Genome-Nuclear Lamina Interactions Regulate Cardiac Stem Cell Lineage Restriction.

Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin, and independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.

Targeting cardiac fibrosis with engineered T cells.

Fibrosis is observed in nearly every form of myocardial disease1. Upon injury, cardiac fibroblasts in the heart begin to remodel the myocardium by depositing excess extracellular matrix, resulting in increased stiffness and reduced compliance of the tissue. Excessive cardiac fibrosis is an important factor in the progression of various forms of cardiac disease and heart failure2. However, clinical interventions and therapies that target fibrosis remain limited3. Here we demonstrate the efficacy of redirected T cell immunotherapy to specifically target pathological cardiac fibrosis in mice. We find that cardiac fibroblasts that express a xenogeneic antigen can be effectively targeted and ablated by adoptive transfer of antigen-specific CD8+ T cells. Through expression analysis of the gene signatures of cardiac fibroblasts obtained from healthy and diseased human hearts, we identify an endogenous target of cardiac fibroblasts-fibroblast activation protein. Adoptive transfer of T cells that express a chimeric antigen receptor against fibroblast activation protein results in a significant reduction in cardiac fibrosis and restoration of function after injury in mice. These results provide proof-of-principle for the development of immunotherapeutic drugs for the treatment of cardiac disease.

Author Correction: Targeting cardiac fibrosis with engineered T cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

FLT3-TKD Measurable Residual Disease Detection Using Droplet Digital PCR and Clinical Applications in Acute Myeloid Leukemia.

The tyrosine kinase domain of the FMS-Like tyrosine kinase 3 (FLT3-TKD) is recurrently mutated in acute myeloid leukemia (AML). Common molecular techniques used in its detection include PCR and capillary electrophoresis, Sanger sequencing and next-generation sequencing with recognized sensitivity limitations. This study aims to validate the use of droplet digital PCR (ddPCR) in the detection of measurable residual disease (MRD) involving the common FLT3-TKD mutations (D835Y, D835H, D835V, D835E). Twenty-two diagnostic samples, six donor controls, and a commercial D835Y positive control were tested using a commercial Bio-rad® ddPCR assay. All known variants were identified, and no false positives were detected in the wild-type control (100% specificity and sensitivity). The assays achieved a limit of detection suitable for MRD testing at 0.01% variant allelic fraction. Serial samples from seven intensively-treated patients with FLT3-TKD variants at diagnosis were tested. Five patients demonstrated clearance of FLT3-TKD clones, but two patients had FLT3-TKD persistence in the context of primary refractory disease. In conclusion, ddPCR is suitable for the detection and quantification of FLT3-TKD mutations in the MRD setting; however, the clinical significance and optimal management of MRD positivity require further exploration.

Glucocorticoid prescribing in neurology.

Glucocorticoids are commonly used for neurological disorders, but they can have significant adverse effects, including adrenal insufficiency, hyperglycaemia, osteoporosis and increased infection risk. Long-term use of corticosteroids requires the prescriber to plan risk mitigation, including monitoring and often coprescribing. This article highlights the potential risks of corticosteroid prescribing and draws together up-to-date guidance with multispecialty input to clarify ways of reducing those risks. We discuss home blood glucose monitoring and consider a steroid safety checklist to promote safer steroid prescribing.

Experiences of implementing the 'Making Every Contact Count' initiative into a UK integrated care system: an interview study.

BACKGROUND: The 'Making Every Contact Count' (MECC) approach is in line with the current National Health Service (NHS) strategy to improve and prevent health conditions in England. Despite its importance and value for preventative healthcare, implementation of MECC varies. The aim of this study was to explore the barriers and facilitators of implementing MECC and MECC training into an integrated care system (ICS). METHODS: Remote semi-structured interviews were conducted with staff across an ICS in the North West of England who were involved in implementing and delivering MECC across the region. Data were analysed initially using an inductive thematic analysis approach and then interpreted using the 'Capability, Opportunity, Motivation = Behaviour' (COM-B) model of behaviour change. RESULTS: We interviewed nine stakeholders and identified three superordinate themes: (1) macro-level barriers and facilitators, e.g. funding; (2) organizational level barriers and facilitators, e.g. time and resource; and (3) individual-level barriers/facilitators for both MECC trainers and MECC agents. CONCLUSIONS: MECC has potential to meet the needs of the public's health, but barriers to its implementation exist. MECC must be successfully embedded into organizations and regions in which it is implemented, which relies on further development of an appropriate infrastructure including sustainable funding and a shift in culture to value preventative healthcare.

The nuclear periphery is a scaffold for tissue-specific enhancers.

Nuclear architecture influences gene regulation and cell identity by controlling the three-dimensional organization of genes and their distal regulatory sequences, which may be far apart in linear space. The genome is functionally and spatially segregated in the eukaryotic nucleus with transcriptionally active regions in the nuclear interior separated from repressive regions, including those at the nuclear periphery. Here, we describe the identification of a novel type of nuclear peripheral chromatin domain that is enriched for tissue-specific transcriptional enhancers. Like other chromatin at the nuclear periphery, these regions are marked by H3K9me2. But unlike the nuclear peripheral Lamina-Associated Domains (LADs), these novel, enhancer-rich domains have limited Lamin B interaction. We therefore refer to them as H3K9me2-Only Domains (KODs). In mouse embryonic stem cells, KODs are found in Hi-C-defined A compartments and feature relatively accessible chromatin. KODs are characterized by low gene expression and enhancers located in these domains bear the histone marks of an inactive or poised state. These results indicate that KODs organize a subset of inactive, tissue-specific enhancers at the nuclear periphery. We hypothesize that KODs may play a role in facilitating and perhaps constraining the enhancer-promoter interactions underlying spatiotemporal regulation of gene expression programs in differentiation and development.

The YGetIt? Program: A Mobile Application, PEEP, and Digital Comic Intervention to Improve HIV Care Outcomes for Young Adults.

INTRODUCTION: In New York State (NYS), young adults account for the largest number of new human immunodeficiency virus (HIV) infections and struggle to seek and remain in HIV care. Digital interventions and access to peer support have demonstrated positive influences on the HIV care continuum and health outcomes. The New York State Department of Health (NYS DOH) developed YGetIt? (YGI) that combines a mobile application, GET!, peer navigation (PEEPs), and a compelling digital comic series, "Tested," to facilitate the timely entry of young people into HIV care, to prevent vulnerable youth from dropping out of care, and to achieve sustained viral load suppression among those in care. This article describes the development and early implementation of the YGI digital intervention. Intervention Design. GET! provided a high level of confidentiality and security, ease of access, and Wi-Fi accessibility. YGI enrolled 113 HIV-positive participants from a clinical setting who were individually randomized at a 1:1 ratio to receive access to GET! plus PEEPs (n = 53) or the app alone (n = 60). LESSONS LEARNED: For recruitment, staff and organization buy-in was essential to the success of the intervention, and building relationships was critical. GET! development was an iterative process. Peer Engagement Educator Professionals (PEEPs) who were tech savvy, representative of the priority population, and had shared life experience with participants were most impactful. Interest in apps declines over time and participants in the APP alone arm were less engaged. CONCLUSION: GET! is a communication and engagement tool that supports HIV care and may serve as a model for like digital interventions.

Recompleting the Caenorhabditis elegans genome.

Caenorhabditis elegans was the first multicellular eukaryotic genome sequenced to apparent completion. Although this assembly employed a standard C. elegans strain (N2), it used sequence data from several laboratories, with DNA propagated in bacteria and yeast. Thus, the N2 assembly has many differences from any C. elegans available today. To provide a more accurate C. elegans genome, we performed long-read assembly of VC2010, a modern strain derived from N2. Our VC2010 assembly has 99.98% identity to N2 but with an additional 1.8 Mb including tandem repeat expansions and genome duplications. For 116 structural discrepancies between N2 and VC2010, 97 structures matching VC2010 (84%) were also found in two outgroup strains, implying deficiencies in N2. Over 98% of N2 genes encoded unchanged products in VC2010; moreover, we predicted ≥53 new genes in VC2010. The recompleted genome of C. elegans should be a valuable resource for genetics, genomics, and systems biology.

Lineage-specific reorganization of nuclear peripheral heterochromatin and H3K9me2 domains.

Dynamic organization of chromatin within the three-dimensional nuclear space has been postulated to regulate gene expression and cell fate. Here, we define the genome-wide distribution of nuclear peripheral heterochromatin as a multipotent P19 cell adopts either a neural or a cardiac fate. We demonstrate that H3K9me2-marked nuclear peripheral heterochromatin undergoes lineage-specific reorganization during cell-fate determination. This is associated with spatial repositioning of genomic loci away from the nuclear periphery as shown by 3D immuno-FISH. Locus repositioning is not always associated with transcriptional changes, but a subset of genes is upregulated. Mef2c is specifically repositioned away from the nuclear periphery during early neurogenic differentiation, but not during early cardiogenic differentiation, with associated transcript upregulation. Myocd is specifically repositioned during early cardiogenic differentiation, but not during early neurogenic differentiation, and is transcriptionally upregulated at later stages of cardiac differentiation. We provide experimental evidence for lineage-specific regulation of nuclear architecture during cell-fate determination in a mouse cell line.

Evaluation of Disease Severity and Molecular Relationships Between Peronospora variabilis Isolates on Chenopodium Species in New Hampshire.

Quinoa is a potential new crop for New England; however, its susceptibility to downy mildew, caused by Peronospora variabilis, is a key obstacle for cultivation. The objectives of this study were to evaluate differential resistance within the Chenopodium genus, identify novel sources of resistance for use in future genetic studies or breeding programs, and investigate phylogenetic relationships of P. variabilis isolates from different Chenopodium hosts. The long-term goal of this research is to develop a resistant variety of quinoa to be grown in New England. Field trials conducted at the University of New Hampshire evaluated downy mildew disease severity on 10 Chenopodium accessions representing four species. Disease severity for each treatment was compared and significant differences in disease severity were observed between accessions. C. berlandieri var. macrocalycium ecotypes collected from Rye Beach, New Hampshire and Appledore Island, Maine exhibited the lowest disease severity over the growing season. P. variabilis was isolated from each accession, and COX2 sequences were compared. Phylogenetic analyses suggest no effect of host species on P. variabilis sequence similarity; however, isolates are shown to cluster by geographic location. This research provides the first step in identifying potential New England native sources of resistance to downy mildew within the genus Chenopodium and provides preliminary information needed to further investigate resistance at the genomic level in Chenopodium spp.

Histone methyltransferase activity programs nuclear peripheral genome positioning.

Spatial organization of the genome in the nucleus plays a critical role in development and regulation of transcription. A genomic region that resides at the nuclear periphery is part of the chromatin layer marked with histone H3 lysine 9 dimethyl (H3K9me2), but chromatin reorganization during cell differentiation can cause movement in and out of this nuclear compartment with patterns specific for individual cell fates. Here we describe a CRISPR-based system that allows visualization coupled with forced spatial relocalization of a target genomic locus in live cells. We demonstrate that a specified locus can be tethered to the nuclear periphery through direct binding to a dCas9-Lap2ß fusion protein at the nuclear membrane, or via targeting of a histone methyltransferase (HMT), G9a fused to dCas9, that promotes H3K9me2 labeling and localization to the nuclear periphery. The enzymatic activity of the HMT is sufficient to promote this repositioning, while disruption of the catalytic activity abolishes the localization effect. We further demonstrate that dCas9-G9a-mediated localization to the nuclear periphery is independent of nuclear actin polymerization. Our data suggest a function for epigenetic histone modifying enzymes in spatial chromatin organization and provide a system for tracking and labeling targeted genomic regions in live cells.

Genetically increasing flux through ß-oxidation in skeletal muscle increases mitochondrial reductive stress and glucose intolerance.

Elevated mitochondrial hydrogen peroxide (H2O2) emission and an oxidative shift in cytosolic redox environment have been linked to high-fat-diet-induced insulin resistance in skeletal muscle. To test specifically whether increased flux through mitochondrial fatty acid oxidation, in the absence of elevated energy demand, directly alters mitochondrial function and redox state in muscle, two genetic models characterized by increased muscle ß-oxidation flux were studied. In mice overexpressing peroxisome proliferator-activated receptor-α in muscle (MCK-PPARα), lipid-supported mitochondrial respiration, membrane potential (ΔΨm), and H2O2 production rate (JH2O2) were increased, which coincided with a more oxidized cytosolic redox environment, reduced muscle glucose uptake, and whole body glucose intolerance despite an increased rate of energy expenditure. Similar results were observed in lipin-1-deficient, fatty-liver dystrophic mice, another model characterized by increased ß-oxidation flux and glucose intolerance. Crossing MCAT (mitochondria-targeted catalase) with MCK-PPARα mice normalized JH2O2 production, redox environment, and glucose tolerance, but surprisingly, both basal and absolute insulin-stimulated rates of glucose uptake in muscle remained depressed. Also surprising, when placed on a high-fat diet, MCK-PPARα mice were characterized by much lower whole body, fat, and lean mass as well as improved glucose tolerance relative to wild-type mice, providing additional evidence that overexpression of PPARα in muscle imposes more extensive metabolic stress than experienced by wild-type mice on a high-fat diet. Overall, the findings suggest that driving an increase in skeletal muscle fatty acid oxidation in the absence of metabolic demand imposes mitochondrial reductive stress and elicits multiple counterbalance metabolic responses in an attempt to restore bioenergetic homeostasis.NEW & NOTEWORTHY Prior work has suggested that mitochondrial dysfunction is an underlying cause of insulin resistance in muscle because it limits fatty acid oxidation and therefore leads to the accumulation of cytotoxic lipid intermediates. The implication has been that therapeutic strategies to accelerate ß-oxidation will be protective. The current study provides evidence that genetically increasing flux through ß-oxidation in muscle imposes reductive stress that is not beneficial but rather detrimental to metabolic regulation.

A World of Indifference/Un Monde D'Indifférence: Canadian Women's Experiences of Psychiatric Hospitalization/Expériences Canadiennes d'hospitalization psychiatrique au Canada.

This article presents findings and analysis stemming from a two-year qualitative study that explored, in their own voices, women's lived experience of psychiatric hospitalization. Feminist Standpoint Theory and Foucauldian analysis frame and provide the moorings for validity, methodology, and analysis of this woman-centred inquiry. The verbatim representation of lived experience, coupled with the coauthoring of this article with two of the study's participants provides an authentic articulation of participant standpoint. Furthermore, this approach creates a space in nursing scholarship in which the co-development of knowledge is also made possible. Overarching themes of docility-making, harm, betrayal, indifference, and resistance are presented and situated in a broader analysis of the hegemony of bio-psychiatric discourse and its colonization of nursing practice. In order to disrupt systems that fail to meet patient needs and fail to represent nursing's values and ethos, we shed light on the possible systemic factors behind the experience of "indifference".

Endocardial Hippo signaling regulates myocardial growth and cardiogenesis.

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells.

Going for the Gold: A Description of the Centers of Excellence Designation by the Infectious Diseases Society of America.

Antimicrobial stewardship programs (ASPs) are recommended by the Centers for Disease Control and Prevention and World Health Organization and mandated by the Joint Commission to curb antimicrobial resistance. However, <50% of institutions have optimal ASPs in place. Building on its experience of antimicrobial stewardship (AMS) advocacy, the Infectious Diseases Society of America (IDSA) developed the AMS Centers of Excellence (CoE) program, which will serve as a conduit to share best practices and highlight the standards for other hospitals to achieve in order to advance the field of AMS. A designation of CoE signifies that these institutions deliver high-quality care consistently, serve as the "gold" standard for executing novel AMS principles, and demonstrate commitment to their ASP. Here, we describe the process and purpose of designating institutions as AMS CoEs, provide awareness to clinicians on opportunities available through IDSA with this CoE designation, and discuss the evolution of the program.

Analysis of Tumor Biology to Advance Cancer Health Disparity Research.

Cancer mortality rates in the United States continue to decline. Reductions in tobacco use, uptake of preventive measures, adoption of early detection methods, and better treatments have resulted in improved cancer outcomes for men and women. Despite this progress, some population groups continue to experience an excessive cancer burden when compared with other population groups. One of the most prominent cancer health disparities exists in prostate cancer. Prostate cancer mortality rates are highest among men of African ancestry when compared with other men, both in the United States and globally. This disparity and other cancer health disparities are largely explained by differences in access to health care, diet, lifestyle, cultural barriers, and disparate exposures to carcinogens and pathogens. Dietary and lifestyle factors, pathogens, and ancestry-related factors can modify tumor biology and induce a more aggressive disease. There are numerous examples of how environmental exposures, like tobacco, chronic stress, or dietary factors, induce an adverse tumor biology, leading to a more aggressive disease and decreased patient survival. Because of population differences in the exposure to these risk factors, they can be the cause of cancer disparities. In this review, we will summarize recent advances in our understanding of prostate and breast cancer disparities in the United States and discuss how the analysis of tumor biology can advance health disparity research.

Alternative acetate production pathways in Chlamydomonas reinhardtii during dark anoxia and the dominant role of chloroplasts in fermentative acetate production.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Emergence of white pine needle damage in the northeastern United States is associated with changes in pathogen pressure in response to climate change.

The defoliation of the eastern white pine (Pinus strobus) across the northeastern United States is an escalating concern threatening the ecological health of northern forests and economic vitality of the region's lumber industry. First documented in the spring of 2010 affecting 24 328 hectares in the state of Maine, white pine needle damage (WPND) has continued to spread and is now well established in all New England states. While causal agents of WPND are known, current research is lacking in both sampling distribution and the specific environmental factor(s) that affect the development and spread of this disease complex. This study aims to construct a more detailed distribution map of the four primary causal agents within the region, as well as utilize long-term WPND monitoring plots and data collected from land-based weather stations to develop a climatic model to predict the severity of defoliation events in the proceeding year. Sampling results showed a greater distribution of WPND than previously reported. WPND was generally found in forest stands that compromised >50% eastern white pine by basal area. No single species, nor a specific combination of species had a dominating presence in particular states or regions, thus supporting the disease complex theory that WPND is neither caused by an individual species nor by a specific combination of species. In addition, regional weather data confirmed the trend of increasing temperature and precipitation observed in this region with the previous year's May, June, and July rainfall being the best predictor of defoliation events in the following year. Climatic models were developed to aid land managers in predicting disease severity and accordingly adjust their management decisions. Our results clearly demonstrate the role changing climate patterns have on the health of eastern white pine in the northeastern United States.

Determinants of nucleosome organization in primary human cells.

Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.