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DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reactionor high-throughput RNA sequencingmethods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

This review highlights recent advances in the understanding of eosinophils and eosinophilic diseases, particularly eosinophilic gastrointestinal diseases during the last year. The increasing incidence of diseases marked by eosinophilia has been documented and highlighted the need to understand eosinophil biology and eosinophilic contributions to disease. Significant insight into the nature of eosinophilic diseases has been achieved using next-generation sequencing technologies, proteomic analysis, and machine learning to analyze tissue biopsies. These technologies have elucidated mechanistic underpinnings of eosinophilic inflammation, delineated patient endotypes, and identified patient responses to therapeutic intervention. Importantly, recent clinical studies using monoclonal antibodies that interfere with type 2 cytokine signaling or deplete eosinophils point to multiple and complex roles of eosinophils in tissues. Several studies identified distinct activation features of eosinophils in different tissues and disease states. The confluence of these studies supports a new paradigm of tissue-resident eosinophils that have pro- and anti-inflammatory immunomodulatory roles in allergic disease. Improved understanding of unique eosinophil activation states is now poised to identify novel therapeutic targets for eosinophilic diseases.

Subclinical mastitis (SCM) is a very common disease of dairy cows. Currently, somatic cell count (SCC) is used for SCM diagnoses. There are no prognostic tests to detect which cows may develop SCM during the dry-off period. Therefore, the objectives of this study were to identify metabolic alterations in the serum of pre-SCM cows during the dry-off period, at −8 and −4 weeks before calving, through a targeted mass spectrometry (MS) assay. Fifteen cows, free of any disease, and 10 cows affected only by SCM postpartum served as controls (CON) and the SCM group, respectively. Results showed 59 and 47 metabolites that differentiated (p ≤ 0.05) CON and pre-SCM cows at –8 and −4 weeks prior to the expected date of parturition, respectively. Regression analysis indicated that a panel of four serum metabolites (AUC = 0.92, p < 0.001) at −8 weeks and another four metabolites (AUC = 0.92, p < 0.01) at −4 weeks prior to parturition might serve as predictive biomarkers for SCM. Early identification of susceptible cows can enable development of better preventive measurements ahead of disease occurrence.

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Summary of adverse events with possible relation to study.

Ex vivo analysis of CMV pp65-specific CD8+ T cells in circulation before and after immunotherapy.

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The serine palmitoyltransferase (SPT) complex catalyzes the first and rate-limiting step in sphingolipid biosynthesis in all eukaryotes. ORM/ORMDL proteins are negative regulators of SPT that respond to cellular sphingolipid levels. However, the molecular basis underlying ORM/ORMDL-dependent homeostatic regulation of SPT is not well understood. We determined the cryo–electron microscopy structure of Arabidopsis SPT-ORM1 complex, composed of LCB1, LCB2a, SPTssa, and ORM1, in an inhibited state. A ceramide molecule is sandwiched between ORM1 and LCB2a in the cytosolic membrane leaflet. Ceramide binding is critical for the ORM1-dependent SPT repression, and dihydroceramides and phytoceramides differentially affect this repression. A hybrid β sheet, formed by the amino termini of ORM1 and LCB2a and induced by ceramide binding, stabilizes the amino terminus of ORM1 in an inhibitory conformation. Our findings provide mechanistic insights into sphingolipid homeostatic regulation via the binding of ceramide to the SPT-ORM/ORMDL complex that may have implications for plant-specific processes such as the hypersensitive response for microbial pathogen resistance.

Purpose:To determine whether inhibition of mTOR kinase-mediated signaling represents a valid therapeutic approach for chronic lymphocytic leukemia (CLL).Experimental Design:Stratification of mTOR activity was carried out in patients with primary CLL samples and an aggressive CLL-like mouse model. The potency of dual mTOR inhibitor AZD8055 to induce apoptosis in primary CLL cells was assessed in the presence/absence of B-cell receptor (BCR) ligation. Furthermore, we addressed the molecular and functional impact of dual mTOR inhibition in combination with BTK inhibitor ibrutinib.Results:Differential regulation of basal mTORC1 activity was observed in poor prognostic CLL samples, with elevated p4EBP1T37/46 and decreased p70S6 kinase activity, suggesting that dual mTORC1/2 inhibitors may exhibit improved response in poor prognostic CLL compared with rapalogs. AZD8055 treatment of primary CLL cells significantly reduced CLL survival in vitro compared with rapamycin, preferentially targeting poor prognostic subsets and overcoming BCR-mediated survival advantages. Furthermore, AZD8055, and clinical analog AZD2014, significantly reduced CLL tumor load in mice. AKT substrate FOXO1, while overexpressed in CLL cells of poor prognostic patients in LN biopsies, peripheral CLL cells, and mouse-derived CLL-like cells, appeared to be inactive. AZD8055 treatment partially reversed FOXO1 inactivation downstream of BCR crosslinking, significantly inhibiting FOXO1T24 phosphorylation in an mTORC2-AKT–dependent manner, to promote FOXO1 nuclear localization, activity, and FOXO1-mediated gene regulation. FOXO1 activity was further significantly enhanced on combining AZD8055 with ibrutinib.Conclusions:Our studies demonstrate that dual mTOR inhibitors show promise as future CLL therapies, particularly in combination with ibrutinib.

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Median survival for glioblastoma (GBM) remains ex vivo analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8+ T-cell polyfunctionality in vivo when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8+ T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ+, TNFα+, and CCL3+ polyfunctional, CMV-specific CD8+ T cells. These increases in polyfunctional CMV-specific CD8+ T cells correlated (R = 0.7371, P = 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.Significance: A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy. Cancer Res; 78(1); 256–64. ©2017 AACR.

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Allogeneic “off-the-shelf” mesenchymal stem/stromal cell (MSC) therapy requires scalable, quality-controlled cell manufacturing and distribution systems to provide clinical-grade products using cryogenic cell banking. However, previous studies report impaired cell function associated with administering freeze-thawed MSCs as single cell suspensions, potentially compromising reliable therapeutic efficacy. Using long-term culture-adapted clinical-grade clonal human bone marrow MSCs (cBMSCs) in this study, we engineered cBMSC sheets in 24 h to provide rapid preparation. We then sought to determine the influence of cBMSC freeze-thawing on both in vitro production of pro-regenerative factors and in vivo ability to reduce renal fibrosis in a rat model compared to freshly harvested cBMSCs. Sheets from freeze-thawed cBMSCs sheets exhibited comparable in vitro protein production and gene expression of pro-regenerative factors [e.g., hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and interleukin 10 (IL-10)] to freshly harvested cBMSC sheets. Additionally, freeze-thawed cBMSC sheets successfully suppressed renal fibrosis in vivo in an established rat ischemia–reperfusion injury model. Despite previous studies reporting that freeze-thawed MSCs exhibit impaired cell functions compared to fresh MSC single cell suspensions, cell sheets engineered from freeze-thawed cBMSCs do not exhibit impaired cell functions, supporting critical steps toward future clinical translation of cBMSC-based kidney disease treatment.

The ability of EBV to switch between latent and lytic infection is key to its long-term persistence in memory B cells, and its ability to persist in proliferating cells is strongly linked to oncogenesis. During latency, most viral genes are epigenetically silenced, and the virus must overcome this repression to reactivate lytic replication.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the chromosome 4q telomere allowing ectopic expression of theDUX4gene in skeletal muscle. Genetic analysis is difficult due to the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding theDUX4transcriptional start site. Within theDUX4exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

The volume of high-quality evidence supporting exercise as beneficial to cancer survivors has grown exponentially; however, the potential harms of exercise remain understudied. Consequently, the trade-off between desirable and undesirable outcomes of engaging in exercise remains unclear to clinicians and people with cancer. Practical guidance on collecting and reporting harms in exercise oncology is lacking. We present a harms reporting protocol developed and refined through exercise oncology trials since 2015.Development of the Exercise Harms Reporting Method (ExHaRM) was informed by national and international guidelines for harms reporting in clinical trials involving therapeutic goods or medical devices, with adaptations to enhance applicability to exercise. The protocol has been adjusted via an iterative process of implementation and adjustment through use in multiple exercise oncology trials involving varied cancer diagnoses (types: breast, brain, gynaecological; stages at diagnosis I–IV; primary/recurrent), and heterogeneous exercise intervention characteristics (face to face/telehealth delivery; supervised/unsupervised exercise). It has also involved the development of terms (such as, adverse outcomes, which capture all undesirable physical, psychological, social and economic outcomes) that facilitate the harms assessment process in exercise.ExHaRM involves: step 1: Monitor occurrence of adverse outcomes through systematic and non-systematic surveillance; step 2: Assess and record adverse outcomes, including severity, causality, impact on intervention and type; step 3: Review of causality by harms panel (and revise as necessary); and step 4: Analyse and report frequencies, rates and clinically meaningful details of all-cause and exercise-related adverse outcomes.ExHaRM provides guidance to improve the quality of harms assessment and reporting immediately, while concurrently providing a framework for future refinement. Future directions include, but are not limited to, standardising exercise-specific nomenclature and methods of assessing causality.