacing parameters, and echocardiographic data were perioperatively assessed and at 3-month follow-up.HBP was successful in 19 of 25 (76.0%) patients, whereas LBBP was successful in 22 of 25 (88.0%) patients. Compared with HBP, LBBP capture threshold was significantly lower (0.76 ± 0.25 V/0.4 ms vs. 1.27 ± 0.61 V/1.0 ms, P = 0.003) and R-wave amplitude was significantly higher with LBBP (11.7 ± 6.6 vs. 4.9 ± 2.4 mV, P 0.001) at implant. The mean procedural time (74.3 ± 17.8 vs. 63.2 ± 12.3 min, P = 0.029) and fluoroscopy duration (10.3 ± 4.5 vs. 6.8 ± 2.2 min, P = 0.005) were significantly longer in the HBP group compared to LBBP. At 3-month follow-up, pacing capture threshold remained more stable in LBBP than in HBP group while left ventricular synchrony was similar between both groups.Despite similar impact on ventricular synchrony compared with HBP, LBBP featured a significantly lower pacing capture threshold, higher R-wave amplitude, and less time to achieve similar success rate in patients with AVB. These findings indicate LBBP as a physiological pacing strategy for AVB patients.

High mortality events due to Streptococcus equi subspecies zooepidemicus (Streptococcus zooepidemicus) in swine have not previously been reported in the United States. In September and October 2019, outbreaks with swine mortality up to 50% due to S. zooepidemicus septicaemia were reported in Ohio and Tennessee. Genomic epidemiological analysis revealed that the eight outbreak isolates were clustered together with ATCC 35246, a Chinese strain caused outbreaks with high mortality, also closely related to three isolates from human cases from Virginia, but significantly different from an outbreak-unrelated swine isolate from Arizona and most isolates from other animal species. Comparative genomic analysis on two outbreak isolates and another outbreak-unrelated isolate identified several genomic islands and virulence genes specifically in the outbreak isolates only, which are likely associated with the high mortality observed in the swine population. These findings have implications for understanding, tracking and possibly preventing diseases caused by S. zooepidemicus in swine.

Objectives. To examine the association between the echocardiographic parameters measured as left atrial diameter (LAD) and left ventricular end-diastolic diameter (LVEDD) and long-term risk of all-cause mortality in adults with hypertrophic cardiomyopathy (HCM) following pacemaker implantation. Methods. A total of 94 adult patients with HCM who underwent pacemaker implantation from November 2002 to June 2013 in our Arrhythmia Center for symptomatic bradycardia and did not receive an implantable cardiac defibrillator (ICD) or cardiac resynchronization therapy (CRT) during follow-up were retrospectively extracted. Results. After careful examination of the medical records, we retrospectively evaluated the clinical characteristics of 74 patients with LAD records (58.1 ± 14.9 years) and 76 patients with LVEDD records (57.6 ± 15.2 years). Based on the receiver-operating characteristic (ROC) curve, the values of LAD = 44 mm and LVEDD = 43 mm were identified to predict the all-cause mortality. In the Kaplan–Meier survival, LAD ≥44 mm and LVEDD ≥43 mm were both significantly associated with all-cause mortality (log-rank test P<0.05). Cox regression analysis indicated that LAD ≥44 mm (HR 3.580; 95% CI = 1.055–12.148; P=0.041) was an independent predictor of all-cause mortality, while LVEDD ≥43 mm was not significantly associated with all-cause mortality. LVOTO was also significantly associated with all-cause mortality (HR = 0.166; 95% CI = 0.036–0.771; P=0.022). Conclusions. In HCM patients with pacemaker implantation, LAD ≥44 mm was an independent predictor of all-cause mortality.

Supplemental material, Supplemental_material for Cases of high mortality in cull sows and feeder pigs associated with Streptococcus equi subsp. zooepidemicus septicemia by Panchan Sitthicharoenchai, Rachel Derscheid, Kent Schwartz, Nubia Macedo, Orhan Sahin, Xuhua Chen, Ganwu Li, Rodger Main and Eric Burrough in Journal of Veterinary Diagnostic Investigation

High mortality events due toStreptococcus equisubspecieszooepidemicus(S. zooepidemicus) in swine have not previously been reported in the United States. In September and October 2019, outbreaks with swine mortality up to 50% due toS. zooepidemicussepticemia were reported in Ohio and Tennessee. Genomic epidemiological analysis revealed that the eight outbreak isolates were clustered together with ATCC 36246, a Chinese strain caused outbreaks with high mortality, also closely related to three isolates from human cases from Virginia, but significantly different from an outbreak-unrelated swine isolate from Arizona and most isolates from other animal species. Comparative genomic analysis on two outbreak isolates and another outbreak-unrelated isolate identified several genomic islands and virulence genes specifically in the outbreak isolates only, which are likely associated with the high mortality observed in the swine population. These findings have implications for understanding, tracking, and possibly preventing diseases caused byS. zooepidemicusin swine.

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.

Permanent His bundle pacing (HBP) has emerged as a new, promising approach to deliver physiological pacing, maintaining long-term ventricular synchrony and left ventricular performance unlike conventional right ventricular apical pacing, for treatment of patients with symptomatic bradycardia.1 Traditionally HBP is performed under fluoroscopic guidance, which entails significant exposure to radiation. A novel nonfluoroscopic three-dimensional (3D) cardiac imaging and navigation system, the KODEX-EPD system (EPD Solutions, Philips, Best, The Netherlands), offers an efficient way of guidance via high-definition (HD) computed tomography–like cardiac imaging and visualization of the His bundle region. It allows precision navigation with spatial resolution of 0.27 mm, HD anatomical imaging, tissue imaging, and physical property imaging. Less complicated than other 3D mapping systems, this electroanatomic mapping system was automatic without an electrophysiology (EP) catheter. We present a case of a 76-year-old man with sick sinus syndrome, in whom the KODEX-EPD system was successfully used to guide an implant of a dual-chamber pacemaker with HBP.

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.

Conventional fluoroscopy guidance, permanent His-bundle pacing (HBP) usually involves high fluoroscopy exposure. This study aims to explore the feasibility of Ensite NavX system and compare its pacing parameters and fluoroscopy doses (FD) with that of conventional fluoroscopy guiding HBP. A total of 30 patients receiving HBP from June to January 2019 were prospectively enrolled into this study (15 patients guided by conventional fluoroscopy and 15 patients by NavX system). Procedure time, FD, and pacing parameters were recorded at implant and 3-month follow-up. Fourteen patients (93.3%) underwent successful HBP using Ensite NavX system. The total FD was 1.4 ± 0.5 mGy in NavX group and 16.1 ± 2.2 mGy in conventional group (P < 0.001). The His lead FD was 0.4 ± 0.3 mGy in NavX group and 12.5 ± 2.2 mGy in conventional group (P < 0.001). There was no significant difference in pacing parameters at implant and follow-up between the two groups. Guided by Ensite NavX system, HBP is efficient with a significant reduction in fluoroscopy exposure.

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.

BackgroundA transcription-factor (TF) network map indicates the direct, functional targets of each TF -- the genes it regulates by binding to their cis-regulatory DNA. Data on the genomic binding locations of each TF and the transcriptional responses to perturbations of its activity, such as overexpressing it, could support TF network mapping. Systematic data sets of both types exist for yeast and for human K562 and HEK293 cells.ResultsIn previous data, most TF binding sites appear to be non-functional, so one cannot take the genes in whose promoters a TF binds as its direct, functional (DF) targets. Taking the genes that are both bound by a TF and responsive to a perturbation of it as its DF targets (intersection algorithm) is also not safe, as we show by deriving a new lower bound on the expected false discovery rate of the intersection algorithm. When there are many non-functional binding sites and many indirect targets, non-functional sites are expected to occur in the cis-regulatory DNA of indirect targets by chance. Dual threshold optimization, a new method for setting significance thresholds on binding and response data, improves the intersection algorithm, as does post-processing perturbation-response data with NetProphet 2.0. A comprehensive new data set measuring the transcriptional response shortly after inducing overexpression of a TF also helps, as does transposon calling cards, a new method for identifying TF binding locations.ConclusionsThe combination of dual threshold optimization and NetProphet greatly expands the high-confidence TF network map in both yeast and human. In yeast, measuring the response shortly after inducing TF overexpression and measuring binding locations by using transposon calling cards improve the network synergistically.

This protocol describes how to create calling card libraries from bulk RNA. This protocol assumes you have successfully transformed cells with piggyBac self-reporting transposons and either undirected piggyBac transposase or your favorite transcription factor (YFTF) fused to piggyBac. Your cells are now ready for RNA extraction, SRT amplification, and library preparation.

Transposon calling cards can identify transcription factor (TF) binding sites. This involves fusing your favorite TF (YFTF) to the hyperactive piggyBac transposase (HyPBase). This is delivered to cells in conjunction with a piggyBac transposon. The TF will visit sites in the genome and YFTF-HyPBase will deposit transposons near binding sites. We then generate sequencing libraries to map the genome-wide localization of transposons. Finally, we identify significant clusters of insertions to identify TF binding sites.

In situ measurements of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ.

Transcription factors (TFs) play a central role in the regulation of gene expression, controlling everything from cell fate decisions to activity dependent gene expression. However, widely-used methods for TF profiling in vivo (e.g. ChIP-seq) yield only an aggregated picture of TF binding across all cell types present within the harvested tissue; thus, it is challenging or impossible to determine how the same TF might bind different portions of the genome in different cell types, or even to identify its binding events at all in rare cell types in a complex tissue such as the brain. Here we present a versatile methodology, FLEX Calling Cards, for the mapping of TF occupancy in specific cell types from heterogenous tissues. In this method, the TF of interest is fused to a hyperactive piggyBac transposase (hypPB), and this bipartite gene is delivered, along with donor transposons, to mouse tissue via a Cre-dependent adeno-associated virus (AAV). The fusion protein is expressed in Cre-expressing cells where it inserts transposon “Calling Cards” near to TF binding sites. These transposons permanently mark TF binding events and can be mapped using high-throughput sequencing. Alternatively, unfused hypPB interacts with and records the binding of the super enhancer (SE)-associated bromodomain protein, Brd4. To demonstrate the FLEX Calling Card method, we first show that donor transposon and transposase constructs can be efficiently delivered to the postnatal day 1 (P1) mouse brain with AAV and that insertion profiles report TF occupancy. Then, using a Cre-dependent hypPB virus, we show utility of this tool in defining cell type-specific TF profiles in multiple cell types of the brain. This approach will enable important cell type-specific studies of TF-mediated gene regulation in the brain and will provide valuable insights into brain development, homeostasis, and disease.

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

Cholesterol efflux capacity has been shown to have an inverse relation with coronary artery disease (CAD) and may overcome the limitations of high-density lipoprotein (HDL) cholesterol levels as a predictor for CAD risks. We investigated the predictive value of cholesterol efflux capacity for the prognosis of CAD. Serum cholesterol efflux capacity in 313 patients newly diagnosed with CAD by coronary angiography was measured, and all patients completed a 3-year follow-up. The primary clinical end points were nonfatal myocardial infarction, nonfatal stroke, and cardiovascular mortality. The secondary clinical end points were class IV heart failure requiring hospitalization and coronary artery revascularization. Cholesterol efflux capacity was lower in patients with CAD compared with control group, and decreased cholesterol efflux capacity was associated with an increased risk of acute coronary syndrome (odds ratios, 0.25; 95% confidence interval, 0.14 to 0.46; p

Sex can be an important determinant of cancer phenotype and exploring sex-specific tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established model of glioblastoma, we discovered transcriptome-wide sexual dimorphism in gene-expression that was concordant with sex differences in H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 co-localization with Myc and p53. The sex-specific enhancer usage drove sex differences in stem cell function and tumorigenicity. Moreover, male and female GBM cells exhibited opposing responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or 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. Thus, for the first time, Brd4 activity is revealed to drive a sexually dimorphic stem cell and tumorigenic phenotype, resulting in diametrically opposite responses to BET inhibition in male and female glioblastoma cells. This has critical implications for the clinical evaluation and use of BET inhibitors. Citation Format: Najla Kfoury-Beaumont, Zongtai Qi, Michael Wilkinson, Lauren Broestl, Kristopher Berrett, Arnav Moudgil, Sumithra Sankararaman, Xuhua Chen, Jay Gertz, Robi Mitra, Joshua B. Rubin. Brd4-bound enhancers drive critical sex differences in glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3658.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12920-018-0424-0) contains supplementary material, which is available to authorized users.

Pathways associated with BAP1 target genes. The ten most significant pathways associated with BAP1 calling-card target genes that were up-regulated (positive response) or down-regulated (negative response) in response to BAP1 depletion. Pathways were identified by GSEA. (PDF 49 kb)

DNA motifs associated with BAP1 binding. HOMER was used to identify DNA motifs that were significantly enriched in genomic regions containing BAP1 calling card insertions. Calling card loci were divided into three categories based on response of associated genes to BAP1 depletion, and analyzed separately. (PDF 25 kb)

Background: Premature ventricular contractions (PVCs) are common in the general population, and frequent PVCs may result in the poor quality of life or even the damage of cardiac function. We examined the efficacy and safety of a traditional Chinese medicine Wenxin Keli for the treatment of frequent PVCs among a relatively large Chinese cohort. Methods: We performed a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 1200 eligible participants were randomly assigned in a ratio of 1:1 to receive Wenxin Keli or the placebo for 4 weeks. The primary and secondary endpoint was the change of PVC numbers and PVC-related symptoms after a 4-week treatment compared with baseline, respectively. In addition, vital signs, laboratory values, and electrocardiographic parameters were assessed in a safety analysis. Results: At the initial evaluation, no significant differences in the baseline characteristics were observed between the Wenxin Keli group and the placebo group. A smaller number of PVCs was observed after the 4-week treatment than at baseline, in both the Wenxin Keli group (5686 ± 5940 vs. 15,138 ± 7597 beats/d, P < 0.001) and the placebo group (10,592 ± 8009 vs. 14,529 ± 5929 beats/d, P < 0.001); moreover, the Wenxin Keli group demonstrated a significantli greater reduction in the frequency of PVCs than the placebo group (P < 0.001). In a full analysis set, patients in the Wenxin Keli group exhibited significantly higher total effective responses in the reduction of PVCs compared to those in the placebo group (83.8% vs. 43.5%, P < 0.001). The per-protocol analysis yielded similar results (83.0% vs. 39.3%, P < 0.001). Treatment with Wenxin Keli also demonstrated superior performance compared to the placebo with respect to PVC-related symptoms. No severe adverse effects attributable to Wenxin Keli were reported. Conclusions: Wenxin Keli treatment effectively reduced the overall number of PVCs and alleviated PVC-related symptoms in patients without structural heart diseases and had no severe side effects.

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, p0.001). 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 p0.001), 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, p0.001). 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.

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, 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 opposing 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 inhibitors than are female cells. Thus, for the first time, Brd4 activity is revealed to drive a sex differences in stem cell and tumorigenic phenotype, resulting in diametrically opposite responses to BET inhibition in male and female GBM cells. This has important implications for the clinical evaluation and use of BET inhibitors.SignificanceConsistent sex differences in incidence and outcome have been reported in numerous cancers including brain tumors. GBM, the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity, including that of GBM. We report that sex-biased Brd4 activity drive sex differences in GBM and render male and female tumor cells differentially sensitive to BET inhibitors. The observed sex differences in BETi treatment strongly indicate that sex differences in disease biology translate into sex differences in therapeutic responses. This has critical implications for clinical use of BET inhibitors further affirming the importance of inclusion of sex as a biological variable.

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.

To investigate whether the sphingomyelin content of erythrocyte membranes (SEM) is changed in patients with acute coronary syndrome (ACS) and determine the correlation between SEM and the total cholesterol content of erythrocyte membranes (CEM).The SEM and CEM levels were measured in 354 patients undergoing coronary artery angiography in three different groups: ACS patients (n=199), patients with stable angina pectoris (SAP) (n=82), and controls (n=73).The SEM levels in the ACS group were significantly higher than those of the SAP group. The SEM levels were correlated positively with the CEM levels in patients with coronary artery disease. Multivariable logistic regression analysis showed that patients with higher levels of both SEM and CEM had an 8.569-fold greater risk of developing ACS than other patients, after adjusting for all potential confounding variables.Elevated SEM and CEM levels showed both independent and combined correlations with the occurrence of ACS and were positively correlated with each other in patients with coronary artery disease. These data suggest that the increased levels of SEM may play a role in the progression to plaque instability in ACS and may be the mechanisms underlying elevated levels of CEM in patients with ACS.

The ability to chronicle transcription-factor binding events throughout the development of an organism would facilitate mapping of transcriptional networks that control cell-fate decisions. We describe a method for permanently recording protein–DNA interactions in mammalian cells. We endow transcription factors with the ability to deposit a transposon into the genome near to where they bind. The transposon becomes a “calling card” that the transcription factor leaves behind to record its visit to the genome. The locations of the calling cards can be determined by massively parallel DNA sequencing. We show that the transcription factor SP1 fused to the piggyBac transposase directs insertion of the piggyBac transposon near SP1 binding sites. The locations of transposon insertions are highly reproducible and agree with sites of SP1-binding determined by ChIP-seq. Genes bound by SP1 are more likely to be expressed in the HCT116 cell line we used, and SP1-bound CpG islands show a strong preference to be unmethylated. This method has the potential to trace transcription-factor binding throughout cellular and organismal development in a way that has heretofore not been possible.

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.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 SBtheir respective medians), subgroup II (DS of one of the MV or the SB ≥its respective median), and subgroup III (DS of both the MV and the SB ≥their respective medians). The incidences of SB occlusion in these three subgroups were compared.Multivariate logistic regression analysis showed that TBL occurrence was not independently predictive of SB occlusion. Based on subgroup analysis, the incidence of SB occlusion in subgroup I (7.4%) was significantly lower than that in subgroup II (13.0%) and subgroup III (22.0%) (P = 0.025). However, there was no significant difference in the incidence of SB occlusion between TBL subgroup I (7.4%) and the non-TBL group (5.1%) (P = 0.53).TBL occurrence cannot be regarded as an independent predictor of acute SB occlusion after MV stenting.

Several genetic variants have been associated with early repolarization syndrome (ERS). However, the lack of functional validations of the mutant effects has limited the interpretation of genetic tests. In the present study, we identified and characterized a novel sodium channel, voltage gated, type V alpha subunit (SCN5A) mutation that was associated with ERS. A 67-year-old male proband suffering from recurrent syncope underwent a documented electrocardiogram (ECG) for polymorphic ventricular tachycardia (VT). It was noted that baseline 12-lead ECG exhibited a predominantly elevated ST-segment which mimicked acute myocardial ischemia in lead V2–V6, and the ECG also demonstrated J waves in lead II, III, aVF and V2–V6. Using genetic analysis, we noted that the proband carried a novel heterozygous missense mutation of A1055G in the SCN5A gene. Whole-cell configuration of patch-clamp analysis revealed that the mutation significantly decreased peak sodium current (INa) density and shifted the steady-state inactivation curve of INa to a more negative potential. Confocal imaging suggested that in the mutant channel a defect of protein expression both on the cell membrane and in cytoplasm was present. The present study demonstrated that a novel heterozygous missense mutation of A1055G in SCN5A led to 'loss-of function' of the sodium channels, and we suggest that it accounts for the arrhythmogenic characteristics of ERS.

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

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.

The tensile creep behavior of a Co-Al-W-base single crystal alloy containing Ta and Ti was investigated at 982 ∘ C and 248 MPa. The lattice misfit of experimental alloy was measured to be positive by synchrotron X-ray diffraction at high temperature, and long term heat treatment at 1000 ∘ C for 1000 h revealed a γ′ volume fraction of 75% without secondary phases. The creep test indicated that the creep properties of experimental alloy exceeded commercial 1st generation Ni-base single crystal superalloy CMSX-3 with respect to the rupture life. The initial cuboidal γ′ precipitates directionally coarsened parallel to the applied stress axis during the creep process. The stacking faults in {111} planes within γ′ rafts were the primary creep deformation mode by TEM investigation.