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Increased DUOX1 and DUOX2 expression is reversed by catalase.

Radiation induced intestinal damage histology and collagen deposition information.

The effect of ascorbate and catalase on clonogenic cell survival in PDAC.

Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH−, i.e., intravenous infusions of ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH− decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H2O2)-mediated mechanism. In this study, we demonstrate that P-AscH− radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH−, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH− in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH− during the radiotherapy “beam on.” Specifically, treatment with P-AscH− increased median overall survival compared with our institutional average (21.7 vs. 12.7 months, P = 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH−–treated subjects was also greater than our institutional average (13.7 vs. 4.6 months, P < 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH− in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH− efficacy is warranted in a phase II clinical trial.Significance:These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.

DOCK (dedicator of cytokinesis) is an 11-member family of typical guanine nucleotide exchange factors (GEFs) expressed in the brain, spinal cord, and skeletal muscle. Several DOCK proteins have been implicated in maintaining several myogenic processes such as fusion. We previously identified DOCK3 as being strongly upregulated in Duchenne muscular dystrophy (DMD), specifically in the skeletal muscles of DMD patients and dystrophic mice.Dock3ubiquitous KO mice on the dystrophin-deficient background exacerbated skeletal muscle and cardiac phenotypes. We generatedDock3conditional skeletal muscle knockout mice (Dock3mKO) to characterize the role of DOCK3 protein exclusively in the adult muscle lineage.Dock3mKO mice presented with significant hyperglycemia and increased fat mass, indicating a metabolic role in the maintenance of skeletal muscle health.Dock3mKO mice had impaired muscle architecture, reduced locomotor activity, impaired myofiber regeneration, and metabolic dysfunction. We identified a novel DOCK3 interaction with SORBS1 through the C-terminal domain of DOCK3 that may account for its metabolic dysregulation. Together, these findings demonstrate an essential role for DOCK3 in skeletal muscle independent of DOCK3 function in neuronal lineages.

Duchenne muscular dystrophy (DMD) is a progressive, X-linked childhood neuromuscular disorder that results from loss-of-function mutations in the DYSTROPHIN gene. DMD patients exhibit muscle necrosis, cardiomyopathy, respiratory failure, and loss of ambulation. One of the major driving forces of DMD disease pathology is chronic inflammation. The current DMD standard of care is corticosteroids; however, there are serious side effects with long-term use, thus identifying novel anti-inflammatory and anti-fibrotic treatments for DMD is of high priority. We investigated the next-generation SINE compound, KPT−8602 (eltanexor) as an oral therapeutic to alleviate dystrophic symptoms. We performed pre-clinical evaluation of the effects of KPT−8602 in DMD zebrafish (sapje) and mouse (D2-mdx) models. KPT−8602 improved dystrophic skeletal muscle pathologies, muscle architecture and integrity, and overall outcomes in both animal models. KPT−8602 treatment ameliorated DMD pathology in D2-mdx mice, with increased locomotor behavior and improved muscle histology. KPT−8602 altered the immunological profile of the dystrophic mice, and reduced circulating osteopontin serum levels. These findings demonstrate KPT−8602 as an effective therapeutic in DMD through by promotion of an anti-inflammatory environment and overall improvement of DMD pathological outcomes.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease characterized by skeletal muscle instability, progressive muscle wasting, and fibrosis. A major driver of DMD pathology stems from aberrant upregulation of transforming growth factor β (TGFβ) signaling. In this report, we investigated the major transducers of TGFβ signaling, i.e., receptor Smads (R-Smads), in DMD patient skeletal muscle and observed a 48-fold increase in Smad8 mRNA. Smad1, Smad2, Smad3, and Smad5 mRNA were only minimally increased. A similar pattern was observed in the muscle from the mdx5cv mouse. Western blot analysis showed upregulation of phosphorylated Smad1, Smad5, and Smad8 compared to total Smad indicating activation of this pathway. In parallel, we observed a profound diminishment of muscle-enriched microRNAs (myomiRs): miR-1, miR-133a, and miR-133b. The pattern of Smad8 induction and myomiR suppression was recapitulated in C2C12 muscle cells after stimulation with bone morphogenetic protein 4 (BMP4), a signaling factor that we found upregulated in DMD muscle. Silencing Smad8 in C2C12 myoblasts derepressed myomiRs and promoted myoblast differentiation; there was also a concomitant upregulation of myogenic regulatory factors (myogenin and myocyte enhancer factor 2D) and suppression of a pro-inflammatory cytokine (interleukin-6). Our data suggest that Smad8 is a negative regulator of miR-1, miR-133a, and miR-133b in muscle cells and that the BMP4-Smad8 axis is a driver of dystrophic pathology in DMD.

Introduction RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. Methods Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. Results Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. Discussion Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.

The Dedicator of Cytokinesis (DOCK) family (DOCK1-11) of genes are essential mediators of cellular migration, growth, and fusion in a variety of cell types and tissues. Recent advances in whole-genome sequencing of patients with undiagnosed genetic disorders have identified several rare pathogenic variants in DOCK genes. We conducted a systematic review and performed a patient database and literature search of reported DOCK pathogenic variants that have been identified in association with clinical pathologies such as global developmental delay, immune cell dysfunction, muscle hypotonia, and muscle ataxia among other categories. We then categorized these pathogenic DOCK variants and their associated clinical phenotypes under several unique categories: developmental, cardiovascular, metabolic, cognitive, or neuromuscular. Our systematic review of DOCK variants aims to identify and analyze potential DOCK-regulated networks associated with neuromuscular diseases and other disease pathologies, which may identify novel therapeutic strategies and targets. This systematic analysis and categorization of human-associated pathologies with DOCK pathogenic variants is the first report to the best of our knowledge for a unique class in this understudied gene family that has important implications in furthering personalized genomic medicine, clinical diagnoses, and improve targeted therapeutic outcomes across many clinical pathologies.

The incidence of pancreatic cancer increases with age, suggesting that chronological aging is a significant risk factor for this disease. Fibroblasts are the major nonmalignant cell type in the stroma of human pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated whether the chronological aging of normal human fibroblasts (NHFs), a previously underappreciated area in pancreatic cancer research, influences the progression and therapeutic outcomes of PDAC. Results from experiments with murine xenografts and 2D and 3D co-cultures of NHFs and PDAC cells revealed that older NHFs stimulate proliferation of and confer resistance to radiation therapy of PDAC. MS-based metabolite analysis indicated that older NHFs have significantly increased arachidonic acid 12-lipoxygenase (ALOX12) expression and elevated levels of its mitogenic metabolite, 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-(S)-HETE) compared with their younger counterparts. In co-cultures with older rather than with younger NHFs, PDAC cells exhibited increases in mitogen-activated protein kinase signaling and cellular metabolism, as well as a lower oxidation state that correlated with their enhanced proliferation and resistance to radiation therapy. Expression of ALOX12 was found to be significantly lower in PDAC cell lines and tumor biopsies, suggesting that PDAC cells rely on a stromal supply of mitogens for their proliferative needs. Pharmacological (hydroxytyrosol) and molecular (siRNA) interventions of ALOX12 in older NHFs suppressed their ability to stimulate proliferation of PDAC cells. We conclude that chronological aging of NHFs contributes to PDAC progression and that ALOX12 and 12-(S)-HETE may be potential stromal targets for interventions that seek to halt progression and improve therapy outcomes.

Personalised orodispersible films (ODFs) manufactured at the point of care offer the possibility of adapting the dosing requirements for individual patients. Inkjet printing was extensively explored as a tool to produce personalised ODFs, but it is extensively limited to dispensing liquid with low viscosity and the interaction between ink and edible substrate complicates the fabrication process. In this study, we evaluated the feasibility of using a micro-dispensing (MD) jet system capable of accurately dispensing viscous liquid to fabricate substrate-free ODFs on-demand. The model inks containing hydroxypropyl methylcellulose (HPMC) and paracetamol were used to prepare personalised ODFs by expanding the film area. Cast films were used as the control sample to benchmark the mechanical properties, disintegration time, and dosing accuracy of MD printed ODFs. Both the cast and printed films showed smooth surface morphology without any bubbles. No significant difference was found in the disintegration time of the MD printed films compared to the cast films. High precision in dosing by MD printing was achieved. The dose of paracetamol had a linear correlation with the dimension of the printed films (R2 = 0.995). The results provide clear evidence of the potential of MD printing to fabricate ODFs and the knowledge foundation of advancing MD printing to a point-of-care small-batch manufacturing technology of personalised ODFs.

miR-486 is a myogenic microRNA, and its reduced skeletal muscle expression is observed in muscular dystrophy. Transgenic overexpression of miR-486 using muscle creatine kinase promoter (MCK-miR-486) partially rescues muscular dystrophy phenotype. We had previously demonstrated reduced circulating and skeletal muscle miR-486 levels with accompanying skeletal muscle defects in mammary tumor models. To determine whether skeletal muscle miR-486 is functionally similar in dystrophies and cancer, we performed functional limitations and biochemical studies of skeletal muscles of MMTV-Neu mice that mimic HER2+ breast cancer and MMTV-PyMT mice that mimic luminal subtype B breast cancer and these mice crossed to MCK-miR-486 mice. miR-486 significantly prevented tumor-induced reduction in muscle contraction force, grip strength, and rotarod performance in MMTV-Neu mice. In this model, miR-486 reversed cancer-induced skeletal muscle changes, including loss of p53, phospho-AKT, and phospho-laminin alpha 2 (LAMA2) and gain of hnRNPA0 and SRSF10 phosphorylation. LAMA2 is a part of the dystrophin-associated glycoprotein complex, and its loss of function causes congenital muscular dystrophy. Complementing these beneficial effects on muscle, miR-486 indirectly reduced tumor growth and improved survival, which is likely due to systemic effects of miR-486 on production of pro-inflammatory cytokines such as IL-6. Thus, similar to dystrophy, miR-486 has the potential to reverse skeletal muscle defects and cancer burden.

miR-486 is a muscle-enriched microRNA, or “myomiR,” that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized mir-486 knockout mice (mir-486 KO). mir-486 KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in mir-486 KO:mdx5cv (DKO) mice. To identify direct in vivo miR-486 muscle target transcripts, we integrated RNA sequencing and chimeric miRNA eCLIP sequencing to identify key transcripts and pathways that contribute towards mir-486 KO and dystrophic disease pathologies. These targets included known and novel muscle metabolic and dystrophic structural remodeling factors of muscle and skeletal muscle contractile transcript targets. Together, our studies identify miR-486 as essential for normal muscle function, a driver of pathological remodeling in dystrophin-deficient muscle, a useful biomarker for dystrophic disease progression, and highlight the use of multiple omic platforms to identify in vivo microRNA target transcripts.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease caused by a pathogenic disruption of the DYSTROPHIN gene that results in non-functional dystrophin protein. DMD patients experience loss of ambulation, cardiac arrhythmia, metabolic syndrome, and respiratory failure. At the molecular level, the lack of dystrophin in the muscle results in myofiber death, fibrotic infiltration, and mitochondrial dysfunction. There is no cure for DMD, although dystrophin-replacement gene therapies and exon-skipping approaches are being pursued in clinical trials. Mitochondrial dysfunction is one of the first cellular changes seen in DMD myofibers, occurring prior to muscle disease onset and progresses with disease severity. This is seen by reduced mitochondrial function, abnormal mitochondrial morphology and impaired mitophagy (degradation of damaged mitochondria). Dysfunctional mitochondria release high levels of reactive oxygen species (ROS), which can activate pro-inflammatory pathways such as IL-1β and IL-6. Impaired mitophagy in DMD results in increased inflammation and further aggravates disease pathology, evidenced by increased muscle damage and increased fibrosis. This review will focus on the critical interplay between mitophagy and inflammation in Duchenne muscular dystrophy as a pathological mechanism, as well as describe both candidate and established therapeutic targets that regulate these pathways.

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease that is caused by the loss of functional dystrophin protein in cardiac and skeletal muscles. DMD patient muscles become weakened, leading to eventual myofiber breakdown and replacement with fibrotic and adipose tissues. Inflammation drives the pathogenic processes through releasing inflammatory cytokines and other factors that promote skeletal muscle degeneration and contributing to the loss of motor function. Selective inhibitors of nuclear export (SINEs) are a class of compounds that function by inhibiting the nuclear export protein exportin 1 (XPO1). The XPO1 protein is an important regulator of key inflammatory and neurological factors that drive inflammation and neurotoxicity in various neurological and neuromuscular diseases. Here, we demonstrate that SINE compound KPT-350 can ameliorate dystrophic-associated pathologies in the muscles of DMD models of zebrafish and mice. Thus, SINE compounds are a promising novel strategy for blocking dystrophic symptoms and could be used in combinatorial treatments for DMD., Hightower et al. demonstrate the therapeutic use of a SINE (selective inhibitor of nuclear export) compound, KPT-350, to treat Duchenne muscular dystrophy (DMD) pathologies in two vertebrate animal models. The authors demonstrate these effects are due to an increase in a pro-regenerative macrophage population in dystrophic skeletal muscles. KPT-350 is in clinical trials for other neuromuscular disorders and may hold promise to ameliorate DMD symptoms.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle disorder resulting in muscle weakness and cardiomyopathy. MicroRNAs have been shown to play essential roles in muscle development, metabolism, and disease pathologies. We demonstrated that miR-486 expression is reduced in DMD muscles and its expression levels correlate with dystrophic disease severity. MicroRNA-486 knockout mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects that were exacerbated on the dystrophic mdx5cv background. We integrated RNA-sequencing and chimeric eCLIP-sequencing data to identify direct in vivo targets of miR-486 and associated dysregulated gene signatures in skeletal muscle. In comparison to our DMD mouse muscle transcriptomes, we identified several of these miR-486 muscle targets including known modulators of dystrophinopathy disease symptoms. Together, our studies identify miR-486 as a driver of muscle remodeling in DMD, a useful biomarker for dystrophic disease progression, and highlight chimeric eCLIP-sequencing as a useful tool to identify direct in vivo microRNA target transcripts.Abstract Figure

The MDX mouse is an animal model of Duchenne muscular dystrophy, a human disease marked by an absence of the cytoskeletal protein, dystrophin. We hypothesized that 1) dystrophin serves a complex mechanical role in skeletal muscles by contributing to passive compliance, viscoelastic properties, and contractile force production and 2) age is a modulator of passive mechanics of skeletal muscles of the MDX mouse. Using an in vitro biaxial mechanical testing apparatus, we measured passive length-tension relationships in the muscle fiber direction as well as transverse to the fibers, viscoelastic stress-relaxation curves, and isometric contractile properties. To avoid confounding secondary effects of muscle necrosis, inflammation, and fibrosis, we used very young 3-wk-old mice whose muscles reflected the prefibrotic and prenecrotic state. Compared with controls, 1) muscle extensibility and compliance were greater in both along fiber direction and transverse to fiber direction in MDX mice and 2) the relaxed elastic modulus was greater in dystrophin-deficient diaphragms. Furthermore, isometric contractile muscle stress was reduced in the presence and absence of transverse fiber passive stress. We also examined the effect of age on the diaphragm length-tension relationships and found that diaphragm muscles from 9-mo-old MDX mice were significantly less compliant and less extensible than those of muscles from very young MDX mice. Our data suggest that the age of the MDX mouse is a determinant of the passive mechanics of the diaphragm; in the prefibrotic/prenecrotic stage, muscle extensibility and compliance, as well as viscoelasticity, and muscle contractility are altered by loss of dystrophin.

Pharmacological ascorbate (P-AscH−, high-dose, intravenous vitamin C) is cytotoxic to tumor cells in doses achievable in humans. Phase I studies in pancreatic cancer (PDAC) utilizing P-AscH− have demonstrated increases in progression free survival, suggesting a reduction in metastatic disease burden. The purpose of this study was to determine the effects of P-AscH− on metastatic PDAC. Several in vitro and in vivo mechanisms involved in PDAC metastases were investigated following treatment with P-AscH−. Serum from PDAC patients in clinical trials with P-AscH− were tested for the presence and quantity of circulating tumor cell-derived nucleases. P-AscH− inhibited invasion, basement membrane degradation, decreased matrix metalloproteinase expression, as well as clonogenic survival and viability during exposure to fluid shear stress. In vivo, P-AscH− significantly decreased formation of ascites, tumor burden over time, circulating tumor cells, and hepatic metastases. Both in vitro and in vivo findings were reversed with the addition of catalase suggesting that the effect of P-AscH− on metastatic disease is mediated by hydrogen peroxide. Finally, P-AscH− decreased CTC-derived nucleases in subjects with stage IV PDAC in a phase I clinical trial. We conclude that P-AscH− attenuates the metastatic potential of PDAC and may prove to be effective for treating advanced disease.

DOCK3 is a member of the DOCK family of guanine nucleotide exchange factors that function to regulate cell migration, fusion, and overall viability. Previously, we identified a miR-486/Dock3 signaling cascade that was dysregulated in dystrophin-deficient muscle which resulted in the overexpression ofDOCK3, however not much else is known about the role of DOCK3 in muscle. In this work, we characterize the functional role of DOCK3 in normal and dystrophic skeletal muscle. By utilizingDock3global knockout (Dock3KO) mice, we found reducingDock3gene via haploinsufficiency in DMD mice improved dystrophic muscle histology, however complete loss ofDock3worsened overall muscle function on a dystrophin-deficient background. Consistent with this,Dock3KO mice have impaired muscle architecture and myogenic differentiation defects. Moreover, transcriptomic analyses ofDock3knockout muscles reveal a decrease in factors known for myogenesis, suggesting a possible mechanism of action. These studies identifyDOCK3as a novel modulator of muscle fusion and muscle health and may yield additional therapeutic targets for treating dystrophic muscle symptoms.

Esophageal atresia (EA/TEF) is a common congenital abnormality present in 1 of 4000 births. Here we show that atretic esophagi lack Noggin (NOG) expression, resulting in immature esophagus that contains respiratory glands. Moreover, when using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOUs) as a model of foregut development and differentiation in vitro, NOG determines whether foregut progenitors differentiate toward esophageal or tracheal epithelium. These results indicate that NOG is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis, and its lack of expression results in EA/TEF.

Transgenic animals are widely used for research and for most of them, genotyping is unavoidable. Published protocols may be powerful but may also present disadvantages such as their cost or the requirement of additional steps/equipment. Moreover, if more than one strain must be genotyped, several protocols may need to be developed.we adapted the existing amplification-resistant mutation protocol to develop the 1-h universal genotyping protocol (1-HUG), which allows the robust genotyping of genetically modified mice in 1 h from sample isolation to polymerase chain reaction gel running.This protocol allows the genotyping of different mouse models including mdx mouse, and FLExDUX4 and HSA-MerCreMer alone or in combination. It can be applied to different types of genomic modifications and to sexing.The 1-HUG protocol can be used routinely in any laboratory using mouse models for neuromuscular diseases.

There is a tremendous clinical need to improve local therapies for pancreatic cancer, with one-third of patients dying due to the consequences of local disease. Local therapy includes ionizing radiation either alone or in combination with chemotherapy. Thus, there are significant numbers of patients who would benefit from improvements in the efficacy of radio- and chemotherapy of pancreatic cancer. Aberrant redox conditions are observed more frequently in pancreatic cancer cells than in their normal counterparts. Exposure of dividing cells to low concentrations of oxidants actually stimulates cell growth and division, while higher concentrations result in growth arrest and necrosis. Pharmacologic ascorbate (P-AscH−) given intravenously produces high concentrations of hydrogen peroxide (H2O2) that are cytotoxic to tumor cells, whereas normal cells are resistant to this oxidative insult. In addition, P-AscH− selectively sensitizes pancreatic cancer cells to ionizing radiation but protects normal pancreatic ductal epithelia, intestinal epithelia, and vascular endothelium against radiation-induced injury. P-AscH− combined with chemoradiation is safe and well-tolerated and may lead to overall clinical benefit in patients with pancreatic cancer. The fundamental difference in redox conditions of tumor versus normal cells enables P-AscH− radiosensitization in tumor cells and radioprotection of normal cells.

HIF-1α is a transcriptional regulator that functions in the adaptation of cells to hypoxic conditions; it strongly impacts the prognosis of patients with cancer. High-dose, intravenous, pharmacological ascorbate (P-AscH-), induces cytotoxicity and oxidative stress selectively in cancer cells by acting as a pro-drug for the delivery of hydrogen peroxide (H2O2); early clinical data suggest improved survival and inhibition of metastasis in patients being actively treated with P-AscH-. Previous studies have demonstrated that activation of HIF-1α is necessary for P-AscH- sensitivity. We hypothesized that pancreatic cancer (PDAC) progression and metastasis could be be targeted by P-AscH- via H2O2-mediated inhibition of HIF-1α stabilization. Our study demonstrates an oxygen- and prolyl hydroxylase-independent regulation of HIF-1α by P-AscH-. Additionally, P-AscH- decreased VEGF secretion in a dose-dependent manner that was reversible with catalase, consistent with an H2O2-mediated mechanism. Pharmacological and genetic manipulations of HIF-1α did not alter P-AscH--induced cytotoxicity. In vivo, P-AscH- inhibited tumor growth and VEGF expression. We conclude that P-AscH- suppresses the levels of HIF-1α protein in hypoxic conditions through a post-translational mechanism. These findings suggest potential new therapies specifically designed to inhibit the mechanisms that drive metastases as a part of PDAC treatment.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the DYSTROPHIN (DMD) gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α (PITPNA) in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of PITPNA expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of Pitpna was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. PITPNA knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, PITPNA knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest PIPTNA as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.

Summary Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)—a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 up-regulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future., Graphical abstract Tumor-propagating cells (TPCs) drive cancer growth, yet mechanisms regulating TPC self-renewal and maintenance are largely unknown. Ignatius et al. show that the NOTCH1/SNAIL1 pathway synergizes with RAS to expand TPCs in embryonal rhabdomyosarcoma. This pathway blocks MEF2C-induced differentiation and enables the de-differentiation of ERMS cells into self-renewing TPCs.

Pharmacologic ascorbate treatment (P-AscH−, high-dose, intravenous vitamin C) results in a transient short-term increase in the flux of hydrogen peroxide that is preferentially cytotoxic to cancer cells versus normal cells. This study examines whether an increase in hydrogen peroxide is sustained posttreatment and potential mechanisms involved in this process. Cellular bioenergetic profiling following treatment with P-AscH− was examined in tumorigenic and nontumorigenic cells. P-AscH− resulted in sustained increases in the rate of cellular oxygen consumption (OCR) and reactive oxygen species (ROS) in tumor cells, with no changes in nontumorigenic cells. Sources for this increase in ROS and OCR were DUOX 1 and 2, which are silenced in pancreatic ductal adenocarcinoma, but upregulated with P-AscH− treatment. An inducible catalase system, to test causality for the role of hydrogen peroxide, reversed the P-AscH−–induced increases in DUOX, whereas DUOX inhibition partially rescued P-AscH−–induced toxicity. In addition, DUOX was significantly downregulated in pancreatic cancer specimens compared with normal pancreas tissues. Together, these results suggest that P-AscH−–induced toxicity may be enhanced by late metabolic shifts in tumor cells, resulting in a feed-forward mechanism for generation of hydrogen peroxide and induction of metabolic stress through enhanced DUOX expression and rate of oxygen consumption. Significance: A high dose of vitamin C, in addition to delivering an acute exposure of H2O2 to tumor cells, activates DUOX in pancreatic cancer cells, which provide sustained production of H2O2.

The recent availability and development of mutant and transgenic zebrafish strains that model human muscular dystrophies has created new research opportunities for therapeutic development. Not only do these models mimic many pathological aspects of human dystrophies, but their small size, large clutch sizes, rapid ex utero development, body transparency, and genetic tractability enable research approaches that would be inconceivable with mammalian model systems. Here we discuss the use of zebrafish models of muscular dystrophy to rapidly screen hundreds to thousands of bioactive compounds in order to identify novel therapeutic candidates that modulate pathologic phenotypes. We review the justification and rationale behind this unbiased approach, including how zebrafish screens have identified FDA-approved drugs that are candidates for treating Duchenne and limb girdle muscular dystrophies. Not only can these drugs be re-purposed for treating dystrophies in a fraction of the time and cost of new drug development, but their identification has revealed novel, unexpected directions for future therapy development. Phenotype-driven zebrafish drug screens are an important compliment to the more established mammalian, target-based approaches for rapidly developing and validating therapeutics for muscular dystrophies.

Printed electronics (PE) technology shows huge promise for the realisation of low-cost and flexible electronics, with the ability to pattern heat- or pressure-sensitive materials. In future developments of the PE market, the ability to produce highly conductive, high-resolution patterns using low-cost and roll-to-roll processes, such as inkjet printing, is a critical technology component for the fabrication of printed electronics and displays. Here, we demonstrate inkjet printing of polyacrylic acid (PAA) capped silver nanoparticle dispersions onto paper for high-conductivity electronic interconnects. We characterise the resulting print quality, feature geometry and electrical performance of inkjet patterned features and demonstrate the high-resolution printing, sub-100 micron feature size, of silver nanoparticle materials onto flexible paper substrate. Printed onto photo-paper, these materials then undergo chemically triggered sintering on exposure to chloride contained in the paper. We investigated the effect of substrate temperature on the properties of printed silver material from room temperature to 50 &deg, C. At room temperature, the resistivity of single layer printed features, of average thickness of 500 nm and width 85 &micro, m, was found to be 2.17 &times, 10&minus, 7 &Omega, &middot, m or 13 times resistivity of bulk silver (RBS). The resistivity initially decreased with an increase in material thickness, when achieved by overprinting successive layers or by decreasing print pitch, and a resistivity of around 10 times RBS was observed after overprinting two times at pitch 75 &micro, m and with single pass print pitch of between 60 and 80 &micro, m, resulting in line thickness up to 920 nm. On further increases in thickness the resistivity increased and reached 27 times RBS at print pitch of 15 &micro, m. On moderate heating of the substrate to 50 &deg, C, more compact silver nanoparticle films were formed, reducing thickness to 200 nm from a single pass print, and lower material resistivity approaching five times RBS was achieved.

Objective Skeletal muscle regeneration relies on muscle-specific adult stem cells (MuSCs), MuSC progeny, muscle progenitor cells (MPCs), and a coordinated myogenic program that is influenced by the extracellular environment. Following injury, MPCs undergo a transient and rapid period of population expansion, which is necessary to repair damaged myofibers and restore muscle homeostasis. Certain pathologies (e.g., metabolic diseases and muscle dystrophies) and advanced age are associated with dysregulated muscle regeneration. The availability of serine and glycine, two nutritionally non-essential amino acids, is altered in humans with these pathologies, and these amino acids have been shown to influence the proliferative state of non-muscle cells. Our objective was to determine the role of serine/glycine in MuSC/MPC function. Methods Primary human MPCs (hMPCs) were used for in vitro experiments, and young (4–6 mo) and old (>20 mo) mice were used for in vivo experiments. Serine/glycine availability was manipulated using specially formulated media in vitro or dietary restriction in vivo followed by downstream metabolic and cell proliferation analyses. Results We identified that serine/glycine are essential for hMPC proliferation. Dietary restriction of serine/glycine in a mouse model of skeletal muscle regeneration lowered the abundance of MuSCs 3 days post-injury. Stable isotope-tracing studies showed that hMPCs rely on extracellular serine/glycine for population expansion because they exhibit a limited capacity for de novo serine/glycine biosynthesis. Restriction of serine/glycine to hMPCs resulted in cell cycle arrest in G0/G1. Extracellular serine/glycine was necessary to support glutathione and global protein synthesis in hMPCs. Using an aged mouse model, we found that reduced serine/glycine availability augmented intermyocellular adipocytes 28 days post-injury. Conclusions These studies demonstrated that despite an absolute serine/glycine requirement for MuSC/MPC proliferation, de novo synthesis was inadequate to support these demands, making extracellular serine and glycine conditionally essential for efficient skeletal muscle regeneration., Highlights • Extracellular serine and glycine are necessary for muscle stem/progenitor cell population expansion. • Human muscle progenitor cells possess limited capacity for de novo serine/glycine biosynthesis. • Extracellular serine/glycine restriction increases reactive oxygen species and reduces intracellular glutathione. • Extracellular serine/glycine inhibits protein synthesis and cell cycle arrest. • Reduced extracellular serine and glycine in old mice impairs skeletal muscle regeneration.

Narrowing of the thoracic inlet leading to airway compression is a rare and challenging condition in the pediatric population. Reports in the literature have described this variant related to multiple conditions including double crush phenomenon following repair of pectus excavatum, anterior spinal displacement, and straight back syndrome. Underlying genetic conditions such as Marfan's Syndrome and Hurler's Syndrome have also been reported to contribute to clinically significant airway compression independent of dynamic tracheal collapse such as tracheomalacia. The borders of the thoracic inlet are anatomically bound by the body of the first thoracic vertebrae (T1) posteriorly, the posterior surface of the manubrium anteriorly, and the medial aspects of the first ribs on either side laterally. Relief of tracheal compression in this location is complicated by the rigidity of the boney thoracic inlet and limited space for lifting procedures such as anterior aortopexy. Several operative approaches to treat this condition have been described including manubrial/sternal resection, first rib resection, and reconstruction of the thoracic inlet. Described here are three patients where successful reconstruction of the thoracic inlet was achieved using autologous rib graft sternoplasty and a titanium sternal plating system to widen the thoracic inlet and eliminate external compression on the trachea.

Chemoradiation has remained the standard of care treatment for many of the most aggressive cancers. However, despite effective toxicity to cancer cells, current chemoradiation regimens are limited in efficacy due to significant normal cell toxicity. Thus, efforts have been made to identify agents demonstrating selective toxicity, whereby treatments simultaneously sensitize cancer cells to and protect normal cells from chemoradiation. Pharmacological ascorbate (intravenous infusions of vitamin C resulting in plasma ascorbate concentrations >20 mM; P-AscH(−)) has demonstrated selective toxicity in a variety of pre-clinical tumor models and is currently being assessed as an adjuvant to standard-of-care therapies in several early phase clinical trials. This review summarizes the most current pre-clinical and clinical data available demonstrating the multidimensional role of P-AscH(−) in cancer therapy including: selective toxicity to cancer cells via a hydrogen peroxide (H(2)O(2))-mediated mechanism; action as a sensitizing agent of cancer cells to chemoradiation; a protectant of normal tissues exposed to chemoradiation; and it’s safety and tolerability in clinical trials.

Health status is determined by the balance of oxidants and antioxidants which protects healthy cells against the threat of internal and external risk factors. Antioxidants such as ascorbate (vitamin C, ascorbic acid) are of fundamental importance in this respect. Ascorbate neutralizes potential damage caused by cellular oxidative stress which may be the greatest risk of damage to healthy tissue. Cellular oxidative stress is mediated by external factors (e.g. psychological stress, physical exertion, drugs, various diseases, environmental pollution, preservatives, smoking, and alcohol) and internal factors (products of cellular metabolism including reactive oxygen species). When the products of oxidative stress are not sufficiently neutralized, healthy cells are at risk for both mitochondrial and DNA damage. In the short term, cell function may deteriorate, while an increased production of proinflammatory cytokines over time may lead to the development of chronic inflammatory changes and diseases, including cancer. Although pharmaceutical research continues to bring effective chemotherapeutic agents to the market, a limiting factor is often the normal tissue and organ toxicity of these substances, which leads to oxidative stress on healthy tissue. There is increasing interest and imperative to protect healthy tissues from the negative effects of radio-chemotherapeutic treatment. The action of ascorbate against the development of oxidative stress may justify its use not only in the prevention of carcinogenesis, but as a part of supportive or complementary therapy during treatment. Ascorbate (particularly when administered parentally at high doses) may have antioxidant effects that work to protect healthy cells and improve patient tolerability to some toxic radio-chemotherapy regimens. Additionally, ascorbate has demonstrated an immunomodulatory effect by supporting mechanisms essential to anti-tumor immunity. Intravenous administration of gram doses of vitamin C produce high plasma levels immediately, but the levels drop rapidly. Following oral vitamin C administration, plasma levels increase slowly to relatively low values, and then gradually decay. With an oral liposomal formulation, significantly higher levels are attainable than with standard oral formulations. Therefore, oral administration of liposomal vitamin C appears to be an optimal adjunct to intravenous administration. In this review, the basic mechanisms and clinical benefits of ascorbate as an antioxidant that may be useful as complementary therapy to chemotherapeutic regimens will be discussed.

Pharmacologic ascorbate (P-AscH(–)) is emerging as a promising adjuvant for advanced pancreatic cancer. P-AscH(–)generates hydrogen peroxide (H(2)O(2)), leading to selective cancer cell cytotoxicity. Catalytic manganoporphyrins, such as MnT4MPyP, can increase the rate of oxidation of P-AscH(–), thereby increasing the flux of H(2)O(2), resulting in increased cytotoxicity. We hypothesized that a multimodal treatment approach, utilizing a combination of P-AscH(–), ionizing radiation and MnT4MPyP, would result in significant flux of H(2)O(2) and pancreatic cancer cytotoxicity. P-AscH(–) with MnT4MPyP increased the rate of oxidation of P-AscH(–)and produced radiosensitization in all pancreatic cancer cell lines tested. Three-dimensional (3D) cell cultures demonstrated resistance to P-AscH(–), radiation or MnT4MPyP treatments alone; however, combined treatment with P-AscH(–) and MnT4MPyP resulted in the inhibition of tumor growth, particularly when also combined with radiation. In vivo experiments using a murine model demonstrated an increased rate of ascorbate oxidation when combinations of P-AscH(–) with MnT4MPyP were given, thus acting as a radiosensitizer. The translational potential was demonstrated by measuring increased ascorbate oxidation ex vivo, whereby MnT4MPyP was added exogenously to plasma samples from patients treated with P-AscH(–) and radiation. Combination treatment utilizing P-AscH(–), manganoporphyrin and radiation results in significant cytotoxicity secondary to enhanced ascorbate oxidation and an increased flux of H(2)O(2). This multimodal approach has the potential to be an effective treatment for pancreatic ductal adenocarcinoma.

Purpose We sought to evaluate value impact of transition from an adult trauma center treating children (ATC) to a verified pediatric trauma center (PTC) in children with blunt splenic injury (BSI). Methods Children with BSI from FY 2005 to FY 2017 were extracted from the hospital trauma registry. February 2009 distinguished “ATC” treated children from “PTC” treated children. Cohorts were subcategorized into “isolated injury” and “multisystem injury”. Quality and financial characteristics were statistically compared. Analysis of covariance was used to evaluate changes in quality and financial trends over the transition period. A multiple linear regression was performed to identify variables independently predictive of hospital and professional charges. Results 126 children with BSI were identified (ATC, n = 56; PTC, n = 70). Splenic procedure rates and hospital charges decreased. Quality and cost metrics for isolated BSI remained unchanged while multisystem BSI children experienced improvements. PTC designation, ISS, splenic procedure, isolated BSI, average hospital LOS, and mortality were all independently predictive of hospital and professional charges. Conclusions PTC verification improves the value of BSI management, but the associated decrease in operative rate is only partially responsible. Multisystem injury children experience the greatest value benefit from PTC verification. Type of study Treatment and cost-effectiveness study. Level of evidence Level III.

Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH−, i.e., intravenous infusions of ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH− decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H2O2)-mediated mechanism. In this study, we demonstrate that P-AscH− radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH−, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH− in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH− during the radiotherapy “beam on.” Specifically, treatment with P-AscH− increased median overall survival compared with our institutional average (21.7 vs. 12.7 months, P = 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH−–treated subjects was also greater than our institutional average (13.7 vs. 4.6 months, P < 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH− in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH− efficacy is warranted in a phase II clinical trial. Significance: These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.

Metastatic disease is the leading cause of pancreatic ductal adenocarcinoma (PDAC) associated death. PDAC cells invade and enter the bloodstream early, before frank malignancy can be detected. Our objective was to develop an in vivo assay enabling the identification and quantification of circulating tumor cells (CTCs) from primary orthotopic PDAC tumors. Human PDAC cells expressing luciferase and green fluorescent protein were orthotopically injected into the pancreas of mice utilizing ultrasound guidance. Bioluminescent imaging was conducted to identify and track tumor growth. CTCs were then isolated and analyzed by flow cytometry to detect GFP-expressing cancer cells. Tumor growth as measured by bioluminescent imaging increased over time. The concentration of CTCs correlated with the strength of bioluminescent imaging signal. In addition, livers bearing macroscopic disease were harvested for further imaging under fluorescence stereomicroscopy and confocal microscopy, which confirmed the presence of metastases. This study represents an orthotopic animal model that reliably detects the presence of CTCs from PDAC. There is a positive correlation between the concentrations of CTCs with overall tumor burden.

MicroRNAs (miRNAs) are small 21-24 nucleotide RNAs that are capable of regulating multiple signaling pathways across multiple tissues. MicroRNAs are dynamically regulated and change in expression levels during periods of early development, tissue regeneration, cancer, and various other disease states. Recently, microRNAs have been isolated from whole serum and muscle biopsies to identify unique diagnostic signatures for specific neuromuscular disease states. Functional studies of microRNAs in cell lines and animal models of neuromuscular diseases have elucidated their importance in contributing to neuromuscular disease progression and pathologies. The ability of microRNAs to alter the expression of an entire signaling pathway opens up their unique ability to be used as potential therapeutic entry points for the treatment of disease. Here, we will review the recent findings of key microRNAs and their dysregulation in various neuromuscular diseases. Additionally, we will highlight the current strategies being used to regulate the expression of key microRNAs as they have become important players in the clinical treatment of some of the neuromuscular diseases.