Placeholder Nucleosomes Underlie Germline-to-Embryo DNA Methylation Reprogramming.

The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.

The chromatin source-sink hypothesis: a shared mode of chromatin-mediated regulations.

We propose that several chromatin-mediated regulatory processes are dominated by source-sink relationships in which factors operate as 'sources' to produce or provide a resource and compete with each other to occupy separate 'sinks'. In this model, large portions of genomic DNA operate as 'sinks', which are filled by 'sources', such as available histone variants, covalent modifications to histones, the readers of these modifications and non-coding RNAs. Competing occupation for the sinks by different sources leads to distinct states of genomic equilibrium in differentiated cells. During dynamic developmental events, such as sexual reproduction, we propose that dramatic and rapid reconfiguration of source-sink relationships modifies chromatin states. We envision that re-routing of sources could occur by altering the dimensions of the sink, by reconfiguration of existing sink occupation or by varying the size of the source, providing a central mechanism to explain a plethora of epigenetic phenomena, which contribute to phenotypic variegation, zygotic genome activation and nucleolar dominance.

Competition for H2A.Z underlies the developmental impacts of repetitive element de-repression.

The histone variant H2A.Z is central to early embryonic development, determining transcriptional competency through chromatin regulation of gene promoters and enhancers. In addition to genic loci, we find that H2A.Z resides at a subset of evolutionarily young repetitive elements, including DNA transposons, long interspersed nuclear elements and long terminal repeats, during early zebrafish development. Moreover, increases in H2A.Z occur when repetitive elements become transcriptionally active. Acquisition of H2A.Z corresponds with a reduction in the levels of the repressive histone modification H3K9me3 and a moderate increase in chromatin accessibility. Notably, however, de-repression of repetitive elements also leads to a significant reduction in H2A.Z over non-repetitive genic loci. Genic loss of H2A.Z is accompanied by transcriptional silencing at adjacent coding sequences, but remarkably, these impacts are mitigated by augmentation of total H2A.Z protein via transgenic overexpression. Our study reveals that levels of H2A.Z protein determine embryonic sensitivity to de-repression of repetitive elements, that repetitive elements can function as a nuclear sink for epigenetic factors and that competition for H2A.Z greatly influences overall transcriptional output during development. These findings uncover general mechanisms in which counteractive biological processes underlie phenotypic outcomes.

The Implant-Induced Foreign Body Response Is Limited by CD13-Dependent Regulation of Ubiquitination of Fusogenic Proteins.

Implanted medical devices, from artificial heart valves and arthroscopic joints to implantable sensors, often induce a foreign body response (FBR), a form of chronic inflammation resulting from the inflammatory reaction to a persistent foreign stimulus. The FBR is characterized by a subset of multinucleated giant cells (MGCs) formed by macrophage fusion, the foreign body giant cells (FBGCs), accompanied by inflammatory cytokines, matrix deposition, and eventually deleterious fibrotic implant encapsulation. Despite efforts to improve biocompatibility, implant-induced FBR persists, compromising the utility of devices and making efforts to control the FBR imperative for long-term function. Controlling macrophage fusion in FBGC formation presents a logical target to prevent implant failure, but the actual contribution of FBGCs to FBR-induced damage is controversial. CD13 is a molecular scaffold, and in vitro induction of CD13KO bone marrow progenitors generates many more MGCs than the wild type, suggesting that CD13 regulates macrophage fusion. In the mesh implant model of FBR, CD13KO mice produced significantly more peri-implant FBGCs with enhanced TGF-ß expression and increased collagen deposition versus the wild type. Prior to fusion, increased protrusion and microprotrusion formation accompanies hyperfusion in the absence of CD13. Expression of fusogenic proteins driving cell-cell fusion was aberrantly sustained at high levels in CD13KO MGCs, which we show is due to a novel CD13 function, to our knowledge, regulating ubiquitin/proteasomal protein degradation. We propose CD13 as a physiologic brake limiting aberrant macrophage fusion and the FBR, and it may be a novel therapeutic target to improve the success of implanted medical devices. Furthermore, our data directly implicate FBGCs in the detrimental fibrosis that characterizes the FBR.

Anp32e protects against accumulation of H2A.Z at Sox motif containing promoters during zebrafish gastrulation.

Epigenetic regulation of chromatin states is crucial for proper gene expression programs and progression during development, but precise mechanisms by which epigenetic factors influence differentiation remain poorly understood. Here we find that the histone variant H2A.Z accumulates at Sox motif-containing promoters during zebrafish gastrulation while neighboring genes become transcriptionally active. These changes coincide with reduced expression of anp32e, the H2A.Z histone removal chaperone, suggesting that loss of Anp32e may lead to increases in H2A.Z binding during differentiation. Remarkably, genetic removal of Anp32e in embryos leads to H2A.Z accumulation prior to gastrulation and developmental genes become precociously active. Accordingly, H2A.Z accumulation occurs most extensively at Sox motif-associated genes, including many which are normally activated following gastrulation. Altogether, our results provide compelling evidence for a mechanism in which Anp32e preferentially restricts H2A.Z accumulation at Sox motifs to regulate the initial phases of developmental differentiation in zebrafish.

The Transplant Bellwether: Endothelial Cells in Antibody-Mediated Rejection.

Ab-mediated rejection of organ transplants remains a stubborn, frequent problem affecting patient quality of life, graft function, and grant survival, and for which few efficacious therapies currently exist. Although the field has gained considerable knowledge over the last two decades on how anti-HLA Abs cause acute tissue injury and promote inflammation, there has been a gap in linking these effects with the chronic inflammation, vascular remodeling, and persistent alloimmunity that leads to deterioration of graft function over the long term. This review will discuss new data emerging over the last 5 y that provide clues into how ongoing Ab-endothelial cell interactions may shape vascular fate and propagate alloimmunity in organ transplants.

Splice factor polypyrimidine tract-binding protein 1 (Ptbp1) primes endothelial inflammation in atherogenic disturbed flow conditions.

NF-κB-mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through adhesion molecules Icam1 and Vcam1. The endothelium is primed for cytokine activation of NF-κB by exposure to low and disturbed blood flow (LDF)but the molecular underpinnings are not fully understood. In an experimental in vivo model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NF-κB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam1 induction by tumor necrosis factor (TNF)-α stimulation as a readout, we performed a CRISPR Cas9 knockout screen and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no effect on cell growth or response to apoptotic stimuli, but reversed LDF splicing patterns and inhibited NF-κB nuclear translocation and transcriptional activation of downstream targets, including Icam1 and Vcam1. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque. In vivo, endothelial-specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice, limiting atherosclerosis. This may be mediated, in part, by allowing inclusion of a conserved alternative exon in Ripk1 leading to a reduction in Ripk1 protein. Our data show that Ptbp1, which is induced in a subset of the endothelium by platelet recruitment at regions of LDF, is required for priming of the endothelium for subsequent NF-κB activation, myeloid cell recruitment and atherosclerosis.

Development of respiratory care guidelines for Duchenne muscular dystrophy in the UK: key recommendations for clinical practice.

Significant inconsistencies in respiratory care provision for Duchenne muscular dystrophy (DMD) are reported across different specialist neuromuscular centres in the UK. The absence of robust clinical evidence and expert consensus is a barrier to the implementation of care recommendations in public healthcare systems as is the need to increase awareness of key aspects of care for those living with DMD. Here, we provide evidenced-based and/or consensus-based best practice for the respiratory care of children and adults living with DMD in the UK, both as part of routine care and in an emergency. METHODOLOGY: Initiated by an expert working group of UK-based respiratory physicians (including British Thoracic Society (BTS) representatives), neuromuscular clinicians, physiotherapist and patient representatives, draft guidelines were created based on published evidence, current practice and expert opinion. After wider consultation with UK respiratory teams and neuromuscular services, consensus was achieved on these best practice recommendations for respiratory care in DMD. RESULT: The resulting recommendations are presented in the form of a flow chart for assessment and monitoring, with additional guidance and a separate chart setting out key considerations for emergency management. The recommendations have been endorsed by the BTS. CONCLUSIONS: These guidelines provide practical, reasoned recommendations for all those managing day-to-day and acute respiratory care in children and adults with DMD. The hope is that this will support patients and healthcare professionals in accessing high standards of care across the UK.

Generation, Characterization and Reactivity of a High-Valent Mononuclear Cobalt(IV)-Diazide Complex.

High-valent Fe(IV)=O intermediates of metalloenzymes have inspired numerous efforts to generate synthetic analogs to mimic and understand their substrate oxidation reactivities. However, high-valent M(IV) complexes of late transition metals are rare. We have recently reported a novel Co(IV)-dinitrate complex (1-NO3) that activates sp3 C-H bonds up to 87 kcal/mol. In this work, we have shown that the nitrate ligands in 1-NO3 can be replaced by azide, a more basic coordinating base, resulting in the formation of a more potent Co(IV)-diazide species (1-N3) that reacts with substrates (hydrocarbons and phenols) at faster rate constants and activates stronger C-H bonds than the parent complex 1-NO3. We have characterized 1-N3 employing a combination of spectroscopic and computational approaches. Our results clearly show that the coordination of azide leads to the modulation of the Co(IV) electronic structure and the Co(IV/III) redox potential. Together with the higher basicity of azide, these thermodynamic parameters contribute to the higher driving forces of 1-N3 than 1-NO3 for C-H bond activation. Our discoveries are thus insightful for designing more reactive bio-inspired high-valent late transition metal complexes for activating inert aliphatic hydrocarbons.

Patient Perspectives in the Diagnosis and Treatment of Gallstone Disease: A Qualitative Analysis.

INTRODUCTION: Gallstone disease is one of the most common surgical diagnoses in the United States. Notably absent from the literature is the patient's perspective on priorities in management. Understanding patient values will assist surgeons and systems in achieving high-quality, patient-focused care for biliary disease. METHODS: Patients who underwent elective or urgent cholecystectomy were invited to participate in a semistructured interview to assess their experience. Interviews were performed over the phone or in person and recordings were transcribed. Each transcription was analyzed independently by two authors using the MAXQDA software, and a mixed deductive-inductive approach was used to develop themes. Anonymized quotes were used to illustrate themes and subthemes regarding the patient's experiences and priorities surrounding gallstone disease. RESULTS: A total of 29 interviews were completed. Most participants were female, but represented a diverse racial and educational group. The most common diagnosis was acute cholecystitis (48%), and 76% of patients underwent an emergency operation. Patients indicated that their main priority regarding treatment was prompt pain control with definitive management so they could return to their previous quality of life. Most patients wanted face-to-face communication with the surgical team both pre and postoperatively. Patients wished they had more information about postoperative care and expectations in the preoperative setting. CONCLUSIONS: Patients' priorities in their care for gallstone disease are centered around definitive management and quality of life. Improvements in communication were identified regarding meeting the surgeon, and postoperative communication. These results can inform surgeons how to prioritize patient perspectives in an acute care surgical system that was not designed with patient input.