A calcineurin-Hoxb13 axis regulates growth mode of mammalian cardiomyocytes.

A major factor in the progression to heart failure in humans is the inability of the adult heart to repair itself after injury. We recently demonstrated that the early postnatal mammalian heart is capable of regeneration following injury through proliferation of preexisting cardiomyocytes1,2 and that Meis1, a three amino acid loop extension (TALE) family homeodomain transcription factor, translocates to cardiomyocyte nuclei shortly after birth and mediates postnatal cell cycle arrest3. Here we report that Hoxb13 acts as a cofactor of Meis1 in postnatal cardiomyocytes. Cardiomyocyte-specific deletion of Hoxb13 can extend the postnatal window of cardiomyocyte proliferation and reactivate the cardiomyocyte cell cycle in the adult heart. Moreover, adult Meis1-Hoxb13 double-knockout hearts display widespread cardiomyocyte mitosis, sarcomere disassembly and improved left ventricular systolic function following myocardial infarction, as demonstrated by echocardiography and magnetic resonance imaging. Chromatin immunoprecipitation with sequencing demonstrates that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and cell cycle. Finally, we show that the calcium-activated protein phosphatase calcineurin dephosphorylates Hoxb13 at serine-204, resulting in its nuclear localization and cell cycle arrest. These results demonstrate that Meis1 and Hoxb13 act cooperatively to regulate cardiomyocyte maturation and proliferation and provide mechanistic insights into the link between hyperplastic and hypertrophic growth of cardiomyocytes.

Asparagine Synthetase Marks a Distinct Dependency Threshold for Cardiomyocyte Dedifferentiation.

BACKGROUND: Adult mammalian cardiomyocytes have limited proliferative capacity, but in specifically induced contexts they traverse through cell-cycle reentry, offering the potential for heart regeneration. Endogenous cardiomyocyte proliferation is preceded by cardiomyocyte dedifferentiation (CMDD), wherein adult cardiomyocytes revert to a less matured state that is distinct from the classical myocardial fetal stress gene response associated with heart failure. However, very little is known about CMDD as a defined cardiomyocyte cell state in transition. METHODS: Here, we leveraged 2 models of in vitro cultured adult mouse cardiomyocytes and in vivo adeno-associated virus serotype 9 cardiomyocyte-targeted delivery of reprogramming factors (Oct4, Sox2, Klf4, and Myc) in adult mice to study CMDD. We profiled their transcriptomes using RNA sequencing, in combination with multiple published data sets, with the aim of identifying a common denominator for tracking CMDD. RESULTS: RNA sequencing and integrated analysis identified Asparagine Synthetase (Asns) as a unique molecular marker gene well correlated with CMDD, required for increased asparagine and also for distinct fluxes in other amino acids. Although Asns overexpression in Oct4, Sox2, Klf4, and Myc cardiomyocytes augmented hallmarks of CMDD, Asns deficiency led to defective regeneration in the neonatal mouse myocardial infarction model, increased cell death of cultured adult cardiomyocytes, and reduced cell cycle in Oct4, Sox2, Klf4, and Myc cardiomyocytes, at least in part through disrupting the mammalian target of rapamycin complex 1 pathway. CONCLUSIONS: We discovered a novel gene Asns as both a molecular marker and an essential mediator, marking a distinct threshold that appears in common for at least 4 models of CMDD, and revealing an Asns/mammalian target of rapamycin complex 1 axis dependency for dedifferentiating cardiomyocytes. Further study will be needed to extrapolate and assess its relevance to other cell state transitions as well as in heart regeneration.

A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardiotoxicity.

AIMS: Cardiotoxicity leading to heart failure (HF) is a growing problem in many cancer survivors. As specific treatment strategies are not available, RNA discovery pipelines were employed and a new and powerful circular RNA (circRNA)-based therapy was developed for the treatment of doxorubicin-induced HF. METHODS AND RESULTS: The circRNA sequencing was applied and the highly species-conserved circRNA insulin receptor (Circ-INSR) was identified, which participates in HF processes, including those provoked by cardiotoxic anti-cancer treatments. Chemotherapy-provoked cardiotoxicity leads to the down-regulation of Circ-INSR in rodents and patients, which mechanistically contributes to cardiomyocyte cell death, cardiac dysfunction, and mitochondrial damage. In contrast, Circ-INSR overexpression prevented doxorubicin-mediated cardiotoxicity in both rodent and human cardiomyocytes in vitro and in a mouse model of chronic doxorubicin cardiotoxicity. Breast cancer type 1 susceptibility protein (Brca1) was identified as a regulator of Circ-INSR expression. Detailed transcriptomic and proteomic analyses revealed that Circ-INSR regulates apoptotic and metabolic pathways in cardiomyocytes. Circ-INSR physically interacts with the single-stranded DNA-binding protein (SSBP1) mediating its cardioprotective effects under doxorubicin stress. Importantly, in vitro transcribed and circularized Circ-INSR mimics also protected against doxorubicin-induced cardiotoxicity. CONCLUSION: Circ-INSR is a highly conserved non-coding RNA which is down-regulated during cardiotoxicity and cardiac remodelling. Adeno-associated virus and circRNA mimics-based Circ-INSR overexpression prevent and reverse doxorubicin-mediated cardiomyocyte death and improve cardiac function. The results of this study highlight a novel and translationally important Circ-INSR-based therapeutic approach for doxorubicin-induced cardiac dysfunction.

Analysis and Validation of Human Targets and Treatments Using a Hepatocellular Carcinoma-Immune Humanized Mouse Model.

BACKGROUND AND AIMS: Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions. APPROACH AND RESULTS: We established cell line-based or patient-derived xenograft-based humanized-immune-system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human-specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real-time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual-combination, and triple-combination treatment using N-(1',2-Dihydroxy-1,2'-binaphthalen-4'-yl)-4-methoxybenzenesulfonamide (C188-9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL-6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized-immune-system mice with HCC. In particular, intratumor human cluster of differentiation-positive (hCD14+ ) cells could produce IL-33 through damage-associated molecular pattern/Toll-like receptor 4/activator protein 1, which up-regulated IL-6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL-33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti-HCC effect of C188-9, bevacizumab, and pembrolizumab. The results showed that the anti-HCC effect of triple-combination therapy was superior to that of single or dual treatments. CONCLUSIONS: Humanized-immune-system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.

Epigenomes of Human Hearts Reveal New Genetic Variants Relevant for Cardiac Disease and Phenotype.

RATIONALE: Identifying genetic markers for heterogeneous complex diseases such as heart failure is challenging and requires prohibitively large cohort sizes in genome-wide association studies to meet the stringent threshold of genome-wide statistical significance. On the other hand, chromatin quantitative trait loci, elucidated by direct epigenetic profiling of specific human tissues, may contribute toward prioritizing subthreshold variants for disease association. OBJECTIVE: Here, we captured noncoding genetic variants by performing epigenetic profiling for enhancer H3K27ac chromatin immunoprecipitation followed by sequencing in 70 human control and end-stage failing hearts. METHODS AND RESULTS: We have mapped a comprehensive catalog of 47 321 putative human heart enhancers and promoters. Three thousand eight hundred ninety-seven differential acetylation peaks (FDR [false discovery rate], 5%) pointed to pathways altered in heart failure. To identify cardiac histone acetylation quantitative trait loci (haQTLs), we regressed out confounding factors including heart failure disease status and used the G-SCI (Genotype-independent Signal Correlation and Imbalance) test1 to call out 1680 haQTLs (FDR, 10%). RNA sequencing performed on the same heart samples proved a subset of haQTLs to have significant association also to gene expression (expression quantitative trait loci), either in cis (180) or through long-range interactions (81), identified by Hi-C (high-throughput chromatin conformation assay) and HiChIP (high-throughput protein centric chromatin) performed on a subset of hearts. Furthermore, a concordant relationship between the gain or disruption of TF (transcription factor)-binding motifs, inferred from alternative alleles at the haQTLs, implied a surprising direct association between these specific TF and local histone acetylation in human hearts. Finally, 62 unique loci were identified by colocalization of haQTLs with the subthreshold loci of heart-related genome-wide association studies datasets. CONCLUSIONS: Disease and phenotype association for 62 unique loci are now implicated. These loci may indeed mediate their effect through modification of enhancer H3K27 acetylation enrichment and their corresponding gene expression differences (bioRxiv: https://doi.org/10.1101/536763). Graphical Abstract: A graphical abstract is available for this article.

Modeling Susceptibility to Cardiotoxicity in Cancer Therapy Using Human iPSC-Derived Cardiac Cells and Systems Biology.

The development of human-induced pluripotent stem cell-derived cardiac cell types has created a new paradigm in assessing drug-induced cardiotoxicity. Advances in genomics and epigenomics have also implicated several genomic loci and biological pathways that may contribute to susceptibility to cancer therapies. In this review, we first provide a brief overview of the cardiotoxicity associated with chemotherapy. We then provide a detailed summary of systems biology approaches being applied to elucidate potential molecular mechanisms involved in cardiotoxicity. Finally, we discuss combining systems biology approaches with iPSC technology to help discover molecular mechanisms associated with cardiotoxicity.

Dimethyl sulfoxide (DMSO) enhances direct cardiac reprogramming by inhibiting the bromodomain of coactivators CBP/p300.

AIMS: Direct cardiac reprogramming represents an attractive way to reversing heart damage caused by myocardial infarction because it removes fibroblasts, while also generating new functional cardiomyocytes. Yet, the main hurdle for bringing this technique to the clinic is the lack of efficacy with current reprogramming protocols. Here, we describe our unexpected discovery that DMSO is capable of significantly augmenting direct cardiac reprogramming in vitro. METHODS AND RESULTS: Upon induction with cardiac transcription factors- Gata4, Hand2, Mef2c and Tbx5 (GHMT), the treatment of mouse embryonic fibroblasts (MEFs) with 1% DMSO induced ~5 fold increase in Myh6-mCherry+ cells, and significantly upregulated global expression of cardiac genes, including Myh6, Ttn, Nppa, Myh7 and Ryr2. RNA-seq confirmed upregulation of cardiac gene programmes and downregulation of extracellular matrix-related genes. Treatment of TGF-ß1, DMSO, or SB431542, and the combination thereof, revealed that DMSO most likely targets a separate but parallel pathway other than TGF-ß signalling. Subsequent experiments using small molecule screening revealed that DMSO enhances direct cardiac reprogramming through inhibition of the CBP/p300 bromodomain, and not its acetyltransferase property. CONCLUSION: In conclusion, our work points to a direct molecular target of DMSO, which can be used for augmenting GHMT-induced direct cardiac reprogramming and possibly other cell fate conversion processes.

Hypertrophic signaling compensates for contractile and metabolic consequences of DNA methyltransferase 3A loss in human cardiomyocytes.

The role of DNA methylation in cardiomyocyte physiology and cardiac disease remains a matter of controversy. We have recently provided evidence for an important role of DNMT3A in human cardiomyocyte cell homeostasis and metabolism, using engineered heart tissue (EHT) generated from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes carrying a knockout of the de novo DNA methyltransferase DNMT3A. Unlike isogenic control EHT, knockout EHT displayed morphological abnormalities such as lipid accumulations inside cardiomyocytes associated with impaired mitochondrial metabolism, as well as functional defects and impaired glucose metabolism. Here, we analyzed the role of DNMT3A in the setting of cardiac hypertrophy. We induced hypertrophic signaling by treatment with 50 nM endothelin-1 and 20 µM phenylephrine for one week and assessed EHT contractility, morphology, DNA methylation, and gene expression. While both knockout EHTs and isogenic controls showed the expected activation of the hypertrophic gene program, knockout EHTs were protected from hypertrophy-related functional impairment. Conversely, hypertrophic treatment prevented the metabolic consequences of a loss of DNMT3A, i.e. abolished lipid accumulation in cardiomyocytes likely by partial normalization of mitochondrial metabolism and restored glucose metabolism and metabolism-related gene expression of knockout EHT. Together, these data suggest an important role of DNA methylation not only for cardiomyocyte physiology, but also in the setting of cardiac disease.

Mechanism of Eccentric Cardiomyocyte Hypertrophy Secondary to Severe Mitral Regurgitation.

BACKGROUND: Primary valvular heart disease is a prevalent cause of morbidity and mortality in both industrialized and developing countries. Although the primary consequence of valvular heart disease is myocardial dysfunction, treatment of valvular heart diseases centers around valve repair or replacement rather than prevention or reversal of myocardial dysfunction. This is particularly evident in primary mitral regurgitation (MR), which invariably results in eccentric hypertrophy and left ventricular (LV) failure in the absence of timely valve repair or replacement. The mechanism of LV dysfunction in primary severe MR is entirely unknown. METHODS: Here, we developed the first mouse model of severe MR. Valvular damage was achieved by severing the mitral valve leaflets and chords with iridectomy scissors, and MR was confirmed by echocardiography. Serial echocardiography was performed to follow up LV morphology and systolic function. Analysis of cardiac tissues was subsequently performed to evaluate valve deformation, cardiomyocyte morphology, LV fibrosis, and cell death. Finally, dysregulated pathways were assessed by RNA-sequencing analysis and immunofluorescence. RESULTS: In the ensuing 15 weeks after the induction of MR, gradual LV dilatation and dysfunction occurred, resulting in severe systolic dysfunction. Further analysis revealed that severe MR resulted in a marked increase in cardiac mass and increased cardiomyocyte length but not width, with electron microscopic evidence of sarcomere disarray and the development of sarcomere disruption. From a mechanistic standpoint, severe MR resulted in activation of multiple components of both the mammalian target of rapamycin and calcineurin pathways. Inhibition of mammalian target of rapamycin signaling preserved sarcomeric structure and prevented LV remodeling and systolic dysfunction. Immunohistochemical analysis uncovered a differential pattern of expression of the cell polarity regulator Crb2 (crumbs homolog 2) along the longitudinal axis of cardiomyocytes and close to the intercalated disks in the MR hearts. Electron microscopy images demonstrated a significant increase in polysome localization in close proximity to the intercalated disks and some areas along the longitudinal axis in the MR hearts. CONCLUSIONS: These results indicate that LV dysfunction in response to severe MR is a form of maladaptive eccentric cardiomyocyte hypertrophy and outline the link between cell polarity regulation and spatial localization protein synthesis as a pathway for directional cardiomyocyte growth.

An Important Role for DNMT3A-Mediated DNA Methylation in Cardiomyocyte Metabolism and Contractility.

BACKGROUND: DNA methylation acts as a mechanism of gene transcription regulation. It has recently gained attention as a possible therapeutic target in cardiac hypertrophy and heart failure. However, its exact role in cardiomyocytes remains controversial. Thus, we knocked out the main de novo DNA methyltransferase in cardiomyocytes, DNMT3A, in human induced pluripotent stem cells. Functional consequences of DNA methylation-deficiency under control and stress conditions were then assessed in human engineered heart tissue from knockout human induced pluripotent stem cell-derived cardiomyocytes. METHODS: DNMT3A was knocked out in human induced pluripotent stem cells by CRISPR/Cas9gene editing. Fibrin-based engineered heart tissue was generated from knockout and control human induced pluripotent stem cell-derived cardiomyocytes. Development and baseline contractility were analyzed by video-optical recording. Engineered heart tissue was subjected to different stress protocols, including serum starvation, serum variation, and restrictive feeding. Molecular, histological, and ultrastructural analyses were performed afterward. RESULTS: Knockout of DNMT3A in human cardiomyocytes had three main consequences for cardiomyocyte morphology and function: (1) Gene expression changes of contractile proteins such as higher atrial gene expression and lower MYH7/MYH6 ratio correlated with different contraction kinetics in knockout versus wild-type; (2) Aberrant activation of the glucose/lipid metabolism regulator peroxisome proliferator-activated receptor gamma was associated with accumulation of lipid vacuoles within knockout cardiomyocytes; (3) Hypoxia-inducible factor 1α protein instability was associated with impaired glucose metabolism and lower glycolytic enzyme expression, rendering knockout-engineered heart tissue sensitive to metabolic stress such as serum withdrawal and restrictive feeding. CONCLUSION: The results suggest an important role of DNA methylation in the normal homeostasis of cardiomyocytes and during cardiac stress, which could make it an interesting target for cardiac therapy.

Robust CTCF-Based Chromatin Architecture Underpins Epigenetic Changes in the Heart Failure Stress-Gene Response.

BACKGROUND: The human genome folds in 3 dimensions to form thousands of chromatin loops inside the nucleus, encasing genes and cis-regulatory elements for accurate gene expression control. Physical tethers of loops are anchored by the DNA-binding protein CTCF and the cohesin ring complex. Because heart failure is characterized by hallmark gene expression changes, it was recently reported that substantial CTCF-related chromatin reorganization underpins the myocardial stress-gene response, paralleled by chromatin domain boundary changes observed in CTCF knockout. METHODS: We undertook an independent and orthogonal analysis of chromatin organization with mouse pressure-overload model of myocardial stress (transverse aortic constriction) and cardiomyocyte-specific knockout of Ctcf. We also downloaded published data sets of similar cardiac mouse models and subjected them to independent reanalysis. RESULTS: We found that the cardiomyocyte chromatin architecture remains broadly stable in transverse aortic constriction hearts, whereas Ctcf knockout resulted in ≈99% abolition of global chromatin loops. Disease gene expression changes correlated instead with differential histone H3K27-acetylation enrichment at their respective proximal and distal interacting genomic enhancers confined within these static chromatin structures. Moreover, coregulated genes were mapped out as interconnected gene sets on the basis of their multigene 3D interactions. CONCLUSIONS: This work reveals a more stable genome-wide chromatin framework than previously described. Myocardial stress-gene transcription responds instead through H3K27-acetylation enhancer enrichment dynamics and gene networks of coregulation. Robust and intact CTCF looping is required for the induction of a rapid and accurate stress response.

Persistent changes in liver methylation and microbiome composition following reversal of diet-induced non-alcoholic-fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic liver disease that is thought to be reversible by changing the diet. To examine the impact of dietary changes on progression and cure of NAFLD, we fed mice a high-fat diet (HFD) or high-fructose diet (HFrD) for 9 weeks, followed by an additional 9 weeks, where mice were given normal chow diet. As predicted, the diet-induced NAFLD elicited changes in glucose tolerance, serum cholesterol, and triglyceride levels in both diet groups. Moreover, the diet-induced NAFLD phenotype was reversed, as measured by the recovery of glucose intolerance and high cholesterol levels when mice were given normal chow diet. However, surprisingly, the elevated serum triglyceride levels persisted. Metagenomic analysis revealed dietary-induced changes of microbiome composition, some of which remained altered even after reversing the diet to normal chow, as illustrated by species of the Odoribacter genus. Genome-wide DNA methylation analysis revealed a "priming effect" through changes in DNA methylation in key liver genes. For example, the lipid-regulating gene Apoa4 remained hypomethylated in both groups even after introduction to normal chow diet. Our results support that dietary change, in part, reverses the NAFLD phenotype. However, some diet-induced effects remain, such as changes in microbiome composition, elevated serum triglyceride levels, and hypomethylation of key liver genes. While the results are correlative in nature, it is tempting to speculate that the dietary-induced changes in microbiome composition may in part contribute to the persistent epigenetic modifications in the liver.

Exploring emotion regulation as a mediator of the relationship between resilience and distress in cancer.

OBJECTIVES: Distress in patients with cancer is a significant problem that affects up to 32% of patients. Yet research indicates that 35% of cancer patients do maintain high levels of well-being. Resilience is one psychological factor implicated as being protective against distress; however, the mechanisms for this relationship are currently unknown. The present study aimed to explore emotion regulation as a potential mediator of the relationship between resilience and distress. METHODS: A cross-sectional survey examining emotional regulation, resilience, and distress was completed by 227 patients from two hospitals with heterogeneous cancer types. Measures included the Difficulties in Emotion Regulation Scale (DERS), the Connor Davidson Resilience Scale, and the Depression, Anxiety, Stress Scale. RESULTS: Difficulties in emotion regulation and resilience explained 33.2% of the variance in distress. Resilience had a significant direct effect on distress, accounting for 15.8% of the variance. However, this effect was no longer significant when difficulties in emotion regulation were controlled for. The indirect effect through difficulties in emotion regulation was significant, b = 0.009, 95% CI [-0.013,-0.007], suggesting that the effect of resilience on distress was fully mediated by emotion regulation. Parallel mediation analyses also examined the differential effects of the six DERS subscales on the relationship between resilience and distress. CONCLUSION: These findings suggest that emotion regulation is an important mediator of resilience in cancer. Hence, in patients with cancer, difficulties in emotion regulation (and the DERS specifically) might be a useful focus for screening for patients at risk of distress.

Pharmacological inhibition of DNA methylation attenuates pressure overload-induced cardiac hypertrophy in rats.

BACKGROUND: Heart failure is associated with altered gene expression and DNA methylation. De novo DNA methylation is associated with gene silencing, but its role in cardiac pathology remains incompletely understood. We hypothesized that inhibition of DNA methyltransferases (DNMT) might prevent the deregulation of gene expression and the deterioration of cardiac function under pressure overload (PO). To test this hypothesis, we evaluated a DNMT inhibitor in PO in rats and analysed DNA methylation in cardiomyocytes. METHODS AND RESULTS: Young male Wistar rats were subjected to PO by transverse aortic constriction (TAC) or to sham surgery. Rats from both groups received solvent or 12.5 mg/kg body weight of the non-nucleosidic DNMT inhibitor RG108, initiated on the day of the intervention. After 4 weeks, we analysed cardiac function by MRI, fibrosis with Sirius Red staining, gene expression by RNA sequencing and qPCR, and DNA methylation by reduced representation bisulphite sequencing (RRBS). RG108 attenuated the ~70% increase in heart weight/body weight ratio of TAC over sham to 47% over sham, partially rescued reduced contractility, diminished the fibrotic response and the downregulation of a set of genes including Atp2a2 (SERCA2a) and Adrb1 (beta1-adrenoceptor). RG108 was associated with significantly lower global DNA methylation in cardiomyocytes by ~2%. The differentially methylated pathways were "cardiac hypertrophy", "cell death" and "xenobiotic metabolism signalling". Among these, "cardiac hypertrophy" was associated with significant methylation differences in the group comparison sham vs. TAC, but not significant between sham+RG108 and TAC+RG108 treatment, suggesting that RG108 partially prevented differential methylation. However, when comparing TAC and TAC+RG108, the pathway cardiac hypertrophy was not significantly differentially methylated. CONCLUSIONS: DNMT inhibitor treatment is associated with attenuation of cardiac hypertrophy and moderate changes in cardiomyocyte DNA methylation. The potential mechanistic link between these two effects and the role of non-myocytes need further clarification.

Ischemic Stroke in Nonvalvular Atrial Fibrillation at Warfarin Initiation: Assessment via a Large Insurance Database.

BACKGROUND AND PURPOSE: Stroke risk may increase shortly after warfarin initiation in nonvalvular atrial fibrillation patients. Because of the brief period and limited number of events, large samples are needed to study this effect. We compared 30-day rates of ischemic stroke between nonvalvular atrial fibrillation patients initiating warfarin to nonwarfarin comparators using an insurance claims database. METHODS: We identified nonvalvular atrial fibrillation patients via 1 inpatient/2 outpatient diagnosis claims from the MarketScan database, January 1, 2009, to December 31, 2010. We studied patients initiating warfarin using prescription claims and 1:1 matched 22 669 initiators to comparators based on age, sex, diagnosis date, and warfarin propensity score. Follow-up began on initiation date; patients were censored at discontinuation/switch of therapy, disenrollment, or end of the study. The median follow-up was 415 days. Cox regression was used to study differences in ischemic stroke risks between warfarin initiators and comparators while controlling for important prognostic factors. RESULTS: Warfarin initiators were generally similar to comparators in clinical features but had higher CHADS2 scores (1.26 versus 1.19). The first 30-day ischemic stroke rate was higher among warfarin initiators than comparators (1.47%/y (27/1836) versus 0.98%/y (18/1837); P=0.18) but lower subsequently (0.81%/y [134/16 543] versus 1.09%/y [406/37 248]; P=0.002). Multivariable regression confirmed a significant interaction between follow-up and warfarin use with the adjusted hazard ratios (95% confidence intervals) for warfarin/comparator as 1.46 (0.80-2.65) in the first 30 days and 0.70 (0.57-0.85) afterward. CONCLUSIONS: Warfarin effect was qualitatively different in the first 30 days after initiation than subsequently. This is consistent with a modest increase in stroke risk occurring briefly after starting warfarin.

Physician and Patient Preferences for Nonvalvular Atrial Fibrillation Therapies.

OBJECTIVES: The objective of this study was to compare patient and physician preferences for different antithrombotic therapies used to treat nonvalvular atrial fibrillation (NVAF). METHODS: Patients diagnosed with NVAF and physicians treating such patients completed 12 discrete choice questions comparing NVAF therapies that varied across five attributes: stroke risk, major bleeding risk, convenience (no regular blood testing/dietary restrictions), dosing frequency, and patients' out-of-pocket cost. We used a logistic regression to estimate the willingness-to-pay (WTP) value for each attribute. RESULTS: The 200 physicians surveyed were willing to trade off $38 (95% confidence interval [CI] $22 to $54] in monthly out-of-pocket cost for a 1% (absolute) decrease in stroke risk, $14 (95% CI $8 to $21) for a 1% decrease in major bleeding risk, and $34 (95% CI $9 to $60) for more convenience. The WTP value among 201 patients surveyed was $30 (95% CI $18 to $42) for reduced stroke risk, $16 (95% CI $9 to $24) for reduced bleeding risk, and -$52 (95% CI -$96 to -6) for convenience. The WTP value for convenience among patients using warfarin was $9 (95% CI $1 to $18) for more convenience, whereas patients not currently on warfarin had a WTP value of -$90 (95% CI -$290 to -$79). Both physicians' and patients' WTP value for once-daily dosing was not significantly different from zero. On the basis of survey results, 85.0% of the physicians preferred novel oral anticoagulants (NOACs) to warfarin. NOACs (73.0%) were preferred among patients using warfarin, but warfarin (78.2%) was preferred among patients not currently using warfarin. Among NOACs, both patients and physicians preferred apixaban. CONCLUSIONS: Both physicians and patients currently using warfarin preferred NOACs to warfarin. Patients not currently using warfarin preferred warfarin over NOACs because of an apparent preference for regular blood testing/dietary restrictions.

Warfarin Discontinuation in Patients With Unprovoked Venous Thromboembolism: A Large US Insurance Database Analysis.

This study examined warfarin therapy discontinuation and its risk factors among patients with unprovoked venous thromboembolism (VTE) in the US clinical practice setting. Adult patients with unprovoked VTE were identified from the MarketScan claims database from January 1, 2006 to December 31, 2012. The index date was defined as the date of first VTE diagnosis. Patients were required to have no VTE diagnosis in the 6 months before index date and continuous health plan enrollment for 6 months before and 12 months after the index date. Warfarin discontinuation rates and adjusted hazard ratios (HRs) were reported. Of 21,163 eligible patients, 15,463 were diagnosed with deep vein thrombosis (DVT) only (73.1%), 5027 with pulmonary embolism (PE) only (23.7%), and 673 with DVT and PE (3.2%). The average duration of warfarin therapy was 5.2 months (SD = 3.0). During 1-year follow-up, 21.4% patients discontinued therapy within 3 months, 42.8% within 6 months, and 70.1% within 12 months. PE versus DVT [HR = 0.77, 95% confidence interval (CI) = 0.74-0.80], comorbid atrial fibrillation (HR = 0.73, 95% CI = 0.66-0.81), thrombophilia (HR = 0.62, 95% CI = 0.54-0.71), and age >40 years (41-65 years: HR = 0.86, 95% CI = 0.81-0.91; >65 years: HR = 0.82, 95% CI = 0.77-0.87) were significantly associated with reduced risk of warfarin discontinuation. Alcohol abuse/dependence (HR = 1.36, 95% CI = 1.20-1.55), cancer history (HR = 1.13, 95% CI = 1.07-1.19), bleeding (HR = 1.07, 95% CI = 1.01-1.15), and catheter ablation (HR = 1.10, 95% CI = 1.00-1.20) in the 6 months before index date were significantly associated with increased risk for warfarin discontinuation. In conclusion, nearly 1 of 4 patients with unprovoked VTE discontinued warfarin within 3 months. Three of 4 patients discontinued therapy within 1 year. Younger age and multiple clinical factors are associated with warfarin therapy discontinuation.

Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons.

Tricho-hepato-enteric syndrome (THE-S): two cases and review of the literature.

UNLABELLED: Tricho-hepato-enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. We report two cases of East Asian descent with THE-S who had remained undiagnosed despite extensive investigations but were diagnosed on whole exome sequencing (WES). Both cases presented with chronic diarrhea, failure to thrive, and recurrent infections. Case 1 had posteriorly rotated low set ears, mild retrognathia, and fine curly hypopigmented hair. She was managed with prolonged total parenteral nutrition and intravenous immunoglobulin infusions. Case 2 had sparse coarse brown hair as well as multiple lentigines and café-au-lait macules. She was managed with amino acid-based formula. For both cases, routine investigations were inconclusive. WES in both cases showed biallelic truncating mutations in TTC37 (c.3507T>G;p.Y1169X and c.3601C>T;p.R1201X in case 1 and c.3507T>G;p.Y1169X and c.154G>T;p.E52X in case 2), suggesting a diagnosis of THE-S. CONCLUSION: We present novel mutations in the TTC37 gene in two individuals of East Asian descent with the rare THE-S, detected by WES. Future identification of patients with THE-S and establishing genotype-phenotype correlations will aid in counseling the patients and their families. WHAT IS KNOWN: • Tricho-Hepato-Enteric syndrome (THE-S) is characterized by severe infantile diarrhea, failure to thrive, dysmorphism, woolly hair, and immune or hepatic dysfunction. • Complex patients with diagnostic dilemmas undergo extensive investigations. What is New: • This is a report of novel mutations in TTC37 in individuals of East Asian descent. • Whole exome sequencing (WES) can be useful in certain complex cases with diagnostic dilemmas.