Embryonic alcohol exposure in zebrafish predisposes adults to cardiomyopathy and diastolic dysfunction.

AIMS: Fetal alcohol spectrum disorders (FASDs) impact up to 0.8% of the global population. However, cardiovascular health outcomes in adult patients, along with predictive biomarkers for cardiac risk stratification, remain unknown. Our aim was to utilize a longitudinal cohort study in an animal model to evaluate the impact of embryonic alcohol exposure (EAE) on cardiac structure, function, and transcriptional profile across the lifespan. METHODS AND RESULTS: Using zebrafish, we characterized the aftereffects of embryonic alcohol exposure (EAE) in adults binned by congenital heart defect (CHD) severity. Chamber sizes were quantified on dissected adult hearts to identify structural changes indicative of cardiomyopathy. Using echocardiography, we quantified systolic function based on ejection fraction and longitudinal strain, and diastolic function based on ventricular filling dynamics, ventricular wall movement, and estimated atrial pressures. Finally, we performed RNA sequencing on EAE ventricles and assessed how differentially expressed genes (DEGs) correlated with cardiac function. Here, we demonstrate that embryonic alcohol exposure (EAE) causes cardiomyopathy and diastolic dysfunction through persistent alterations to ventricular wall structure and gene expression. Following abnormal ventricular morphogenesis, >30% of all EAE adults developed increased atrial-to-ventricular size ratios, abnormal ventricular filling dynamics, and reduced myocardial wall relaxation during early diastole despite preserved systolic function. RNA sequencing of the EAE ventricle revealed novel and heart failure-associated genes (slc25a33, ankrd9, dusp2, dusp4, spry4, eya4, and edn1) whose expression levels were altered across the animal's lifespan or correlated with the degree of diastolic dysfunction detected in adulthood. CONCLUSIONS: Our study identifies EAE as a risk factor for adult-onset cardiomyopathy and diastolic dysfunction, regardless of CHD status, and suggests novel molecular indicators of adult EAE-induced heart disease.

Air pollution accountability research: Moving from a chain to a web.

Air pollution accountability studies examine the relationship(s) between an intervention, regulation, or event and the resulting downstream impacts, if any, on emissions, exposure, and/or health. The sequence of events has been schematically described as an accountability chain. Here, we update the existing framework to capture real-life complexities and to highlight important factors that fall outside the linear chain. This new "accountability web" is intended to convey the intricacies associated with conducting an accountability study to various audiences, including researchers, policy makers, and stakeholders. We also identify data considerations for planning and completing a robust accountability study, including those relevant to novel and innovative air pollution and exposure data. Finally, we present a series of recommendations for the accountability research community that can serve as a guide for the next generation of accountability studies.

Generation and application of endogenously floxed alleles for cell-specific knockout in zebrafish.

The zebrafish is amenable to a variety of genetic approaches. However, lack of conditional deletion alleles limits stage- or cell-specific gene knockout. Here, we applied an existing protocol to establish a floxed allele for gata2a but failed to do so due to off-target integration and incomplete knockin. To address these problems, we applied simultaneous co-targeting with Cas12a to insert loxP sites in cis, together with transgenic counterscreening and comprehensive molecular analysis, to identify off-target insertions and confirm targeted knockins. We subsequently used our approach to establish endogenously floxed alleles of foxc1a, rasa1a, and ruvbl1, each in a single generation. We demonstrate the utility of these alleles by verifying Cre-dependent deletion, which yielded expected phenotypes in each case. Finally, we used the floxed gata2a allele to demonstrate an endothelial autonomous requirement in lymphatic valve development. Together, our results provide a framework for routine generation and application of endogenously floxed alleles in zebrafish.

Comparison of targeted vs. expanded pharmacogenomic testing: What are we missing?

BACKGROUND: Pharmacogenomics (PGx) is used as a medication management strategy by a small but growing number of institutions. PGx allows prescribers to individually treat patients concordant with their genes. Recent litigation for preventable PGx-mediated adverse events highlights the need to accelerate PGx implementation for patient safety. Genetic variations cause drug metabolism, transport, and target changes, affecting medication response and tolerability. PGx testing often consists of targeted testing aimed at specific gene-drug pairs or disease states. Conversely, expanded panel testing can evaluate all known actionable gene-drug interactions, enhancing proactive clarity regarding patient response. OBJECTIVES: Evaluate the divergence of targeted PGx testing with a single gene-drug pair test (cardiac), a two-gene panel, and a focused psychiatric panel compared to expanded PGx testing. METHODS: An expanded PGx panel (≥25 genes) was compared to a single gene-drug pair test of CYP2C19/clopidogrel, a dual gene test of CYP2C19/CYP2D6, a 7-gene psychiatric list, and a 14-gene psychiatric panel to inform specific depression and pain management drugs. The expanded panel provided a baseline to evaluate total PGx variations compared to those possibly missed by targeted testing. RESULTS: Targeted testing did not identify up to 95% of total PGx gene-drug interactions discovered. The expanded panel reported all gene-drug interactions for any medication with Clinical Pharmacogenomics Implementation Consortium (CPIC) guidance or U.S. Food and Drug Administration (FDA) labeling for that gene. Single gene CYP2C19/clopidogrel testing missed or did not report on ∼95% of total interactions, CYP2C19/CYP2D6 testing missed or did not report ∼89%, and the 14-gene panel missed or did not report on ∼73%. The 7-gene list missed ∼20% of discovered potential PGx interactions but was not designed to identify gene-drug interactions. CONCLUSIONS: Targeted PGx testing for limited genes or by specialty may miss or not report significant portions of PGx gene-drug interactions. This can lead to potential patient harm from the missed interactions and subsequent failed therapies and/or adverse reactions.

Implementing comprehensive pharmacogenomics in a community hospital-associated primary care setting.

BACKGROUND: Pharmacogenomics (PGx) is an emerging field. Many drug-gene interactions are known but not yet routinely addressed in clinical practice. Therefore, there is a significant gap in care, necessitating development of implementation strategies. OBJECTIVE: The objective of the study was to assess the impact of implementing a PGx practice model which incorporates comprehensive pharmacogenomic risk evaluation, testing and medication optimization administered by 7 PGx-certified ambulatory care pharmacists embedded across 30 primary care clinic sites. METHODS: Pharmacogenomic services were implemented in 30 primary care clinics within the Cincinnati, Ohio area. Patients are identified for pharmacogenomic testing using a clinical decision support tool (CDST) that is fully integrated in the electronic medical record (EMR) or by provider designation (e.g., psychotropic drug failure). Pharmacogenomic testing is performed via buccal swab using standardized clinic processes. Discrete data results are returned directly into the EMR/CDST for review by PGx-certified ambulatory care pharmacists. Recommendations and prescriptive changes are then discussed and implemented as a collaborative effort between pharmacist, primary care provider, specialists, and patient. RESULTS: A total of 422 unique interactions were assessed by the embedded ambulatory care PGx pharmacists (N = 7) during this interim analysis. About half (213) were pharmacogenomic interactions, and of these, 124 were actionable. When an intervention was actionable, 82% of the time a change in medication was recommended. The underlying reasons for recommending therapy alterations were most commonly ineffective therapy (43%), adverse drug reaction prevented (34%), or adverse drug reaction observed (13%). CONCLUSION: Variations in drug metabolism, response, and tolerability can negatively impact patient outcomes across many disease states and treatment specialties. Incorporation of pharmacogenomic testing with accessible clinical decision support into the team-based care model allows for a truly comprehensive review and optimization of medications. Our initial analysis suggests that comprehensive PGx testing should be considered to enhance medication safety and efficacy in at-risk patients.

Validation of Pharmacogenomic Interaction Probability (PIP) Scores in Predicting Drug-Gene, Drug-Drug-Gene, and Drug-Gene-Gene Interaction Risks in a Large Patient Population.

Utilizing pharmacogenomic (PGx) testing and integrating evidence-based guidance in drug therapy enables an improved treatment response and decreases the occurrence of adverse drug events. We conducted a retrospective analysis to validate the YouScript® PGx interaction probability (PIP) algorithm, which predicts patients for whom PGx testing would identify one or more evidence-based, actionable drug-gene, drug-drug-gene, or drug-gene-gene interactions (EADGIs). PIP scores generated for 36,511 patients were assessed according to the results of PGx multigene panel testing. PIP scores versus the proportion of patients in whom at least one EADGI was found were 22.4% vs. 22.4% (p = 1.000), 23.5% vs. 23.4% (p = 0.6895), 30.9% vs. 29.4% (p = 0.0667), and 27.3% vs. 26.4% (p = 0.3583) for patients tested with a minimum of 3-, 5-, 14-, and 25-gene panels, respectively. These data suggest a striking concordance between the PIP scores and the EAGDIs found by gene panel testing. The ability to identify patients most likely to benefit from PGx testing has the potential to reduce health care costs, enable patient access to personalized medicine, and ultimately improve drug efficacy and safety.

RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.

Intrinsic myocardial defects underlie an Rbfox-deficient zebrafish model of hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is characterized by underdevelopment of left sided structures including the ventricle, valves, and aorta. Prevailing paradigm suggests that HLHS is a multigenic disease of co-occurring phenotypes. Here, we report that zebrafish lacking two orthologs of the RNA binding protein RBFOX2, a gene linked to HLHS in humans, display cardiovascular defects overlapping those in HLHS patients including ventricular, valve, and aortic deficiencies. In contrast to current models, we demonstrate that these structural deficits arise secondary to impaired pump function as these phenotypes are rescued when Rbfox is specifically expressed in the myocardium. Mechanistically, we find diminished expression and alternative splicing of sarcomere and mitochondrial components that compromise sarcomere assembly and mitochondrial respiration, respectively. Injection of human RBFOX2 mRNA restores cardiovascular development in rbfox mutant zebrafish, while HLHS-linked RBFOX2 variants fail to rescue. This work supports an emerging paradigm for HLHS pathogenesis that centers on myocardial intrinsic defects.

Le Fort III fractures: An approach to resuscitation and management.

Introduction: Le Fort fractures occur in approximately 20% of facial fractures and result from a high velocity/force mechanism of injury. In those rare 20% occurrences, the Le Fort III fractures are the least common and are highly associated with injuries of the cervical spine, intracranial, and internal neck structures. Importance: This makes them difficult to manage and requiring a definitive sequence of resuscitation and thorough secondary and tertiary surveys thereafter. The morbidity and mortality of these severe fractures is high but with appropriate resuscitation and adequate stabilization of the fracture, this may be improved on and lowered. Case presentation: A male sustaining multiple stabs to the face presents to a level one trauma emergency unit haemodynamically unstable/abnormal with a threatened airway and stridor. Discussion: This case report walks through step-by-step the management approaches at each stage thereby assessing and managing the outcomes of each diagnosis. Conclusion: Le Fort III fractures are rare but critical injuries that require intensive resuscitation and a multidisciplinary approach to achieve wholistic and appropriate management of these patients. Adequate initial resuscitation and stabilization of fractures may improve the morbidity and mortality of these sever injuries.

Ruvbl2 Suppresses Cardiomyocyte Proliferation During Zebrafish Heart Development and Regeneration.

Cardiomyocyte proliferation is an important source of new myocardium during heart development and regeneration. Consequently, mutations in drivers of cardiomyocyte proliferation cause congenital heart disease, and infarcted human hearts scar because cardiomyocytes exit the cell cycle postnatally. To boost cardiomyocyte proliferation in either setting, critical regulators must be identified. Through an ENU screen in zebrafish, the liebeskummer (lik) mutant was isolated and described as having elevated cardiomyocyte numbers during embryogenesis. The lik mutation results in a three amino acid insertion into Ruvbl2, a highly conserved ATPase. Because both gain- and loss-of-function properties have been described for ruvbl2 lik , it remains unclear whether Ruvbl2 positively or negatively regulates cardiomyocyte proliferation. Here, we demonstrate that Ruvbl2 is a suppressor of cardiomyocyte proliferation during zebrafish heart development and regeneration. First, we confirmed speculation that augmented cardiomyocyte numbers in ruvbl2 lik/lik hearts arise by hyperproliferation. To characterize bona fide ruvbl2 null animals, we created a ruvbl2 locus deletion allele (ruvbl2 Δ ). Like ruvbl2 lik/lik mutants, ruvbl2 Δ/Δ and compound heterozygote ruvbl2 lik/Δ animals display ventricular hyperplasia, demonstrating that lik is a loss of function allele and that ruvbl2 represses cardiomyocyte proliferation. This activity is autonomous because constitutive myocardial overexpression of Ruvbl2 is sufficient to suppress cardiomyocyte proliferation in control hearts and rescue the hyperproliferation observed in ruvbl2 Δ/Δ mutant hearts. Lastly, heat-shock inducible overexpression of Ruvbl2 suppresses cardiomyocyte proliferation during heart regeneration and leads to scarring. Together, our data demonstrate that Ruvbl2 functions autonomously as a suppressor of cardiomyocyte proliferation during both zebrafish heart development and adult heart regeneration.

Latent TGFß-binding proteins 1 and 3 protect the larval zebrafish outflow tract from aneurysmal dilatation.

Aortic root aneurysm is a common cause of morbidity and mortality in Loeys-Dietz and Marfan syndromes, where perturbations in transforming growth factor beta (TGFß) signaling play a causal or contributory role, respectively. Despite the advantages of cross-species disease modeling, animal models of aortic root aneurysm are largely restricted to genetically engineered mice. Here, we report that zebrafish devoid of the genes encoding latent-transforming growth factor beta-binding protein 1 and 3 (ltbp1 and ltbp3, respectively) develop rapid and severe aneurysm of the outflow tract (OFT), the aortic root equivalent. Similar to syndromic aneurysm tissue, the distended OFTs display evidence for paradoxical hyperactivated TGFß signaling. RNA-sequencing revealed significant overlap between the molecular signatures of disease tissue from mutant zebrafish and a mouse model of Marfan syndrome. Moreover, chemical inhibition of TGFß signaling in wild-type animals phenocopied mutants but chemical activation did not, demonstrating that TGFß signaling is protective against aneurysm. Human relevance is supported by recent studies implicating genetic lesions in LTBP3 and, potentially, LTBP1 as heritable causes of aortic root aneurysm. Ultimately, our data demonstrate that zebrafish can now be leveraged to interrogate thoracic aneurysmal disease and identify novel lead compounds through small-molecule suppressor screens. This article has an associated First Person interview with the first author of the paper.

Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Innate Mechanisms of Heart Regeneration.

Heart regeneration is a remarkable process whereby regrowth of damaged cardiac tissue rehabilitates organ anatomy and function. Unfortunately, the human heart is highly resistant to regeneration, which creates a shortage of cardiomyocytes in the wake of ischemic injury, and explains, in part, why coronary artery disease remains a leading cause of death worldwide. Luckily, a detailed blueprint for achieving therapeutic heart regeneration already exists in nature because several lower vertebrate species successfully regenerate amputated or damaged heart muscle through robust cardiomyocyte proliferation. A growing number of species are being interrogated for cardiac regenerative potential, and several commonalities have emerged between those animals showing high or low innate capabilities. In this review, we provide a historical perspective on the field, discuss how regenerative potential is influenced by cardiomyocyte properties, mitogenic signals, and chromatin accessibility, and highlight unanswered questions under active investigation. Ultimately, delineating why heart regeneration occurs preferentially in some organisms, but not in others, will uncover novel therapeutic inroads for achieving cardiac restoration in humans.

MEDLINE search retrieval issues: A longitudinal query analysis of five vendor platforms.

This study compared the results of data collected from a longitudinal query analysis of the MEDLINE database hosted on multiple platforms that include PubMed, EBSCOHost, Ovid, ProQuest, and Web of Science. The goal was to identify variations among the search results on the platforms after controlling for search query syntax. We devised twenty-nine cases of search queries comprised of five semantically equivalent queries per case to search against the five MEDLINE database platforms. We ran our queries monthly for a year and collected search result count data to observe changes. We found that search results varied considerably depending on MEDLINE platform. Reasons for variations were due to trends in scholarly publication such as publishing individual papers online first versus complete issues. Some other reasons were metadata differences in bibliographic records; differences in the levels of specificity of search fields provided by the platforms and large fluctuations in monthly search results based on the same query. Database integrity and currency issues were observed as each platform updated its MEDLINE data throughout the year. Specific biomedical bibliographic databases are used to inform clinical decision-making, create systematic reviews, and construct knowledge bases for clinical decision support systems. They serve as essential information retrieval and discovery tools to help identify and collect research data and are used in a broad range of fields and as the basis of multiple research designs. This study should help clinicians, researchers, librarians, informationists, and others understand how these platforms differ and inform future work in their standardization.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N')-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N'-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N'-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Chronic adolescent stress causes sustained impairment of cognitive flexibility and hippocampal synaptic strength in female rats.

Females that experience chronic stress during development, particularly adolescence, are the most vulnerable group to stress-induced disease. While considerable attention has been devoted to stress-induced manifestation of anxiety, depression, and PTSD, evidence indicates that a history of chronic stress is also a risk factor for cognitive decline and dementia - with females again in a higher risk group. This interplay between sex and stress history indicates specific mechanisms drive neural dysfunction across the lifespan. The presence of sex and stress steroid receptors in the hippocampus provides a point of influence for these variables to drive changes in cognitive function. Here, we used a rodent model of chronic adolescent stress (CAS) to determine the extent to which CAS modifies glutamatergic signaling resulting in cognitive dysfunction. Male and female Wistar rats born in-house remained non-stressed (NS), unmanipulated aside from standard cage cleaning, or were exposed to either physical restraint (60 min) or social defeat (CAS) each day (6 trials each), along with social isolation, throughout the adolescent period (PND 35-47). Cognition was assessed in adult (PND 80-130) male and female rats (n = 10-12) using the Barnes Maze task and the Attention Set-Shift task. Whole hippocampi were extracted from a second cohort of male and female rats (NS and CAS; n = 9-10) and processed for RNA sequencing. Brain tissue from the first cohort (n = 6) was processed for density of glutamatergic synaptic markers (GluA1, NMDA1a, and synaptophysin) or whole-cell patch clamping (n = 4) to determine glutamatergic activity in the hippocampus. Females with a history of chronic stress had shorter latencies to locate the goal box than NS controls during acquisition learning but showed an increased latency to locate the new goal box during reversal learning. This reversal deficit persisted across domains as females with a history of stress required more trials to reach criterion during the reversal phases of the Attention Set-Shift task compared to controls. Ovariectomy resulted in greater performance variability overall during reversal learning with CAS females showing worse performance. Males showed no effects of CAS history on learning or memory performance. Bioinformatic prediction using gene ontology categorization indicated that in females, postsynaptic membrane gene clusters, specifically genes related to glutamatergic synapse remodeling, were enriched with a history of stress. Structural analysis indicated that CAS did not alter glutamate receptor density in females. However, functionally, CAS females had a decreased AMPA/NMDA-dependent current ratio compared to controls indicating a weakening in synaptic strength in the hippocampus. Males showed only a slight change in density of NMDA1a labeling in the CA3 region with a history of stress. The data observed here suggest that females are at risk for impaired cognitive flexibility following a history of adolescent stress, possibly driven by changes in glutamatergic signaling.

Validation of the bag-mediated filtration system for environmental surveillance of poliovirus in Nairobi, Kenya.

AIMS: This study compared the bag-mediated filtration system (BMFS) and standard WHO two-phase separation methods for poliovirus (PV) environmental surveillance, examined factors impacting PV detection and monitored Sabin-like (SL) PV type 2 presence with withdrawal of oral polio vaccine type 2 (OPV2) in April 2016. METHODS AND RESULTS: Environmental samples were collected in Nairobi, Kenya (Sept 2015-Feb 2017), concentrated via BMFS and two-phase separation methods, then assayed using the WHO PV isolation algorithm and intratypic differentiation diagnostic screening kit. SL1, SL2 and SL3 were detected at higher rates in BMFS than two-phase samples (P < 0·05). In BMFS samples, SL PV detection did not significantly differ with volume filtered, filtration time or filter shipment time (P > 0·05), while SL3 was detected less frequently with higher shipment temperatures (P = 0·027). SL2 was detected more frequently before OPV2 withdrawal in BMFS and two-phase samples (P < 1 × 10-5 ). CONCLUSIONS: Poliovirus was detected at higher rates with the BMFS, a method that includes a secondary concentration step, than using the standard WHO two-phase method. SL2 disappearance from the environment was commensurate with OPV2 withdrawal. SIGNIFICANCE AND IMPACT OF THE STUDY: The BMFS offers comparable or improved PV detection under the conditions in this study, relative to the two-phase method.

Research Priorities for Heart Failure With Preserved Ejection Fraction: National Heart, Lung, and Blood Institute Working Group Summary.

Heart failure with preserved ejection fraction (HFpEF), a major public health problem that is rising in prevalence, is associated with high morbidity and mortality and is considered to be the greatest unmet need in cardiovascular medicine today because of a general lack of effective treatments. To address this challenging syndrome, the National Heart, Lung, and Blood Institute convened a working group made up of experts in HFpEF and novel research methodologies to discuss research gaps and to prioritize research directions over the next decade. Here, we summarize the discussion of the working group, followed by key recommendations for future research priorities. There was uniform recognition that HFpEF is a highly integrated, multiorgan, systemic disorder requiring a multipronged investigative approach in both humans and animal models to improve understanding of mechanisms and treatment of HFpEF. It was recognized that advances in the understanding of basic mechanisms and the roles of inflammation, macrovascular and microvascular dysfunction, fibrosis, and tissue remodeling are needed and ideally would be obtained from (1) improved animal models, including large animal models, which incorporate the effects of aging and associated comorbid conditions; (2) repositories of deeply phenotyped physiological data and human tissue, made accessible to researchers to enhance collaboration and research advances; and (3) novel research methods that take advantage of computational advances and multiscale modeling for the analysis of complex, high-density data across multiple domains. The working group emphasized the need for interactions among basic, translational, clinical, and epidemiological scientists and across organ systems and cell types, leveraging different areas or research focus, and between research centers. A network of collaborative centers to accelerate basic, translational, and clinical research of pathobiological mechanisms and treatment strategies in HFpEF was discussed as an example of a strategy to advance research progress. This resource would facilitate comprehensive, deep phenotyping of a multicenter HFpEF patient cohort with standardized protocols and a robust biorepository. The research priorities outlined in this document are meant to stimulate scientific advances in HFpEF by providing a road map for future collaborative investigations among a diverse group of scientists across multiple domains.

Loss of ELK1 has differential effects on age-dependent organ fibrosis.

ETS domain-containing protein-1 (ELK1) is a transcription factor important in regulating αvß6 integrin expression. αvß6 integrins activate the profibrotic cytokine Transforming Growth Factor ß1 (TGFß1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where αvß6 mediated TGFß activation has been implicated. Here we identify that Elk1-knockout (Elk1-/0) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1-/0 aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1-/0 animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1 in human disease we exposed human lung epithelial cells and murine lung slices to cigarette smoke extract, which lead to reduced ELK1 expression andmay explain the loss of ELK1 in human disease. These data support a fundamental role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.