Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models.

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines

Distribution of AMPA Receptor Subunits and Tarps in Synaptic and Extrasynaptic Membranes of the Adult Rat Nucleus Accumbens

We characterized the distribution of AMPA receptor (AMPAR) subunits and the transmembrane AMPA receptor regulatory proteins (TARPs) γ-2 and γ-4 in adult rat nucleus accumbens (NAc) using a method that separates plasma membranes into synaptic membrane-enriched and extrasynaptic membrane-enriched fractions. We also measured GluA1 phosphorylated at serine 845 (pS845 GluA1) and serine 831 (pS831 GluA1). GluA1–3 protein levels and pS831 GluA1/total GluA1 were higher in synaptic membranes. However, pS845 GluA1/total GluA1 was higher in extrasynaptic membranes, consistent with a role for S845 phosphorylation in GluA1 insertion at extrasynaptic sites. Homeric GluA1 receptors were detected in extrasynaptic membranes, consistent with evidence for extrasynaptic Ca2+-permeable AMPARs in other systems. The TARP γ-2 was enriched in synaptic membranes, whereas γ-4 was mainly found in extrasynaptic membranes, suggesting distinct roles for these proteins in the NAc. These experiments provide fundamental information that will aid in the interpretation of studies on AMPAR-related plasticity in the NAc

Estimation of Uncertainty in Air-­Water Exchange Flux 2 and Gross Volatilization Loss of PCBs: a Case Study 3 based on Passive Sampling in the Lower Great Lakes

Compared with dry and wet deposition fluxes, air–water exchange flux cannot be directly measured experimentally. Its model-based calculation contains considerable uncertainty because of the uncertainties in input parameters. To capture the inherent variability of air–water exchange flux of PCBs across the lower Great Lakes and to calculate their annual gross volatilization loss, 57 pairs of air and water samples from 19 sites across Lakes Erie and Ontario were collected using passive sampling technology during 2011–2012. Error propagation analysis and Monte Carlo simulation were applied to estimate uncertainty in the air–water exchange fluxes. Results from both methods were similar, but error propagation analysis estimated a smaller uncertainty than Monte Carlo simulation in cases of net deposition. Maximum likelihood estimations (MLE) of wind speed and air temperature were recommended to quantify the site-specific air–water exchange flux. An assumed 30–40% of relative uncertainty in overall air–water mass transfer velocity was confirmed. MLEs of volatilization fluxes of total PCBs across Lakes Erie and Ontario were 0.78 and 0.53 ng m–2 day–1, respectively, and gross volatilization losses of total PCBs over the whole lakes were 74 kg year–1 for Lake Erie and 63 kg year–1 for Lake Ontario. Mass balance analysis across Lake Ontario indicated that volatilization was the uppermost loss process of aqueous PCBs

First Documentation of the European Gut Fungus, \u3cem\u3eEphemerellomyces\u3c/em\u3e, and Other Insect Associated Endosymbionts in the Dry Creek Drainage, Boise, Idaho

Trichomycetes, a former class of obligate endosymbiotic fungi, are now recognized as an ecological group that inhabits the gut(s) of immature insects. Though the biodiversity and geographical distribution of trichomycetes are worldwide, our knowledge of the group in the Pacific Northwest is limited due to the few researchers conducting studies on them. Dry Creek drainage in Boise, Idaho was selected in the winter of 2009-10 as a potential site to find gut fungi. This initial survey provides the first account of Ephemerellomyces aquilonius, a species previously documented and studied only in Norway. Ephemerellomyces aquilonius (a monotypic genus) is a member of the Harpellales, an order of trichomycetes, and was dissected from the digestive tracts of mayflies (Ephemeroptera) collected from the creek. Amongst the various aquatic habitats surveyed and hosts recovered, gut fungi were also documented in stoneflies (Plecoptera) and black flies (Diptera: Simuliidae). Our investigation provides a significant new record of the very rare and unusual taxon, E. aquilonius, previously only known in Western Europe. The success of this brief survey demonstrates promise for further discoveries of gut fungi at this site, in Idaho and the Pacific Northwest. This disjunct distribution bridges an enormous geographical divide, although the species actually may be more widespread than earlier anticipated. Future collections and research on specimens from Dry Creek, with sequence data to be generated in our laboratory will be eagerly awaited as we place this unusual species in our expanding molecular-based phylogenies. We highlight here the morphology of E. aquilonius and some of the other endosymbionts found there

Meta Gene Set Enrichment Analyses Link miR-137-regulated Pathways With Schizophrenia Risk

Background: A single nucleotide polymorphism (SNP) within MIR137, the host gene for miR-137, has been identified repeatedly as a risk factor for schizophrenia. Previous genetic pathway analyses suggest that potential targets of this microRNA (miRNA) are also highly enriched in schizophrenia-relevant biological pathways, including those involved in nervous system development and function. Methods: In this study, we evaluated the schizophrenia risk of miR-137 target genes within these pathways. Gene set enrichment analysis of pathway-specific miR-137 targets was performed using the stage 1 (21,856 subjects) schizophrenia genome wide association study data from the Psychiatric Genomics Consortium and a small independent replication cohort (244 subjects) from the Mind Clinical Imaging Consortium and Northwestern University. Results: Gene sets of potential miR-137 targets were enriched with variants associated with schizophrenia risk, including target sets involved in axonal guidance signaling, Ephrin receptor signaling, long-term potentiation, PKA signaling, and Sertoli cell junction signaling. The schizophrenia-risk association of SNPs in PKA signaling targets was replicated in the second independent cohort. Conclusions: These results suggest that these biological pathways may be involved in the mechanisms by which this MIR137 variant enhances schizophrenia risk. SNPs in targets and the miRNA host gene may collectively lead to dysregulation of target expression and aberrant functioning of such implicated pathways. Pathway-guided gene set enrichment analyses should be useful in evaluating the impact of other miRNAs and target genes in different diseases

Particulate Matter, DNA Methylation in Nitric Oxide Synthase, and Childhood Respiratory Disease

Background: Air pollutants have been associated with childhood asthma and wheeze. Epigenetic regulation of nitric oxide synthase—the gene responsible for nitric oxide production—may be affected by air pollutants and contribute to the pathogenesis of asthma and wheeze. Objective: Our goal was to investigate the association between air pollutants, DNA methylation, and respiratory outcomes in children. Methods: Given residential address and buccal sample collection date, we estimated 7-day, 1-month, 6-month, and 1-year cumulative average $PM_{2.5}$ and $PM_{10}$ (particulate matter ≤ 2.5 and ≤ 10 µm aerodynamic diameter, respectively) exposures for 940 participants in the Children’s Health Study. Methylation of 12 CpG sites in three NOS (nitric oxide synthase) genes was measured using a bisulfite-polymerase chain reaction Pyrosequencing assay. Beta regression models were used to estimate associations between air pollutants, percent DNA methylation, and respiratory outcomes. Results: A 5-µg/$m^3$ increase in $PM_{2.5}$ was associated with a 0.20% [95% confidence interval (CI): –0.32, –0.07] to 1.0% (95% CI: –1.61, –0.56) lower DNA methylation at NOS2A position 1, 0.06% (95% CI: –0.18, 0.06) to 0.58% (95% CI: –1.13, –0.02) lower methylation at position 2, and 0.34% (95% CI: –0.57, –0.11) to 0.89% (95% CI: –1.57, –0.21) lower methylation at position 3, depending on the length of exposure and CpG locus. One-year $PM_{2.5}$ exposure was associated with 0.33% (95% CI: 0.01, 0.65) higher in average DNA methylation of 4 loci in the NOS2A CpG island. A 5-µg/$m^3$ increase in 7-day and 1-year $PM_{2.5}$ was associated with 0.6% (95% CI: 0.13, 0.99) and 2.8% (95% CI: 1.77, 3.75) higher NOS3 DNA methylation. No associations were observed for NOS1. $PM_{10}$ showed similar but weaker associations with DNA methylation in these genes. Conclusions: $PM_{2.5}$ exposure was associated with percent DNA methylation of several CpG loci in NOS genes, suggesting an epigenetic mechanism through which these pollutants may alter production of nitric oxide

Dietary Cholesterol Concentration and Duration Degrade Long-Term Memory of Classical Conditioning of the Rabbit's Nictitating Membrane Response

A rabbit model of Alzheimer's disease based on feeding a cholesterol diet for eight weeks shows sixteen hallmarks of the disease, including learning and memory changes. Although we have shown 2% cholesterol and copper in water can retard learning, other studies show feeding dietary cholesterol before learning can improve acquisition whereas feeding cholesterol after learning can degrade long-term memory. We explored this issue by manipulating cholesterol concentration and duration following classical trace conditioning of the rabbit's nictitating membrane response and assessed conditioned responding after eight weeks on cholesterol. First, rabbits given trace classical conditioning followed by 0.5%, 1%, or 2% cholesterol for eight weeks showed body weight and serum cholesterol levels that were a function of dietary cholesterol. Although all concentrations of cholesterol showed some sign of retarding long-term memory, the level of memory retardation was correlated with serum cholesterol levels. Second, rabbits given trace conditioning followed by different durations of a 2% cholesterol diet combined with different durations of a 0% control diet for 8 weeks showed duration and timing of a 2% cholesterol diet were important in affecting recall. The data support the idea that dietary cholesterol may retard long-term memory