Alternative Splicing of Four Trafficking Genes Regulates Myofiber Structure and Skeletal Muscle Physiology

During development, transcriptional and post-transcriptional networks are coordinately regulated to drive organ maturation. Alternative splicing contributes by producing temporal-specific protein isoforms. We previously found that genes undergoing splicing transitions during mouse postnatal heart development are enriched for vesicular trafficking and membrane dynamics functions. Here, we show that adult trafficking isoforms are also expressed in adult skeletal muscle and hypothesize that striated muscle utilizes alternative splicing to generate specific isoforms required for function of adult tissue. We deliver morpholinos into flexor digitorum brevis muscles in adult mice to redirect splicing of four trafficking genes to the fetal isoforms. The splicing switch results in multiple structural and functional defects, including transverse tubule (T-tubule) disruption and dihydropyridine receptor alpha (DHPR) and Ryr1 mislocalization, impairing excitation-contraction coupling, calcium handling, and force generation. The results demonstrate a previously unrecognized role for trafficking functions in adult muscle tissue homeostasis and a specific requirement for the adult splice variants

Synthesis and Characterization of \u3cem\u3ed\u3c/em\u3e\u3csub\u3e5\u3c/sub\u3e-Barbarin for Use in Barbarin-Related Research

Based on structural similarities and equine administration experiments, Barbarin, 5-phenyl-2-oxazolidinethione from Brassicaceae plants, is a possible source of equine urinary identifications of aminorex, (R,S)-5-phenyl-4,5-dihydro-1,3-oxazol-2-amine, an amphetamine-related US Drug Enforcement Administration (DEA) controlled substance considered illegal in sport horses. We now report the synthesis and certification of d5-barbarin to facilitate research on the relationship between plant barbarin and such aminorex identifications. D5-barbarin synthesis commenced with production of d5-2-oxo-2-phenylacetaldehyde oxime (d5-oxime) from d5-acetophenone via butylnitrite in an ethoxide/ethanol solution. This d5-oxime was then reduced with lithium aluminum hydride (LiAlH4) to produce the corresponding d5-2-amino-1-phenylethan-1-ol (d5-phenylethanolamine). Final ring closure of the d5-phenylethanolamine was performed by the addition of carbon disulfide (CS2) with pyridine. The reaction product was purified by recrystallization and presented as a stable white crystalline powder. Proton NMR spectroscopy revealed a triplet at 5.88 ppm for one proton, a double doublet at 3.71 ppm for one proton, and double doublet at 4.11 ppm for one proton, confirming d5-barbarin as the product. Further characterization by high resolution mass spectrometry supports the successful synthesis of d5-barbarin. Purity of the recrystallized product was ascertained by High Performance Liquid Chromatography (HPLC) to be greater than 98%. Together, we have developed the synthesis and full characterization of d5-barbarin for use as an internal standard in barbarin-related and equine forensic research

Loss of peroxiredoxin-2 exacerbates eccentric contraction-induced force loss in dystrophin-deficient muscle

Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or βcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions

Comparative Analysis of the Frequency and Distribution of Stem and Progenitor Cells in the Adult Mouse Brain

cells (NSCs) and progenitor cells, but it cannot discriminate between these two populations. Given two assays have purported to overcome this shortfall, we performed a comparative analysis of the distribution and frequency of NSCs and progenitor cells detected in 400 m coronal segments along the ventricular neuraxis of the adult mouse brain using the neurosphere assay, the neural colony forming cell assay (N-CFCA), and label-retaining cell (LRC) approach. We observed a large variation in the number of progenitor/stem cells detected in serial sections along the neuraxis, with the number of neurosphereforming cells detected in individual 400 m sections varying from a minimum of eight to a maximum of 891 depending upon the rostral-caudal coordinate assayed. Moreover, the greatest variability occurred in the rostral portion of the lateral ventricles, thereby explaining the large variation in neurosphere frequency previously reported. Whereas the overall number of neurospheres (3730 276) or colonies (4275 124) we detected along the neuraxis did not differ significantly, LRC numbers were significantly reduced (1186 188, 7 month chase) in comparison to both total colonies and neurospheres. Moreover, approximately two orders of magnitude fewer NSC-derived colonies (50 10) were detected using the N-CFCA as compared to LRCs. Given only 5% of the LRCs are cycling (BrdU/Ki-67) or competent to divide (BrdU/Mcm-2), and proliferate upon transfer to culture, it is unclear whether this technique selectively detects endogenous NSCs. Overall, caution should be taken with the interpretation and employment of all these techniques

Personal Protective Equipment and Antiviral Drug Use during Hospitalization for Suspected Avian or Pandemic Influenza1

In a pandemic, many current national stockpiles of PPE and antiviral medications are likely inadequate

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Imaging for assessment of cancer treatment response to immune checkpoint inhibitors can be complementary in identifying hypophysitis

IntroductionHypophysitis is reported in 8.5%–14% of patients receiving combination immune checkpoint inhibition (cICI) but can be a diagnostic challenge. This study aimed to assess the role of routine diagnostic imaging performed during therapeutic monitoring of combination anti-CTLA-4/anti-PD-1 treatment in the identification of hypophysitis and the relationship of imaging findings to clinical diagnostic criteria.MethodsThis retrospective cohort study identified patients treated with cICI between January 2016 and January 2019 at a quaternary melanoma service. Medical records were reviewed to identify patients with a documented diagnosis of hypophysitis based on clinical criteria. Available structural brain imaging with magnetic resonance imaging (MRI) or computed tomography (CT) of the brain and 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography with computed tomography (FDG-PET/CT) were assessed retrospectively. The main radiological outcome measures were a relative change in pituitary size or FDG uptake temporally attributed to cICI.ResultsThere were 162 patients (median age 60 years, 30% female) included. A total of 100 and 134 had serial CT/MRI of the brain and FDG-PET/CT, respectively. There were 31 patients who had a documented diagnosis of hypophysitis and an additional 20 who had isolated pituitary imaging findings. The pituitary gland enlargement was mild, and the largest absolute gland size was 13 mm, with a relative increase of 7 mm from baseline. There were no cases of optic chiasm compression. Pituitary enlargement and increased FDG uptake were universally transient. High-dose glucocorticoid treatment for concurrent irAEs prevented assessment of the pituitary–adrenal axis in 90% of patients with isolated imaging findings.ConclusionCareful review of changes in pituitary characteristics on imaging performed for assessment of therapeutic response to iICI may lead to increased identification and more prompt management of cICI-induced hypophysitis

Multiethnic Meta-Analysis Identifies Ancestry-Specific and Cross-Ancestry Loci for Pulmonary Function

Nearly 100 loci have been identified for pulmonary function, almost exclusively in studies of European ancestry populations. We extend previous research by meta-analyzing genome-wide association studies of 1000 Genomes imputed variants in relation to pulmonary function in a multiethnic population of 90,715 individuals of European (N = 60,552), African (N = 8429), Asian (N = 9959), and Hispanic/Latino (N = 11,775) ethnicities. We identify over 50 additional loci at genome-wide significance in ancestry-specific or multiethnic meta-analyses. Using recent fine-mapping methods incorporating functional annotation, gene expression, and differences in linkage disequilibrium between ethnicities, we further shed light on potential causal variants and genes at known and newly identified loci. Several of the novel genes encode proteins with predicted or established drug targets, including KCNK2 and CDK12. Our study highlights the utility of multiethnic and integrative genomics approaches to extend existing knowledge of the genetics of lung function and clinical relevance of implicated loci

Genetic variants associated with subjective well-being, depressive symptoms, and neuroticism identified through genome-wide analyses

Very few genetic variants have been associated with depression and neuroticism, likely because of limitations on sample size in previous studies. Subjective well-being, a phenotype that is genetically correlated with both of these traits, has not yet been studied with genome-wide data. We conducted genome-wide association studies of three phenotypes: subjective well-being (n = 298,420), depressive symptoms (n = 161,460), and neuroticism (n = 170,911). We identify 3 variants associated with subjective well-being, 2 variants associated with depressive symptoms, and 11 variants associated with neuroticism, including 2 inversion polymorphisms. The two loci associated with depressive symptoms replicate in an independent depression sample. Joint analyses that exploit the high genetic correlations between the phenotypes (|ρ^| ≈ 0.8) strengthen the overall credibility of the findings and allow us to identify additional variants. Across our phenotypes, loci regulating expression in central nervous system and adrenal or pancreas tissues are strongly enriched for association.</p