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Mitochondria are the primary site of skeletal muscle fuel metabolism and ATP production. Although insulin is a major regulator of fuel metabolism, its effect on mitochondrial ATP production is not known. Here we report increases in vastus lateralis muscle mitochondrial ATP production capacity (32-42%) in healthy humans (P < 0.01) i.v. infused with insulin (1.5 milliunits/kg of fat-free mass per min) while clamping glucose, amino acids, glucagon, and growth hormone. Increased ATP production occurred in association with increased mRNA levels from both mitochondrial (NADH dehydrogenase subunit IV) and nuclear [cytochrome c oxidase (COX) subunit IV] genes (164-180%) encoding mitochondrial proteins (P < 0.05). In addition, muscle mitochondrial protein synthesis, and COX and citrate synthase enzyme activities were increased by insulin (P < 0.05). Further studies demonstrated no effect of low to high insulin levels on muscle mitochondrial ATP production for people with type 2 diabetes mellitus, whereas matched nondiabetic controls increased 16-26% (P < 0.02) when four different substrate combinations were used. In conclusion, insulin stimulates mitochondrial oxidative phosphorylation in skeletal muscle along with synthesis of gene transcripts and mitochondrial protein in human subjects. Skeletal muscle of type 2 diabetic patients has a reduced capacity to increase ATP production with high insulin levels.

TBX21 encodes for the transcription factor T-bet (T-box expressed in T cells), which influences naïve T lymphocyte development and has been implicated in asthma pathogenesis. Specifically, the T-bet knockout mouse spontaneously develops airway hyperresponsiveness and other changes consistent with asthma. Because airway responsiveness is moderated by the use of inhaled corticosteroids in asthma, it is conceivable that genetic variation in TBX21 may alter asthma phenotypes in a treatment-specific fashion. Here we demonstrate that the nonsynonymous variation in TBX21 coding for replacement of histidine 33 with glutamine is associated with significant improvement in the PC20 (a measure of airway responsiveness) of asthmatic children in a large clinical trial spanning 4 years. We note that this increase occurs only in the children randomized to inhaled corticosteroids and that it dramatically enhances the overall improvement in PC20 associated with inhaled corticosteroid usage. The average PC20 at trial end for subjects on inhaled corticosteroids possessing a variant allele was in the normal range for nonasthmatics. In cellular models, we show that the TBX21 variant increases T helper 1 and decreases T helper 2 cytokine expression comparably with wild type. TBX21 may thus be an important determinant pharmacogenetic response to the therapy of asthma with inhaled corticosteroids.

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Pulmonary fibrosis results from dysregulated lung repair and involves multiple cell types. The role of endothelial cells (EC) in lung fibrosis is poorly understood. Using single cell RNA-sequencing we identified endothelial transcription factors involved in lung fibrogenesis, including FOXF1, SMAD6, ETV6 and LEF1. Focusing on FOXF1, we found that FOXF1 is decreased in EC within human idiopathic pulmonary fibrosis (IPF) and mouse bleomycin-injured lungs. Endothelial-specific Foxf1 inhibition in mice increased collagen depositions, promoted lung inflammation, and impaired R-Ras signaling. In vitro, FOXF1-deficient EC increased proliferation, invasion and activation of human lung fibroblasts, and stimulated macrophage migration by secreting IL-6, TNFα, CCL2 and CXCL1. FOXF1 inhibited TNFα and CCL2 through direct transcriptional activation of Rras gene promoter. Transgenic overexpression or endothelial-specific nanoparticle delivery of Foxf1 cDNA decreased pulmonary fibrosis in bleomycin-injured mice. Nanoparticle delivery of FOXF1 cDNA can be considered for future therapies in IPF. Pulmonary fibrosis results from dysregulated lung repair, but the role of endothelial cells (EC) in fibrosis is unclear. Here, the authors show that FOXF1/R-Ras signalling in EC inhibits profibrotic mediators and that ECspecific nanoparticle FOXF1 gene therapy decreases lung fibrosis in mice.

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

Aedes aegypti transmit pathogenic arboviruses while the mosquito itself tolerates the infection. We examine a piRNA-based immunity that relies on the acquisition of viral derived cDNA (vDNA) and how this pathway discriminates between self and non-self. The piRNAs derived from these vDNAs are essential for virus control and Piwi4 has a central role in the pathway. Piwi4 binds preferentially to virus-derived piRNAs but not to transposon-targeting piRNAs. Analysis of episomal vDNA from infected cells reveals that vDNA molecules are acquired through a discriminatory process of reverse-transcription and recombination directed by endogenous retrotransposons. Using a high-resolution Ae. aegypti genomic sequence, we found that vDNAs integrated in the host genome as endogenous viral elements (EVEs), produce antisense piRNAs that are preferentially loaded onto Piwi4. Importantly, EVE-derived piRNAs are specifically loaded onto Piwi4 to inhibit virus replication. Thus, Ae. aegypti employs a sophisticated antiviral mechanism that promotes viral persistence and generates long-lasting adaptive immunity.

NF-kappaB is known to exert a cytoprotective action against TNF-alpha-induced apoptosis. To study the role of NF-kappaB in various TNF-alpha-treated epithelial cell lines, we generated stable transfectants overexpressing a mutated unresponsive form of the IkappaBalpha inhibitor (MT cells). As NF-kappaB prevented TNF-alpha-induced apoptosis in various epithelial cancer cell lines, we searched for NF-kappaB target gene products responsible for this difference of sensitivity. We observed an increased Bcl-X(L) expression level in OVCAR-3 cells compared with OVCAR-3 cells expressing a mutated IkappaBalpha inhibitor (MT cells). Induction of the antioxidant enzyme MnSOD was detected only in TNF-alpha-treated OVCAR, MCF7A/Z and HCT116 cells but not in MT cells. Moreover, reactive oxygen species were involved in TNF-alpha-induced apoptosis, as various antioxidants partially protected these cells from apoptosis. At last, transfection of the MnSOD cDNA in MT cells, which do not express this protein after TNF-alpha stimulation, partially restored resistance to TNF-alpha-induced cell death, as observed by clonogenic assays. However, transfection of the Bcl-X(L) cDNA did not induce any protective effect. Therefore, MnSOD expression is induced by NF-kappaB in epithelial cancer cells in response to TNF-alpha, and is at least partially responsible for their resistance to TNF-alpha-induced apoptosis, presumably through the clearance of death-inducing ROS.

Subclonal architecture and genomic evolution of small-cell lung cancer (SCLC) under treatment has not been well studied primarily due to lack of tumor specimens, particularly longitudinal samples acquired during treatment. SCLC is characterized by early hematogenous spread, which makes circulating cell-free tumor DNA (ctDNA) sequencing a promising modality for genomic profiling. Here, we perform targeted deep sequencing of 430 cancer genes on pre-treatment tumor biopsies, as well as on plasma samples collected prior to and during treatment from 22 SCLC patients. Similar subclonal architecture is observed between pre-treatment ctDNA and paired tumor DNA. Mean variant allele frequency of clonal mutations from pre-treatment ctDNA is associated with progression-free survival and overall survival. Pre- and post-treatment ctDNA mutational analysis demonstrate that mutations of DNA repair and NOTCH signaling pathways are enriched in post-treatment samples. These data suggest that ctDNA sequencing is promising to delineate genomic landscape, subclonal architecture, and genomic evolution of SCLC. Small cell lung cancer (SCLC) may evolve under treatment. But tumor tissues are often not available to study evolution of SCLC. Here, the authors utilize circulating tumor DNA to investigate the genomic evolution and subclonal architecture of SCLC during therapy.

Combined heart and renal failure is associated with high cardiovascular morbidity and mortality. Anti-oxidant and anti-inflammatory, non-hematopoietic effects of erythropoietin (EPO) treatment have been proposed. Monocytes may act as biosensors of the systemic environment. We hypothesized that monocyte transcriptomes of patients with cardiorenal syndrome (CRS) reflect the pathophysiology of the CRS and respond to short-term EPO treatment at a recommended dose for treatment of renal anemia. Patients with CRS and anemia (n = 18) included in the EPOCARES trial were matched to healthy controls (n = 12). Patients were randomized to receive 50 IU/kg/week EPO or not. RNA from CD14(+)-monocytes was subjected to genome wide expression analysis (Illumina) at baseline and 18 days (3 EPO injections) after enrolment. Transcriptomes from patients were compared to healthy controls and effect of EPO treatment was evaluated within patients. In CRS patients, expression of 471 genes, including inflammation and oxidative stress related genes was different from healthy controls. Cluster analysis did not separate patients from healthy controls. The 6 patients with the highest hsCRP levels had more differentially expressed genes than the 6 patients with the lowest hsCRP levels. Analysis of the variation in log(2) ratios of all individual 18 patients indicated that 4 of the 18 patients were different from the controls, whereas the other 14 were quite similar. After short-term EPO treatment, every patient clustered to his or her own baseline transcriptome. Two week EPO administration only marginally affected expression profiles on average, however, individual gene responses were variable. In stable, treated CRS patients with mild anemia, monocyte transcriptomes were modestly altered, and indicated imprints of inflammation and oxidative stress. EPO treatment with a fixed dose has hematopoietic effects, had no appreciable beneficial actions on monocyte transcription profiles, however, could also not be associated with undesirable transcriptional responses.

Many previous studies have provided evidence for genome changes in polyploids, but there are little data on the overall population dynamics of genome change and whether it causes phenotypic variability. We analyzed genetic, epigenetic, gene expression, and phenotypic changes in ~50 resynthesized Brassica napus lines independently derived by hybridizing double haploids of Brassica oleracea and Brassica rapa. A previous analysis of the first generation (S0) found that genetic changes were rare, and cytosine methylation changes were frequent. Our analysis of a later generation found that most S0 methylation changes remained fixed in their S5 progeny, although there were some reversions and new methylation changes. Genetic changes were much more frequent in the S5 generation, occurring in every line with lines normally distributed for number of changes. Genetic changes were detected on 36 of the 38 chromosomes of the S5 allopolyploids and were not random across the genome. DNA fragment losses within lines often occurred at linked marker loci, and most fragment losses co-occurred with intensification of signal from homoeologous markers, indicating that the changes were due to homoeologous nonreciprocal transpositions (HNRTs). HNRTs between chromosomes A1 and C1 initiated in early generations, occurred in successive generations, and segregated, consistent with a recombination mechanism. HNRTs and deletions were correlated with qualitative changes in the expression of specific homoeologous genes and anonymous cDNA amplified fragment length polymorphisms and with phenotypic variation among S5 polyploids. Our data indicate that exchanges among homoeologous chromosomes are a major mechanism creating novel allele combinations and phenotypic variation in newly formed B. napus polyploids.