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Background: Heart failure with reduced ejection fraction (HFrEF) remains a significant public health issue, with the disease advancing despite neurohormonal antagonism. Energetic dysfunction is a likely contributor to residual disease progression, and we have previously reported a strong association of plasma metabolite profiles with survival among patients with HFrEF. However, the genetic and biologic mechanisms that underlie the metabolite-survival association in HFrEF were uncertain., Methods and Results: We performed genetic mapping of the key metabolite parameters, followed by mediation analyses of metabolites and genotypes on survival, and genetic pathway analyses. Patients with HFrEF ( n  = 1,003) in the Henry Ford Pharmacogenomic Registry (HFPGR; 500 self-reported Black/African race patients [AA], 503 self-reported White/European race patients [EA], and 249 deaths over a median of 2.7 years) with genome-wide genotyping and targeted metabolomic profiling of plasma were included. We tested genome-wide association (GWA) of single nucleotide polymorphisms (SNPs) with the prognostic metabolite profile (PMP) and its components; first stratified by race, and then combined via meta-analysis for the entire cohort. Seven independent loci were identified as GWA significant hits in AA patients (3 for PMP and 4 for individual metabolites), one of which was also significant in the entire cohort (rs944469). No genome wide significant hits were found in White/EA patients. Among these SNPs, only rs35792152, (a hit for 3.HBA) tended to be associated with mortality in standard survival analysis (HR = 1.436, p  = 0.052). The mediation analyses indicated several significant associations between SNPs, metabolites, and mortality in AA patients. Functional annotation mapping (FUMA) implicated inflammation, DNA metabolic, and mRNA splicing processes., Conclusions: GWAS of key metabolites and survival along with FUMA pathway analysis revealed new candidate genes which unveiled molecular pathways that contribute to HF disease progression via metabolic and energetic abnormalities., Competing Interests: DL has received research funding or support from Akros, Astra Zeneca, Eli Lilly, Pfizer, Illumina, Janssen and SomaLogic, and has acted as consultant to ACI Clinical (Abbott Laboratories), AstraZeneca, Cytokinetics, Duke Clinical Research Institute (CONNECT-HF), Illumina, Janssen, Martin Pharmaceuticals, Ortho Clinical Diagnostics, Otsuka, and ARMGO. HS has received research funding from Novartis, and Impulse Dynamics Inc. and is consultant to Novartis, Impulse Dynamics, Vascular Dynamics, and ViCardia. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Revathi Venkateswaran, She, Gui, Luzum, Bryson, Malouf, Williams, Sabbah, Gardell and Lanfear.)

Background: Prostaglandin E2 acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that activation of the EP3 receptor reduces cardiac contractility, and its expression increases after a myocardial infarction (MI), mediating the reduction in cardiac function. In contrast, cardiac overexpression of the EP4 receptor in MI substantially improves cardiac function. Moreover, we recently reported that mice overexpressing EP3 have heart failure under basal conditions and worsened cardiac function after MI. Thus, the deleterious effects of the prostaglandin E2 EP receptors in the heart are mediated via its EP3 receptor. We, therefore, hypothesized that cardiomyocyte-specific knockout (CM-EP3 KO) or antagonism of the EP3 receptor protects the heart after MI., Methods: To test our hypothesis, we made the novel CM-EP3 KO mouse and subjected CM-EP3 KO or controls to sham or MI surgery for 2 weeks. In separate experiments, C57BL/6 mice were subjected to 2 weeks of MI and treated with either the EP3 antagonist L798 106 or vehicle starting 3 days post-MI., Results: CM-EP3 KO significantly prevented a decline in cardiac function after MI compared with WT animals and prevented an increase in hypertrophy and fibrosis. Excitingly, mice treated with L798 106 3 days after MI had significantly better cardiac function compared with vehicle-treated mice., Conclusions: Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function, and pharmaceutical targeting of the EP3 receptor may be a therapeutic option in the treatment of heart failure., Competing Interests: Disclosures None.

Prostaglandin E2 (PGE2) signals differently through 4 receptor subtypes (EP1-EP4) to elicit diverse physiologic/pathologic effects. We previously reported that PGE2 via its EP3 receptor reduces cardiac contractility and male mice with cardiomyocyte-specific deletion of the EP4 receptor (EP4 KO) develop dilated cardiomyopathy. The aim of this study was to identify pathways responsible for this phenotype. We performed ingenuity pathway analysis (IPA) and found that genes differentiating WT mice and EP4 KO mice were significantly overrepresented in mitochondrial (adj. p value = 6.28 × 10 -26 ) and oxidative phosphorylation (adj. p value = 1.58 × 10 -27 ) pathways. Electron microscopy from the EP4 KO hearts show substantial mitochondrial disarray and disordered cristae. Not surprisingly, isolated adult mouse cardiomyocytes (AVM) from these mice have reduced ATP levels compared to their WT littermates and reduced expression of key genes involved in the electron transport chain (ETC) in older mice. Moreover, treatment of AVM from C57Bl/6 mice with PGE2 or the EP3 agonist sulprostone resulted in changes of various genes involved in the ETC, measured by the Mitochondrial Energy Metabolism RT 2 -profiler assay. Lastly, the EP4 KO mice have reduced expression of superoxide dismuatse-2 (SOD2), whereas treatment of AVM with PGE2 or sulprostone increase superoxide production, suggesting increased oxidative stress levels in these EP4 KO mice. Altogether the current study supports the premise that PGE2 acting via its EP4 receptor is protective, while signaling through its other receptors, likely EP3, is deleterious., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

This review article summarizes the role of prostaglandin E 2 (PGE 2 ) and its receptors (EP1-EP4) as it relates to the inflammatory cardiomyopathy, myocarditis. During the COVID-19 pandemic, the onset of myocarditis in a subset of patients prompted a debate on the use of nonsteroidal anti-inflammatory drugs (NSAIDs), like ibuprofen, which act to inhibit the actions of prostaglandins. This review aims to further understanding of the role of PGE 2 in the pathogenesis or protection of the myocardium in myocarditis. Inflammatory cardiomyopathies encompass a broad spectrum of disorders, all characterized by cardiac inflammation. Therefore, for the purpose of this review, the authors have placed particular emphasis on etiologies of myocarditis where effects of PGE 2 have been documented., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Aims: Prostaglandin E2 (PGE2) is a lipid hormone that signals through 4 different G-protein coupled receptor subtypes which act to regulate key physiological processes. Our laboratory has previously reported that PGE2 through its EP3 receptor reduces cardiac contractility at the level of isolated cardiomyocytes and in the isolated working heart preparation. We therefore hypothesized that cardiomyocyte specific overexpression of the PGE2 EP3 receptor further decreases cardiac function in a mouse model of heart failure produced by myocardial infarction., Main Methods: Our study tested this hypothesis using EP3 transgenic mice (EP3 TG), which overexpress the porcine analogue of human EP3 in the cardiomyocytes, and their wildtype (WT) littermates. Mice were analyzed 2 wks after myocardial infarction (MI) or sham operation by echocardiography, RT-PCR, immunohistochemistry, and histology., Key Findings: We found that the EP3 TG sham controls had a reduced ejection fraction, reduced fractional shortening, and an increased left ventricular dimension at systole and diastole compared to the WT sham controls. Moreover, there was a further reduction in the EP3 TG mice after myocardial infarction. Additionally, single-cell analysis of cardiomyocytes isolated from EP3 TG mice showed reduced contractility under basal conditions. Overexpression of EP3 significantly increased cardiac hypertrophy, interstitial collagen fraction, macrophage, and T-cell infiltration in the sham operated group. Interestingly, after MI, there were no changes in hypertrophy but there were changes in collagen fraction, and inflammatory cell infiltration., Significance: Overexpression of EP3 reduces cardiac function under basal conditions and this is exacerbated after myocardial infarction., Competing Interests: Declaration of competing interest No conflicts of interest, financial or otherwise, are declared by the authors., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Brain injury is the most common cause of death for patients resuscitated from cardiac arrest. Magnesium is an attractive neuroprotective compound which protects neurons from ischemic injury by reducing neuronal calcium overload via NMDA receptor modulation and preventing calcium-induced mitochondrial permeability transition. Intramuscular (IM) delivery of MgSO 4 during CPR has the potential to target these mechanisms within an early therapeutic window. We hypothesize that IM MgSO 4 administrated during CPR could achieve therapeutic serum magnesium levels within 15 min after ROSC and improve neurologic outcomes in a rat model of asphyxial cardiac arrest. Male Long Evans rats were subjected to 8-min asphyxial cardiac arrest and block randomized to receive placebo, 107 mg/kg, 215 mg/kg, or 430 mg/kg MgSO 4 IM at the onset of CPR. Serum magnesium concentrations increased rapidly with IM delivery during CPR, achieving twofold to fourfold increase by 15 min after ROSC in all magnesium dose groups. Rats subjected to cardiac arrest or sham surgery were block randomized to treatment groups for assessment of neurological outcomes. We found that IM MgSO 4 during CPR had no effect on ROSC rate (p > 0.05). IM MgSO 4 treatment had no statistically significant effect on 10-day survival with good neurologic function or hippocampal CA1 pyramidal neuron survival compared to placebo treatment. In conclusion, a single dose IM MgSO 4 during CPR achieves up to fourfold baseline serum magnesium levels within 15 min after ROSC; however, this treatment strategy did not improve survival, recovery of neurologic function, or neuron survival. Future studies with repeated dosing or in combination with hypothermic targeted temperature management may be indicated., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This review article provides an update for the role of prostaglandin E2 receptors (EP1, EP2, EP3 and EP4) in cardiovascular disease. Where possible we have reported citations from the last decade although this was not possible for all of the topics covered due to the paucity of publications. The authors have attempted to cover the subjects of ischemia-reperfusion injury, arrhythmias, hypertension, novel protein binding partners of the EP receptors and their pathophysiological significance, and cardiac regeneration. These latter two topics bring studies of the EP receptors into new and exciting areas of research that are just beginning to be explored. Where there is peer-reviewed literature, the authors have placed particular emphasis on clinical studies although these are limited in number., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: It remains unclear whether the plasma proteome adds value to established predictors in heart failure (HF) with reduced ejection fraction (HFrEF). We sought to derive and validate a plasma proteomic risk score (PRS) for survival in patients with HFrEF (HFrEF-PRS)., Methods: Patients meeting Framingham criteria for HF with EF<50% were enrolled (N=1017) and plasma underwent SOMAscan profiling (4453 targets). Patients were randomly divided 2:1 into derivation and validation cohorts. The HFrEF-PRS was derived using Cox regression of all-cause mortality adjusted for clinical score and NT-proBNP (N-terminal pro-B-type natriuretic peptide), then was tested in the validation cohort. Risk stratification improvement was evaluated by C statistic, integrated discrimination index, continuous net reclassification index, and median improvement in risk score for 1-year and 3-year mortality., Results: Participants' mean age was 68 years, 48% identified as Black, 35% were female, and 296 deaths occurred. In derivation (n=681), 128 proteins associated with mortality, 8 comprising the optimized HFrEF-PRS. In validation (n=336) the HFrEF-PRS associated with mortality (hazard ratio, 2.27 [95% CI, 1.84-2.82], P =6.3×10 -14 ), Kaplan-Meier curves differed significantly between HFrEF-PRS quartiles ( P =2.2×10 -6 ), and it remained significant after adjustment for clinical score and NT-proBNP (hazard ratio, 1.37 [95% CI, 1.05-1.79], P =0.021). The HFrEF-PRS improved metrics of risk stratification (C statistic change, 0.009, P =0.612; integrated discrimination index, 0.041, P =0.010; net reclassification index=0.391, P =0.078; median improvement in risk score=0.039, P =0.016) and associated with cardiovascular death and HF phenotypes (eg, 6-minute walk distance, EF change). Most HFrEF-PRS proteins had little known connection to HFrEF., Conclusions: A plasma multiprotein score improved risk stratification in patients with HFrEF and identified novel candidates.

Mitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

The mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

Study Objective: To create a preoperative predictive model for prolonged post-anaesthesia care unit (PACU) stay for outpatient surgery and compare with an existing (University of California-San Diego, UCSD) model., Design: Retrospective observational study., Setting: Post-anaesthesia care unit. Patients: Outpatient surgical patients discharged on the same day in a large academic institution. Preoperative data were collected. The study period was three months in 2016. Measurements: Prolonged PACU stay defined as a length of stay longer than the third quartile. We utilized multivariate regression analyses and bootstrapping statistical techniques to create a predictive model for prolonged PACU stay. Main results: Four strong predictors for prolonged PACU stay: general anaesthesia, obstructive sleep apnoea, surgical specialty and scheduled case duration. Our model had an excellent discrimination performance and a good calibration., Conclusion: We developed a predictive model for prolonged PACU stay in our institution. This model is different from the UCSD model probably secondary to local and regional differences in outpatient surgery practice. Therefore, individual practice study outcomes may not apply to other practices without careful consideration of these differences.

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E 2 (PGE 2 ) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP 3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP 4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP 3 and EP 4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP 3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP 3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP 3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP 3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP 3 -transgenic mice, which overexpress EP 3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP 3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP 3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN. NEW & NOTEWORTHY This study is the first to show that systemic treatment with an EP 3 receptor antagonist (L798,106) attenuates the angiotensin II-induced increase in blood pressure in mice. The results from this project could complement existing hypertension therapies by combining blockade of the EP 3 receptor with antihypertensive drugs.

Background: Prostaglandin E2 (PGE 2 ) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We have previously reported that mice lacking the EP4 receptor in the cardiomyocytes develop heart failure with a phenotype of dilated cardiomyopathy. Also, these mice have increased levels of chemokines, like MCP-5, in their left ventricles. We have recently reported that overexpression of the EP4 receptor could improve cardiac function in the myocardial infarction model. Furthermore, we showed that overexpression of EP4 had an anti-inflammatory effect in the whole left ventricle. It has also been shown that PGE 2 can antagonize lipopolysaccharide-induced secretion of chemokines/cytokines in various cell types. We therefore hypothesized that PGE 2 inhibits lipopolysaccharide (LPS)-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor., Methods and Results: Our hypothesis was tested using isolated mouse adult ventricular fibroblasts (AVF) treated with LPS. Pre-treatment of the cells with PGE 2 and the EP4 agonist CAY10598 resulted in reductions of the pro-inflammatory response induced by LPS. Specifically, we observed reductions in MCP-5 secretion. Western blot analysis showed reductions in phosphorylated Akt and IκBα indicating reduced NF-κB activation. The anti-inflammatory effects of PGE 2 and EP4 agonist signaling appeared to be independent of cAMP, p-44/42, or p38 pathways., Conclusion: Exogenous treatment of PGE 2 and the EP4 receptor agonist blocked the pro-inflammatory actions of LPS. Mechanistically, this was mediated via reduced Akt phosphorylation and inhibition of NF-κB., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Micrococcal nuclease (MNase) is widely used to map nucleosomes. However, its aggressive endo-/exo-nuclease activities make MNase-seq unreliable for determining nucleosome occupancies, because cleavages within linker regions produce oligo- and mono-nucleosomes, whereas cleavages within nucleosomes destroy them. Here, we introduce a theoretical framework for predicting nucleosome occupancies and an experimental protocol with appropriate spike-in normalization that confirms our theory and provides accurate occupancy levels over an MNase digestion time course. As with human cells, we observe no overall differences in nucleosome occupancies between Drosophila euchromatin and heterochromatin, which implies that heterochromatic compaction does not reduce MNase accessibility of linker DNA.

Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling., Competing Interests: MM, TB, JH, SH No competing interests declared, (© 2019, Meers et al.)

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.

Background: Prostaglandin E2 (PGE 2 ) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We recently reported that PGE 2 via its EP3 receptor could reduce cardiac contractility of isolated myocytes and the working heart preparation. We thus hypothesized that there is an imbalance in the EP3/EP4 ratio towards EP3 in the failing heart and that overexpression of EP4 in a mouse model of heart failure would improve cardiac function., Methods and Results: Our hypothesis was tested in a mouse model of myocardial infarction (MI) with the use of AAV9-EP4 driven by the myosin heavy chain promoter to overexpress EP4 in the cardiac myocytes. Echocardiography was performed to assess cardiac function. We found that overexpression of EP4 improved shortening fraction (p = 0.0025), ejection fraction (p = 0.0003), and reduced left ventricular dimension at systole (p = 0.0013). Overexpression of EP4 also significantly reduced indices of cardiac hypertrophy and interstitial collagen fraction. Animals treated with AAV9-EP4 also had a significant decrease in TNFα mRNA expression and in the number of macrophages and T cells migrated post MI coupled with a reduction in the expression of iNOS., Conclusion: Overexpression of EP4 improves cardiac function post MI. This may be mediated through reductions in adverse cardiac remodeling or via inhibition of cytokine/chemokine production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We developed a chemical cleavage method that releases single nucleosome dyad-containing fragments, allowing us to precisely map both single nucleosomes and linkers with high accuracy genome-wide in yeast. Our single nucleosome positioning data reveal that nucleosomes occupy preferred positions that differ by integral multiples of the DNA helical repeat. By comparing nucleosome dyad positioning maps to existing genomic and transcriptomic data, we evaluated the contributions of sequence, transcription, and histones H1 and H2A.Z in defining the chromatin landscape. We present a biophysical model that neglects DNA sequence and shows that steric occlusion suffices to explain the salient features of nucleosome positioning.

Even the most innovative healthcare technologies provide patient benefits only when adopted by clinicians and/or patients in actual practice. Yet realizing optimal positive impact from a new technology for the widest range of individuals who would benefit remains elusive. In software and new product development, iterative rapid-cycle "agile" methods more rapidly provide value, mitigate failure risks, and adapt to customer feedback. Co-development between builders and customers is a key agile principle. But how does one accomplish co-development with busy clinicians? In this paper, we discuss four practical agile co-development practices found helpful clinically: (1) User stories for lightweight requirements; (2) Time-boxed development for collaborative design and prompt course correction; (3) Automated acceptance test driven development, with clinician-vetted specifications; and (4) Monitoring of clinician interactions after release, for rapid-cycle product adaptation and evolution. In the coming wave of innovation in healthcare apps ushered in by open APIs to EHRs, learning rapidly what new product features work well for clinicians and patients will become even more crucial.

In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence. DOI: http://dx.doi.org/10.7554/eLife.01861.001.

The expression and genome-wide mapping of epitope-tagged DNA- and chromatin-binding proteins in cultured cells has become a powerful strategy for epigenome characterization, especially in Drosophila, where cell lines derived from numerous tissues are now available. However this strategy relies on establishing transfected cell lines, which is time-consuming and introduces variability. Here we show that baculovirus-encoded proteins can be efficiently produced following infection of Drosophila cell lines of different types. Using chromatin affinity purification, we show that epitope-tagged proteins produced in baculovirus-infected cells provide genome-wide profiles of the histone variant H2Av that are comparable to those produced by plasmid-transfected cells. The ability to express multiple epitope-tagged proteins for epigenome analysis from a single culture, and to do this in a variety of Drosophila cell lines, significantly extends the range of epigenome analysis.

Background: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known., Results: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants., Conclusion: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.

Background: Centromeres represent the last frontiers of plant and animal genomics. Although they perform a conserved function in chromosome segregation, centromeres are typically composed of repetitive satellite sequences that are rapidly evolving. The nucleosomes of centromeres are characterized by a special H3-like histone (CenH3), which evolves rapidly and adaptively in Drosophila and Arabidopsis. Most plant, animal and fungal centromeres also bind a large protein, centromere protein C (CENP-C), that is characterized by a single 24 amino-acid motif (CENPC motif)., Results: Whereas we find no evidence that mammalian CenH3 (CENP-A) has been evolving adaptively, mammalian CENP-C proteins contain adaptively evolving regions that overlap with regions of DNA-binding activity. In plants we find that CENP-C proteins have complex duplicated regions, with conserved amino and carboxyl termini that are dissimilar in sequence to their counterparts in animals and fungi. Comparisons of Cenpc genes from Arabidopsis species and from grasses revealed multiple regions that are under positive selection, including duplicated exons in some grasses. In contrast to plants and animals, yeast CENP-C (Mif2p) is under negative selection., Conclusions: CENP-Cs in all plant and animal lineages examined have regions that are rapidly and adaptively evolving. To explain these remarkable evolutionary features for a single-copy gene that is needed at every mitosis, we propose that CENP-Cs, like some CenH3s, suppress meiotic drive of centromeres during female meiosis. This process can account for the rapid evolution and the complexity of centromeric DNA in plants and animals as compared to fungi.

Lung samples from pneumonic lesions in cattle and goats, naturally or experimentally infected with strains of the Mycoplasma mycoides cluster, were fixed in formalin and embedded in paraffin. An immunohistochemical technique using monoclonal or polyclonal antibodies was performed on tissue sections in order to detect Mycoplasma antigens. Four monoclonal antibodies (MAbs), one (2A3) raised against M. mycoides ssp. mycoides small colony (SC) and large colony (LC), two (1D3 and 5E5) against M. mycoides ssp. capri, and one (5A10) against M. bovis, were used. A range of polyclonal antibodies, raised to the individual subspecies of the M. mycoides cluster, and one to Pasteurella haemolytica, was also used. The MAb 2A3 showed positive immunostaining in lung sections from cattle and goats naturally and experimentally infected with M. mycoides ssp. mycoides SC and LC, but not with pneumonic lesions of cattle and goats due to other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 1D3 showed immunostaining in lung sections from goats naturally and experimentally infected with M. mycoides ssp. capri, but again not with pneumonic lesions caused by other members of the M. mycoides cluster, M. bovis or Pasteurella spp. The MAb 5E5 immunoreacted in sections from pneumonic lesions from all animals infected with one of the three M. mycoides cluster subspecies used in the study, but not with M. bovis or Pasteurella infected tissue. Immunoreaction was mainly found in the cell debris around necrotic areas, as well as in macrophages, neutrophils and epithelial cells. The localization of antigens of the M. mycoides cluster using polyclonal antisera followed basically the same pattern as that obtained with the monoclonals. However, a wide cross reactivity was found between different antisera and relatively high background immunostaining was also seen, especially in necrotic areas. The results suggest that immunohistochemical methods using monoclonal antibodies are useful tools for the diagnosis and study of the pathogenesis of pneumonia caused by the Mycoplasmas of the M. mycoides cluster.