Non-invasive genetic monitoring for the threatened valley elderberry longhorn beetle.

The valley elderberry longhorn beetle (VELB), Desmocerus californicus dimorphus (Coleoptera: Cerambycidae), is a federally threatened subspecies endemic to the Central Valley of California. The VELB range partially overlaps with that of its morphologically similar sister taxon, the California elderberry longhorn beetle (CELB), Desmocerus californicus californicus (Coleoptera: Cerambycidae). Current surveying methods are limited to visual identification of larval exit holes in the VELB/CELB host plant, elderberry (Sambucus spp.), into which larvae bore and excavate feeding galleries. Unbiased genetic approaches could provide a much-needed complementary approach that has more precision than relying on visual inspection of exit holes. In this study we developed a DNA sequencing-based method for indirect detection of VELB/CELB from frass (insect fecal matter), which can be easily and non-invasively collected from exit holes. Frass samples were collected from 37 locations and the 12S and 16S mitochondrial genes were partially sequenced using nested PCR amplification. Three frass-derived sequences showed 100% sequence identity to VELB/CELB barcode references from museum specimens sequenced for this study. Database queries of frass-derived sequences also revealed high similarity to common occupants of old VELB feeding galleries, including earwigs, flies, and other beetles. Overall, this non-invasive approach is a first step towards a genetic assay that could augment existing VELB monitoring and accurately discriminate between VELB, CELB, and other insects. Furthermore, a phylogenetic analysis of 12S and 16S data from museum specimens revealed evidence for the existence of a previously unrecognized, genetically distinct CELB subpopulation in southern California

Recurrent epimutations activate gene body promoters in primary glioblastoma

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Reciprocal co-regulation of EGR2 and MECP2 is disrupted in Rett syndrome and autism

Mutations in MECP2, encoding methyl-CpG-binding protein 2 (MeCP2), cause the neurodevelopmental disorder Rett syndrome (RTT). Although MECP2 mutations are rare in idiopathic autism, reduced MeCP2 levels are common in autism cortex. MeCP2 is critical for postnatal neuronal maturation and a modulator of activity-dependent genes such as Bdnf (brain-derived neurotropic factor) and JUNB. The activity-dependent early growth response gene 2 (EGR2), required for both early hindbrain development and mature neuronal function, has predicted binding sites in the promoters of several neurologically relevant genes including MECP2. Conversely, MeCP2 family members MBD1, MBD2 and MBD4 bind a methylated CpG island in an enhancer region located in EGR2 intron 1. This study was designed to test the hypothesis that MECP2 and EGR2 regulate each other’s expression during neuronal maturation in postnatal brain development. Chromatin immunoprecipitation analysis showed EGR2 binding to the MECP2 promoter and MeCP2 binding to the enhancer region in EGR2 intron 1. Reduction in EGR2 and MeCP2 levels in cultured human neuroblastoma cells by RNA interference reciprocally reduced expression of both EGR2 and MECP2 and their protein products. Consistent with a role of MeCP2 in enhancing EGR2, Mecp2-deficient mouse cortex samples showed significantly reduced EGR2 by quantitative immunofluorescence. Furthermore, MeCP2 and EGR2 show coordinately increased levels during postnatal development of both mouse and human cortex. In contrast to age-matched Controls, RTT and autism postmortem cortex samples showed significant reduction in EGR2. Together, these data support a role of dysregulation of an activity-dependent EGR2/MeCP2 pathway in RTT and autism

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

Sequencing-based DNA methylation profiling methods are comprehensive and, as accuracy and affordability improve, will increasingly supplant microarrays for genome-scale analyses. Here, four sequencing-based methodologies were applied to biological replicates of human embryonic stem cells to compare their CpG coverage genome-wide and in transposons, resolution, cost, concordance and its relationship with CpG density and genomic context. The two bisulfite methods reached concordance of 82% for CpG methylation levels and 99% for non-CpG cytosine methylation levels. Using binary methylation calls, two enrichment methods were 99% concordant, while regions assessed by all four methods were 97% concordant. To achieve comprehensive methylome coverage while reducing cost, an approach integrating two complementary methods was examined. The integrative methylome profile along with histone methylation, RNA, and SNP profiles derived from the sequence reads allowed genome-wide assessment of allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression

Hyperoxemia and excess oxygen use in early acute respiratory distress syndrome : Insights from the LUNG SAFE study

Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Concerns exist regarding the prevalence and impact of unnecessary oxygen use in patients with acute respiratory distress syndrome (ARDS). We examined this issue in patients with ARDS enrolled in the Large observational study to UNderstand the Global impact of Severe Acute respiratory FailurE (LUNG SAFE) study. Methods: In this secondary analysis of the LUNG SAFE study, we wished to determine the prevalence and the outcomes associated with hyperoxemia on day 1, sustained hyperoxemia, and excessive oxygen use in patients with early ARDS. Patients who fulfilled criteria of ARDS on day 1 and day 2 of acute hypoxemic respiratory failure were categorized based on the presence of hyperoxemia (PaO2 > 100 mmHg) on day 1, sustained (i.e., present on day 1 and day 2) hyperoxemia, or excessive oxygen use (FIO2 ≥ 0.60 during hyperoxemia). Results: Of 2005 patients that met the inclusion criteria, 131 (6.5%) were hypoxemic (PaO2 < 55 mmHg), 607 (30%) had hyperoxemia on day 1, and 250 (12%) had sustained hyperoxemia. Excess FIO2 use occurred in 400 (66%) out of 607 patients with hyperoxemia. Excess FIO2 use decreased from day 1 to day 2 of ARDS, with most hyperoxemic patients on day 2 receiving relatively low FIO2. Multivariate analyses found no independent relationship between day 1 hyperoxemia, sustained hyperoxemia, or excess FIO2 use and adverse clinical outcomes. Mortality was 42% in patients with excess FIO2 use, compared to 39% in a propensity-matched sample of normoxemic (PaO2 55-100 mmHg) patients (P = 0.47). Conclusions: Hyperoxemia and excess oxygen use are both prevalent in early ARDS but are most often non-sustained. No relationship was found between hyperoxemia or excessive oxygen use and patient outcome in this cohort. Trial registration: LUNG-SAFE is registered with ClinicalTrials.gov, NCT02010073publishersversionPeer reviewe

Mapping development and health effects of cooking with solid fuels in low-income and middle-income countries, 2000-18 : a geospatial modelling study

Background More than 3 billion people do not have access to clean energy and primarily use solid fuels to cook. Use of solid fuels generates household air pollution, which was associated with more than 2 million deaths in 2019. Although local patterns in cooking vary systematically, subnational trends in use of solid fuels have yet to be comprehensively analysed. We estimated the prevalence of solid-fuel use with high spatial resolution to explore subnational inequalities, assess local progress, and assess the effects on health in low-income and middle-income countries (LMICs) without universal access to clean fuels.Methods We did a geospatial modelling study to map the prevalence of solid-fuel use for cooking at a 5 km x 5 km resolution in 98 LMICs based on 2.1 million household observations of the primary cooking fuel used from 663 population-based household surveys over the years 2000 to 2018. We use observed temporal patterns to forecast household air pollution in 2030 and to assess the probability of attaining the Sustainable Development Goal (SDG) target indicator for clean cooking. We aligned our estimates of household air pollution to geospatial estimates of ambient air pollution to establish the risk transition occurring in LMICs. Finally, we quantified the effect of residual primary solid-fuel use for cooking on child health by doing a counterfactual risk assessment to estimate the proportion of deaths from lower respiratory tract infections in children younger than 5 years that could be associated with household air pollution.Findings Although primary reliance on solid-fuel use for cooking has declined globally, it remains widespread. 593 million people live in districts where the prevalence of solid-fuel use for cooking exceeds 95%. 66% of people in LMICs live in districts that are not on track to meet the SDG target for universal access to clean energy by 2030. Household air pollution continues to be a major contributor to particulate exposure in LMICs, and rising ambient air pollution is undermining potential gains from reductions in the prevalence of solid-fuel use for cooking in many countries. We estimated that, in 2018, 205000 (95% uncertainty interval 147000-257000) children younger than 5 years died from lower respiratory tract infections that could be attributed to household air pollution.Interpretation Efforts to accelerate the adoption of clean cooking fuels need to be substantially increased and recalibrated to account for subnational inequalities, because there are substantial opportunities to improve air quality and avert child mortality associated with household air pollution. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd.Peer reviewe