During muscle atrophy, thick, but not thin, filament components are degraded by MuRF1-dependent ubiquitylation

Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1

Automated Reporter Quantification In Vivo: High-Throughput Screening Method for Reporter-Based Assays in Zebrafish

Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current “high-content” (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current “high-content” whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform

EC86-219 1986 Nebraska Swine Report

This 1986 Nebraska Swine Report was prepared by the staff in Animal Science and cooperating departments for use in the Extension and Teaching programs at the University of Nebraska-Lincoln. Authors from the following areas contributed to this publication: Swine Nutrition, swine diseases, pathology, economics, engineering, swine breeding, meats, agronomy, and diagnostic laboratory. It covers the following areas: breeding, disease control, feeding, nutrition, economics, housing and meats

Transcriptomic and Metabolic Responses to a Live-Attenuated Francisella tularensis Vaccine

The immune response to live-attenuated Francisella tularensis vaccine and its host evasion mechanisms are incompletely understood. Using RNA-Seq and LC–MS on samples collected pre-vaccination and at days 1, 2, 7, and 14 post-vaccination, we identified differentially expressed genes in PBMCs, metabolites in serum, enriched pathways, and metabolites that correlated with T cell and B cell responses, or gene expression modules. While an early activation of interferon α/β signaling was observed, several innate immune signaling pathways including TLR, TNF, NF-κB, and NOD-like receptor signaling and key inflammatory cytokines such as Il-1α, Il-1β, and TNF typically activated following infection were suppressed. The NF-κB pathway was the most impacted and the likely route of attack. Plasma cells, immunoglobulin, and B cell signatures were evident by day 7. MHC I antigen presentation was more actively up-regulated first followed by MHC II which coincided with the emergence of humoral immune signatures. Metabolomics analysis showed that glycolysis and TCA cycle-related metabolites were perturbed including a decline in pyruvate. Correlation networks that provide hypotheses on the interplay between changes in innate immune, T cell, and B cell gene expression signatures and metabolites are provided. Results demonstrate the utility of transcriptomics and metabolomics for better understanding molecular mechanisms of vaccine response and potential host–pathogen interactions

Alterations in the Human Plasma Lipidome in Response to Tularemia Vaccination

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy

Mechanism of DNA Methylation-Directed Histone Methylation by KRYPTONITE

In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation

Correlation between Peripheral Blood and Bone Marrow Somatic Mutations Among Patients with Suspected or Established Myelodysplastic Syndromes from the National MDS Study

INTRODUCTION: Myelodysplastic Syndromes (MDS) are caused by progressive clonal dominance of mutated hematopoietic stem cells. While assessment of mutations in peripheral blood (PB) is an established standard for monitoring patients with some diseases such as chronic myeloid leukemia, the sensitivity and precision of PB screening and monitoring for somatic mutations in patients with MDS and related conditions such as clonal cytopenias of undetermined significance (CCUS) is less certain. Multiple published guidelines require bone marrow (BM) evaluations for the above, an invasive procedure not often performed outside of clinical trials. We compared somatic mutations and variant allele frequencies (VAFs) in paired PB and BM samples to assess the utility of PB results as a surrogate for diagnosis and monitoring. METHODS: The National MDS Natural History Study (National MDS Study) is an observational longitudinal cohort study of cytopenic patients suspected of having a diagnosis of MDS. All patients have PB and BM sampled when entering the study, prior to formal diagnosis. For this study, paired PB and BM baseline DNA samples from 36 patients were assessed for somatic mutations. Only subjects with minimum of one variant detected in their BM were included in this study. DNA was extracted from PB using an automated FlexSTAR (AutoGen) and from BM cell pellets using a QIAamp DNA Mini Kit (QIAGEN). Targeted exon sequencing of 96 genes was performed using a NovaSeq 6000 at a mean coverage of >1,200X and a mean breadth (bases covered at ≥100X) of >99.9%. Reads were aligned against a patched version of the build GRCh38 using BWA-MEM. VarScan2 was used to detect single nucleotide variants and short insertions/deletions with a minimum VAF of 2% and 5%, respectively. Resulting variants from 53 genes were manually reviewed to retain likely disease-causing variants. Correlation analysis between PB and BM VAFs was performed using Pearson correlation and linear regression. To confirm paired samples were sourced from the same subject, the Somalier software using a set of highly polymorphic SNPs across different ancestries was utilized to ensure a high level of relatedness. RESULTS: The 36 patients included 10 (28%) with MDS, 2 (6%) with MDS/MPN, 1 (3%) with AML (<30% blasts), and 23 (64%) with CCUS. The median age was 73 years (Table 1). Five of 36 patients had circulating PB blasts. Among the 191 mutations identified, the most common were TET2 (27%), SRSF2 (13%), DNMT3A (9%), SF3B1 (8%), and ASXL1 (8%). Correlation analysis of mutations (n=180) shared between paired PB and BM samples indicated a linear relationship between VAFs detected in PB and BM (Pearson correlation r=0.95, Figure 1), with a slight increase in variance observed with increasing VAFs. The deviation from the perfect (grey line) to the observed (yellow line) linear fit indicated that PB VAFs underestimated VAFs in BM by approximately 23% overall (Figure 1). For example, a BM VAF of 0.20 translated to a mean PB VAF of 0.16 based on the regression fit. The lower sensitivity was confirmed when looking at the number of total missed variants (present in BM, absent in PB): 9 of 101 (9%). Two mutations were detected in PB, but not BM. These mutations and diagnoses were 1) ETNK1 (VAF 0.026) in a patient with MDS-EB-1; and 2) CBL (VAF 0.021) in a patient with MDS with isolated del5q. Next, we assessed the relationship between percent of detected mutations and minimum VAF that was detected using linear regression and found that the higher the minimum VAF, the lower the percent of missed mutations. For example, for a 0.1, 0.2, and 0.3 minimum VAF, 11%, 9%, and 8% of BM mutations were missed, respectively. There was no difference in concordance between MDS and CCUS. SUMMARY & CONCLUSIONS: PB can be used to reliably identify somatic mutations in patients with suspected or established MDS and related conditions. Linear correlation between PB and BM VAFs showed, unsurprisingly, that BM mutations with higher VAFs are more likely to be detected. If PB is positive, this method of variant detection may be used potentially to diagnose and monitor. However, if negative (or if VAF is less than 0.10), bone marrow genomics are still necessary to exclude detectable clonal mutations. For quantitative predictive tools that use VAFs, PB VAFs would need to be corrected to improve predictive results; our estimated linear regression equation could be used to guide this in a larger prospective study

Utility of Targeted Gene Sequencing to Differentiate Myeloid Malignancies from other Cytopenic Conditions

The National Heart, Lung, and Blood Institute National MDS Natural History Study (NCT02775383) is a prospective cohort study enrolling cytopenic patients with suspected myelodysplastic syndromes (MDS) to evaluate factors associated with disease. Here, we sequenced 53 genes in bone marrow samples harvested from 1,298 patients diagnosed with myeloid malignancy, including MDS and non-MDS myeloid malignancy, or alternative marrow conditions with cytopenia based on concordance between independent histopathologic reviews (local, centralized, and tertiary to adjudicate disagreements when needed). We developed a novel two-stage diagnostic classifier based on mutational profiles in 18 of 53 sequenced genes that were sufficient to best (1) predict a diagnosis of myeloid malignancy and (2) within those with a predicted myeloid malignancy, predict whether they had MDS. The classifier achieved a PPV of 0.84 and NPV of 0.8 with an AUROC of 0.85 when classifying patients as myeloid vs. no myeloid malignancy based on VAFs in 17 genes and a PPV of 0.71 and NPV of 0.64 with an AUROC of 0.73 when classifying patients as MDS vs. non-MDS malignancy based on VAFs in 10 genes. We next assessed how this approach could complement histopathology to improve diagnostic accuracy. For 99 of 139 (71%) patients (PPV of 0.83 and NPV of 0.65) with local and centralized histopathologic disagreement in myeloid vs. no myeloid malignancy, the classifier-predicted diagnosis agreed with the tertiary pathology review (considered the internal gold standard). An online version of the classifier that can be used with either VAFs or binary mutation profiles is available at https://thenationalmdsstudy.net