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A first-in-human trial of diaphragmatic gene therapy (AAV1-CMV-GAA) to treat respiratory and neural dysfunction in early-onset Pompe disease was conducted. The primary objective of this study was to assess the safety of rAAV1-CMV-hGAA vector delivered to the diaphragm muscle of Pompe disease subjects with ventilatory insufficiency. Safety was assessed by measurement of change in serum chemistries and hematology, urinalysis, and immune response to GAA and AAV, as well as change in level of health. The data demonstrate that the AAV treatment was safe and there were no adverse events related to the study agent. Adverse events related to the study procedure were observed in subjects with lower baseline neuromuscular function. All adverse events were resolved before the end of the study, except for one severe adverse event determined not to be related to either the study agent or the study procedure. In addition, an anti-capsid and anti-transgene antibody response was observed in all subjects who received rAAV1-CMV-hGAA, except for subjects who received concomitant immunomodulation to manage reaction to enzyme replacement therapy, as per their standard of care. This observation is significant for future gene therapy studies and serves to establish a clinically relevant approach to blocking immune responses to both the AAV capsid protein and transgene product.

Objective: Infants born to mothers with obesity have greater adiposity, ectopic fat storage, and are at increased risk for childhood obesity and metabolic disease compared with infants of normal weight mothers, though the cellular mechanisms mediating these effects are unclear., Methods: We tested the hypothesis that human, umbilical cord-derived mesenchymal stem cells (MSCs) from infants born to obese (Ob-MSC) versus normal weight (NW-MSC) mothers demonstrate altered fatty acid metabolism consistent with adult obesity. In infant MSCs undergoing myogenesis in vitro, we measured cellular lipid metabolism and AMPK activity, AMPK activation in response to cellular nutrient stress, and MSC DNA methylation and mRNA content of genes related to oxidative metabolism., Results: We found that Ob-MSCs exhibit greater lipid accumulation, lower fatty acid oxidation (FAO), and dysregulation of AMPK activity when undergoing myogenesis in vitro. Further experiments revealed a clear phenotype distinction within the Ob-MSC group where more severe MSC metabolic perturbation corresponded to greater neonatal adiposity and umbilical cord blood insulin levels. Targeted analysis of DNA methylation array revealed Ob-MSC hypermethylation in genes regulating FAO (PRKAG2, ACC2, CPT1A, SDHC) and corresponding lower mRNA content of these genes. Moreover, MSC methylation was positively correlated with infant adiposity., Conclusions: These data suggest that greater infant adiposity is associated with suppressed AMPK activity and reduced lipid oxidation in MSCs from infants born to mothers with obesity and may be an important, early marker of underlying obesity risk., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

TBC1 domain family member 1 (TBC1D1), a Rab GTPase-activating protein and paralogue of Akt substrate of 160 kDa (AS160), has been implicated in both insulin- and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase-mediated glucose transporter type 4 (GLUT4) translocation. However, the role of TBC1D1 in contracting muscle remains ambiguous. We therefore explored the metabolic consequence of ablating TBC1D1 in both resting and contracting skeletal muscles, utilizing a rat TBC1D1 KO model. Although insulin administration rapidly increased ( p < 0.05) plasma membrane GLUT4 content in both red and white gastrocnemius muscles, the TBC1D1 ablation did not alter this response nor did it affect whole-body insulin tolerance, suggesting that TBC1D1 is not required for insulin-induced GLUT4 trafficking events. Consistent with findings in other models of altered TBC1D1 protein levels, whole-animal and ex vivo skeletal muscle fat oxidation was increased in the TBC1D1 KO rats. Although there was no change in mitochondrial content in the KO rats, maximal ADP-stimulated respiration was higher in permeabilized muscle fibers, which may contribute to the increased reliance on fatty acids in resting KO animals. Despite this increase in mitochondrial oxidative capacity, run time to exhaustion at various intensities was impaired in the KO rats. Moreover, contraction-induced increases in sarcolemmal GLUT4 content and glucose uptake were lower in the white gastrocnemius of the KO animals. Altogether, our results highlight a critical role for TBC1D1 in exercise tolerance and contraction-mediated translocation of GLUT4 to the plasma membrane in skeletal muscle., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Purpose of Review: Insulin resistance is an important risk factor for metabolic diseases such as type 2 diabetes, cardiovascular disease and certain cancers. A common characteristic of strategies that improve insulin sensitivity involves the activation of the energy sensing enzyme of the cell, AMP-activated protein kinase (AMPK). The purpose of this review is to explore the mechanisms associated with AMPK activation to improve insulin sensitivity with a focus on fatty acid metabolism. We will also discuss the literature surrounding direct AMPK activators to improve insulin resistance and important considerations for the design of direct AMPK activators., Recent Findings: AMPK activation can decrease de novo lipogenesis, increase fatty acid oxidation and promote mitochondrial integrity to improve insulin sensitivity. Drugs targeted to directly activate AMPK show therapeutic promise, yet in vivo data is lacking., Summary: Designing a drug to directly activate AMPK may improve insulin resistance by reducing liver de novo lipogenesis and increasing brown and white adipose tissue mitochondrial function. However, in vivo experimental procedures to support this notion are not extensive and more research is required.

Intestinal dysbiosis contributes to obesity and insulin resistance, but intervening with antibiotics, prebiotics, or probiotics can be limited by specificity or sustained changes in microbial composition. Postbiotics include bacterial components such as lipopolysaccharides, which have been shown to promote insulin resistance during metabolic endotoxemia. We found that bacterial cell wall-derived muramyl dipeptide (MDP) is an insulin-sensitizing postbiotic that requires NOD2. Injecting MDP lowered adipose inflammation and reduced glucose intolerance in obese mice without causing weight loss or altering the composition of the microbiome. MDP reduced hepatic insulin resistance during obesity and low-level endotoxemia. NOD1-activating muropeptides worsened glucose tolerance. IRF4 distinguished opposing glycemic responses to different types of peptidoglycan and was required for MDP/NOD2-induced insulin sensitization and lower metabolic tissue inflammation during obesity and endotoxemia. IRF4 was dispensable for exacerbated glucose intolerance via NOD1. Mifamurtide, an MDP-based drug with orphan drug status, was an insulin sensitizer at clinically relevant doses in obese mice., (Copyright © 2017 Elsevier Inc. All rights reserved.)

X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Fluazaindolizine is a new highly effective and selective product for the control of plant parasitic nematodes. Specificity for nematodes coupled with absence of activity against the target sites of commercial nematicides suggests that fluazaindolizine has a novel mode of action. The discovery, structure-activity development and biological properties for this new class of nematicides are presented., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pompe disease is an inherited disorder due to a mutation in the gene that encodes acid α-glucosidase (GAA). Children with infantile-onset Pompe disease develop progressive hypotonic weakness and cardiopulmonary insufficiency that may eventually require mechanical ventilation (MV). Our team conducted a first in human trial of diaphragmatic gene therapy (AAV1-CMV-GAA) to treat respiratory neural dysfunction in infantile-onset Pompe. Subjects (aged 2-15years, full-time MV: n=5, partial/no MV: n=4) underwent a period of preoperative inspiratory muscle conditioning exercise. The change in respiratory function after exercise alone was compared to the change in function after intramuscular delivery of AAV1-CMV-GAA to the diaphragm with continued exercise. Since AAV-mediated gene therapy can reach phrenic motoneurons via retrograde transduction, we hypothesized that AAV1-CMV-GAA would improve dynamic respiratory motor function to a greater degree than exercise alone. Dependent measures were maximal inspiratory pressure (MIP), respiratory responses to inspiratory threshold loads (load compensation: LC), and physical evidence of diaphragm activity (descent on MRI, EMG activity). Exercise alone did not change function. After AAV1-CMV-GAA, MIP was unchanged. Flow and volume LC responses increased after dosing (p<0.05 to p<0.005), but only in the subjects with partial/no MV use. Changes in LC tended to occur on or after 180days. At Day 180, the four subjects with MRI evidence of diaphragm descent had greater maximal voluntary ventilation (p<0.05) and tended to be younger, stronger, and use fewer hours of daily MV. In conclusion, combined AAV1-CMV-GAA and exercise training conferred benefits to dynamic motor function of the diaphragm. Children with a higher baseline neuromuscular function may have greater potential for functional gains., (Copyright © 2016 Elsevier Inc. All rights reserved.)

An important step to incriminate a mosquito as a vector of a disease pathogen is finding evidence of direct contact between the mosquito and humans. Typically, this is accomplished through landing/biting catches, or host blood meal analysis in engorged mosquitoes via immunologic assays. An alternate approach is to identify the presence of specific mosquito anti-saliva protein antibodies in the blood of exposed hosts. Following the discovery of dengue infected, free roaming non-human primates in Puerto Rico, we investigated which mosquito species had bitten these primates using a serologic assay. Serum samples from 20 patas monkeys (Erythrocebus patas) and two rhesus macaques (Macaca mulatta) were used to evaluate mosquito bite exposure to Aedes aegypti, Aedes mediovittatus, Aedes taeniorhynchus, and Culex quinquefasciatus mosquitoes. Of 22 non-human primates examined 20 (90%), 17 (77%), 13 (59%), and 7 (31%) were positive for exposure to Ae. mediovittatus, Cx. quinquefasciatus, Ae. taeniorhynchus, and Ae. aegypti, respectively. Our findings indicated that free-roaming primates in Puerto Rico were exposed to the bites of one proven dengue vector, Ae. aegypti and one potential dengue vector, Ae. mediovittatus., (Published by Elsevier B.V.)

Despite widespread use of statins to reduce low-density lipoprotein cholesterol (LDL-C) and associated atherosclerotic cardiovascular risk, many patients do not achieve sufficient LDL-C lowering due to muscle-related side effects, indicating novel treatment strategies are required. Bempedoic acid (ETC-1002) is a small molecule intended to lower LDL-C in hypercholesterolemic patients, and has been previously shown to modulate both ATP-citrate lyase (ACL) and AMP-activated protein kinase (AMPK) activity in rodents. However, its mechanism for LDL-C lowering, efficacy in models of atherosclerosis and relevance in humans are unknown. Here we show that ETC-1002 is a prodrug that requires activation by very long-chain acyl-CoA synthetase-1 (ACSVL1) to modulate both targets, and that inhibition of ACL leads to LDL receptor upregulation, decreased LDL-C and attenuation of atherosclerosis, independently of AMPK. Furthermore, we demonstrate that the absence of ACSVL1 in skeletal muscle provides a mechanistic basis for ETC-1002 to potentially avoid the myotoxicity associated with statin therapy., Competing Interests: S.L.P., R.S.N., C.M.B., S.F., P.H.E.G., G.R.S. and N.D.L. received compensation from Esperion Therapeutics, Inc. The remaining authors declare no competing financial interests.

Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the β1 subunit, but whether salsalate requires AMPK-β1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-β1-knockout (AMPK-β1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (∼1 mmol/L). Salsalate treatment increased VO 2 , lowered fasting glucose, improved glucose tolerance, and led to an ∼55% reduction in liver lipid content. These effects were observed in both WT and AMPK-β1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton conductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling., Competing Interests: Duality of Interest. No potential conflicts of interest relevant to this article were reported., (© 2016 by the American Diabetes Association.)

Background: Mechanical ventilation (MV) during a cardio-thoracic surgery contributes to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction. Especially, it is critical in older adults who have lower muscle reparative capacity following MV. Reports have shown that the intraoperative intermittent hemidiaphragm electrical stimulation can maintain and/or improve post-surgery diaphragm function. In particular, from a molecular point of view, intermittent ES may reduce oxidative stress and increase regulatory autophagy levels, and therefore improve diaphragm function in animal studies. We have recently shown in humans that intraoperative ES attenuates mitochondrial dysfunction and force decline in single diaphragm muscle fibers. The aim of this study was to investigate an effect of ES on oxidative stress, antioxidant status and autophagy biomarker levels in the human diaphragm during surgery., Methods: One phrenic nerve was simulated with an external cardiac pacer in operated older subjects (62.4 ± 12.9 years) (n = 8) during the surgery. The patients received 30 pulses per min every 30 min. The muscle biopsy was collected from both hemidiaphragms and frozen for further analyses. 4-hydroxynonenal (4-HNE), an oxidative stress marker, and autophagy marker levels (Beclin-1 and the ratio of microtubule-associated protein light chain 3, I and II-LC3 II/I) protein concentrations were detected by the western blot technique. Antioxidant enzymatic activity copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutase were analyzed., Results: Levels of lipid peroxidation (4-HNE) were significantly lower in the stimulated side (p < 0.05). The antioxidant enzyme activities (CuZnSOD and MnSOD) in the stimulated side of the diaphragm were not different than in the unstimulated side (p > 0.05). Additionally, the protein concentrations of Beclin-1 and the LC3 II/I ratio were higher in the stimulated side (p < 0.05)., Conclusion: These results suggest that the intraoperative electrical stimulation decreases oxidative stress levels and upregulates autophagy levels in the stimulated hemidiaphragm. These results may contribute future studies and clinical applications on reducing post-operative diaphragm dysfunction.

Nonalcoholic fatty liver disease (NAFLD) is a growing worldwide epidemic and an important risk factor for the development of insulin resistance, type 2 diabetes, nonalcoholic steatohepatitis (NASH), and hepatic cellular carcinoma (HCC). Despite the prevalence of NAFLD, lifestyle interventions involving exercise and weight loss are the only accepted treatments for this disease. Over the last decade, numerous experimental compounds have been shown to improve NAFLD in preclinical animal models, and many of these therapeutics have been shown to increase the activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Because AMPK activity is reduced by inflammation, obesity, and diabetes, increasing AMPK activity has been viewed as a viable therapeutic strategy to improve NAFLD. In this review, we propose three primary mechanisms by which AMPK activation may improve NAFLD. In addition, we examine the mechanisms by which AMPK is activated. Finally, we identify 27 studies that have used AMPK activators to reduce NAFLD. Future considerations for studies examining the relationship between AMPK and NAFLD are highlighted., (Copyright © 2016 the American Physiological Society.)

Canagliflozin, dapagliflozin, and empagliflozin, all recently approved for treatment of type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose reuptake by sodium/glucose cotransporter (SGLT) 2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMPK, an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin, or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of acetyl-CoA carboxylase (ACC) 1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue, or spleen. Because phosphorylation of ACC by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared with other SGLT2 inhibitors., (© 2016 by the American Diabetes Association.)

Objective: The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s) remain unclear., Methods: Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1) was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin., Results: Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions., Conclusion: These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations.

Background: Hemophagocytic lymphohistiocytosis (HLH) is a rare, potentially fatal disorder characterized by fever, pancytopenia, hepatosplenomegaly, and increased serum ferritin. HLH is being increasingly reported as a complication of dengue, a common tropical acute febrile illness., Methodology/principal Findings: After a cluster of pediatric dengue-associated HLH patients was identified during the 2012-2013 dengue epidemic in Puerto Rico, active surveillance and a case-control investigation was conducted at four referral hospitals to determine the incidence of HLH in children and identify risk factors for HLH following dengue. Patients with dengue-associated HLH (cases) were matched by month of illness onset and admission hospital to dengue patients that did not develop HLH (controls). During 2008-2013, a total of 33 HLH patients were identified, of which 22 (67%) were associated with dengue and 1 died (dengue-associated HLH case-fatality rate: 4.5%). Two patients with dengue-associated HLH had illness onset in 2009, none had illness onset during the 2010 dengue epidemic, and 20 had illness onset during the 2012-2013 epidemic. Frequency of infection with either dengue virus (DENV)-1 or DENV-4 did not differ between cases and controls. Cases were younger than controls (median age: 1 vs. 13 years, p < 0.01), were hospitalized longer (18 vs. 5 days, p < 0.01), and were admitted more frequently to pediatric intensive care units (100% vs. 16%, p < 0.01). Cases had co-infection (18.2% vs. 4.5%, p = 0.04), recent influenza-like illness (54.5% vs. 25.0%, p = 0.01), and longer duration of fever (7 vs. 5 days; p < 0.01). Cases were more likely to have lymphadenopathy, hepatomegaly, splenomegaly, anemia, and elevated liver transaminases (p ≤ 0.02)., Conclusions/significance: During this cluster of dengue-associated HLH cases that was temporally associated with the 2012-2013 epidemic, most patients with dengue-associated HLH were infants and had higher morbidity than dengue inpatients. Physicians throughout the tropics should be aware of HLH as a potential complication of dengue, particularly in patients with anemia and severe liver injury., Competing Interests: The authors have declared that no competing interests exist.

Brown (BAT) and white (WAT) adipose tissues play distinct roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes. The AMP-activated protein kinase (AMPK) is a cellular energy sensor, but its role in regulating BAT and WAT metabolism is unclear. We generated an inducible model for deletion of the two AMPK β subunits in adipocytes (iβ1β2AKO) and found that iβ1β2AKO mice were cold intolerant and resistant to β-adrenergic activation of BAT and beiging of WAT. BAT from iβ1β2AKO mice had impairments in mitochondrial structure, function, and markers of mitophagy. In response to a high-fat diet, iβ1β2AKO mice more rapidly developed liver steatosis as well as glucose and insulin intolerance. Thus, AMPK in adipocytes is vital for maintaining mitochondrial integrity, responding to pharmacological agents and thermal stress, and protecting against nutrient-overload-induced NAFLD and insulin resistance., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Integrated (0 + 5) vascular surgery (VS) residency programs must include 24 months of training in core general surgery. The Accreditation Council for Graduate Medical Education currently does not require specific case numbers in general surgery for 0 + 5 trainees; however, program directors have structured this time to optimize operative experience. The aim of this study is to determine the case volume and type of cases that VS residents are exposed to during their core surgery training., Design: Accreditation council for graduate medical education operative logs for current 0 + 5 VS residents were obtained and retrospectively reviewed to determine general surgery case volume and distribution between open and laparoscopic cases performed. Standard statistical methods were applied., Setting: A total of 12 integrated VS residency programs provided operative case logs for current residents., Participants: A total of 41 integrated VS residents in clinical years 2 through 5., Results: During the postgraduate year-1 training year, residents participated in significantly more open than laparoscopic general surgery cases (p < 0.0001). This difference was consistent over the first 3 years of training. The most frequently logged open general surgery cases are hernia repair (20%), skin and soft tissue (7.4%), and breast (6.3%). Residents in programs with core surgery over 3 years participated in significantly more general surgery operations compared with residents in programs with core surgery spread out over 4 years (p = 0.035)., Conclusions: 0 + 5 VS residents perform significantly more open operations than laparoscopic operations during their core surgery training. The majority of these operations are minor, nonabdominal procedures. The 0 + 5 VS residency program general surgery operative training requirements should be reevaluated and case minimums defined. The general surgery training component of 0 + 5 VS residencies may need to be restructured to meet the needs of current and future trainees., (Copyright © 2016 Association of Program Directors in Surgery. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Pompe disease is an inherited disorder notable for severe, progressive ventilatory compromise. Although ventilatory failure has been attributed to myofiber dysfunction secondary to diaphragmatic glycogen accumulation, neural involvement of the phrenic motor system is also a prominent feature. Direct diaphragm pacing supplements respiratory function in other disorders of the phrenic motor system. Accordingly, it is hypothesized that augmented neuromuscular activity via diaphragm pacing would promote weaning from mechanical ventilation in patients with Pompe disease who are unresponsive to conventional, muscle-directed treatments., Case Description: Three patients with Pompe disease developed diaphragm paresis that resulted in chronic mechanical ventilation dependence. After preoperative inspiratory muscle strengthening exercises failed to improve function, fine-wire pacing electrodes were laparoscopically implanted into the diaphragm. Diaphragm conditioning was initiated the first postoperative week and consisted of gradual increases in stimulation parameters, lengthening of stimulation sessions, and ventilator weaning. Ventilation and intramuscular electromyographic activity were recorded periodically during conditioning to quantify diaphragm neuromuscular function., Outcomes: During paced breathing without mechanical ventilation, tidal volumes increased, and 2 patients were weaned from daytime ventilator dependence within the first 3 months of pacing, which has been sustained over the long-term. A third patient reduced reliance on daytime ventilation, but weaning was delayed by malacia of the large airways. In all patients, pacing appeared to facilitate spontaneous phrenic motor unit activity during independent breathing without ventilator or pacer support., Discussion: The findings are consistent with the view that diaphragm pacing has potential rehabilitative value to reduce reliance on mechanical ventilation in people with Pompe disease, but further study is needed. Diaphragm pacing represents a paradigm shift in the management of respiratory insufficiency for Pompe disease that warrants further controlled examination., (© 2016 American Physical Therapy Association.)

Objectives: Investigate both the utility and feasibility of perioperative nutritional supplementation with an arginine-enriched immunonutrition formula to high-risk head and neck cancer surgical patients and examine its effects on acute post-operative clinical outcomes., Materials & Methods: This prospective, non-randomized, interventional cohort study compared high-risk head and neck cancer surgical patients who consumed a pre- and post-operative arginine-based nutritional supplement to those that did not. Outcome measures included post-operative complications, length of hospitalization, readmission rates and measurement of nutritional biomarkers., Results: 195 high-risk head and neck cancer surgical patients were enrolled. 59% of the patients used the nutritional supplement, 41% did not. Of the 80 patients who did not receive the immunonutrition formula, 38 (47.5%) experienced post-operative complications of all types as compared to 29 of the 115 (25.2%) patients who did consume the product (p=0.0021). Pharyngeal leaks or fistulas were the most common post-operative complications in both groups and more common in patients who did not receive supplementation (p=0.007). Length of stay was on average 2.8 days longer in patients who did not have enhanced nutrition (p=0.02), while readmission rates between the two groups were similar (p=0.91). Measurements of nutritional biomarkers were not reported secondary to low collection rates., Conclusion: Enhanced perioperative nutrition may result in significant reductions of post-operative fistula formations and decreased length of stay in a high-risk head and neck cancer population, even in the setting of poor compliance. The potential quality improvement in both patient care and healthcare cost is both real and significant., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pompe disease is an inherited neuromuscular disorder that affects respiratory function and leads to dependence on external ventilatory support. We studied the activation of the diaphragm using bilateral phrenic magnetic stimulation and hypothesized that diaphragm compound muscle action potential (CMAP) amplitude and evoked transdiaphragmatic pressure (Twitch PDI) would correlate to disease severity. Eight patients with late onset Pompe disease (LOPD, aged 14-48 years) and four healthy control subjects completed the tests. Maximal Twitch PDI responses were progressively reduced in patients with LOPD compared to control subjects (1.4-17.1cm H2O, p<0.001) and correlated to voluntary functional tests (p<0.05). Additionally, CMAP amplitude (mA) was lower in the patients who used nighttime or fulltime ventilatory support, when compared to controls and patients who used no ventilatory support (p<0.005). However, the normalized (%peak) Twitch PDI and CMAP responses were similar between patients and controls. This suggests a loss of functional phrenic motor units in patients, with normal recruitment of remaining motor units., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: I NTRODUCTION: Individuals with X-linked myotubular myopathy (XLMTM) and other centronuclear myopathies (CNMs) frequently have profound respiratory insufficiency that requires support early in life. Still, few quantitative data exist to characterize respiratory motor function in CNM., Methods: We evaluated the reliance upon mechanical ventilation (MV), ventilatory kinematics, unassisted tidal volumes, and maximal respiratory pressures in 14 individuals with CNMs, including 10 boys with XLMTM., Results: Thirteen participants required full-time, invasive MV. Maximal inspiratory pressures were higher in subjects who breathed unsupported at least 1 hour/day as compared with 24-hour MV users [33.7 (11.9-42.3) vs. 8.4 (6.0-10.9) cm H(2)O, P < 0.05]. Years of MV dependence correlated significantly with MEP (r = -0.715, P < 0.01)., Conclusions: Respiratory function in CNMs may be related to deconditioning from prolonged MV and/or differences in residual respiratory muscle strength. Results from this study may assist in evaluating severe respiratory insufficiency in neuromuscular clinical care and research., (© 2015 Wiley Periodicals, Inc.)

Since 2000, the UK government has funded surveys aimed at understanding the UK public's attitudes toward science, scientists, and science policy. Known as the Public Attitudes to Science series, these surveys and their predecessors have long been used in UK science communication policy, practice, and scholarship as a source of authoritative knowledge about science-related attitudes and behaviors. Given their importance and the significant public funding investment they represent, detailed academic scrutiny of the studies is needed. In this essay, we critically review the most recently published Public Attitudes to Science survey (2014), assessing the robustness of its methods and claims. The review casts doubt on the quality of key elements of the Public Attitudes to Science 2014 survey data and analysis while highlighting the importance of robust quantitative social research methodology. Our analysis comparing the main sample and booster sample for young people demonstrates that quota sampling cannot be assumed equivalent to probability-based sampling techniques., (© The Author(s) 2016.)

Revascularization for acute mesenteric ischemia (AMI) can be achieved through a bypass from the aorta or iliac arteries, embolectomy, open exposure of SMA and retrograde recanalization and stent, or percutaneous antegrade stenting. Flush occlusion of the SMA can make antegrade recanalization very challenging and is usually unsuccessful. We present a novel approach for recanalization of superior mesenteric artery (SMA) via the celiac artery for acute mesenteric ischemia. A 69-year-old lady with previous endarterectomy of SMA and extensive small bowel resection presented with severe abdominal pain, emesis, leukocytosis, and imaging finding of new SMA flush occlusion. She refused to consent for a laparotomy. Percutaneous retrograde transcollateral recanalization of SMA was performed via the celiac artery through the pancreaticoduodenal arcade, and the SMA then stented. This resulted in subsequent resolution of patient's symptoms and discharge. SMA revascularization with retrograde transcollateral wiring technique is an important tool in the armamentarium of the vascular care specialist when antegrade percutaneous approach and open exposure via laparotomy are not an option., Competing Interests: The authors have no funding and conflicts of interest to disclose.

Background: Loss-of-function mutations in the myotubularin (MTM1) gene cause X-linked myotubular myopathy (XLMTM), a fatal, inherited pediatric disease that affects the entire skeletal musculature. Labrador retriever dogs carrying an MTM1 missense mutation exhibit strongly reduced synthesis of myotubularin, the founder member of a lipid phosphatase required for normal skeletal muscle function. The resulting canine phenotype resembles that of human patients with comparably severe mutations, and survival does not normally exceed 4 months., Methods: We studied MTM1 mutant dogs (n=7) and their age-matched control littermates (n=6) between the ages of 10 and 25 weeks. Investigators blinded to the animal identities sequentially measured limb muscle pathology, fore- and hind limb strength, walking gait, respiratory function and neurological impairment., Results: MTM1-mutant puppies display centrally-nucleated myofibers of reduced size and disrupted sarcotubular architecture progressing until the end of life, an average of 17 weeks. In-life measures of fore- and hind limb strength establish the rate at which XLMTM muscles weaken, and their corresponding decrease in gait velocity and stride length. Pulmonary function tests in affected dogs reveal a right-shifted relationship between peak inspiratory flow (PIF) and inspiratory time (TI); neurological assessments indicate that affected puppies as young as 10 weeks show early signs of neurological impairment (neurological severity score, NSS =8.6±0.9) with progressive decline (NSS =5.6±1.7 at 17 weeks-of-age)., Conclusions: Our findings document the rate of disease progression in a large animal model of XLMTM and lay a foundation for preclinical studies.

Dengue virus (DENV) nonstructural-1 (NS1) glycoprotein is useful for diagnosis of DENV infections in the first 8 days of illness with any of the four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). However, NS1 diagnostics are less sensitive for secondary DENV infections so the utility of NS1 diagnostics in dengue endemic countries where there is predominantly secondary infections is being questioned. Heat-mediated immunecomplex dissociation (ICD) prior to testing serum samples can significantly improve NS1 test sensitivity in secondary infections but requires monoclonal antibodies (MAbs) reactive to heat-denatured NS1. In order to incorporate a simple heat-mediated ICD step, a crucial step was to develop new MAbs with high affinity and specificity to heat-denatured DENV NS1 protein. In the present study, six new MAbs were isolated from BALB/c mice immunized with recombinant monomeric NS1 of DENV-1 and DENV-2. Characterization using three different methods: indirect ELISA, fixed cell ELISA and western blot revealed that all six MAbs are serotype-cross-reactive and capable of recognizing dimeric and hexameric isoforms as well as heat-denatured NS1 from all four DENV serotypes. No cross-reactivity to NS1 of West Nile virus and Yellow fever virus was observed on western blot and indirect ELISA. Five of the six MAbs mapped to the DENV NS1 region of 105-119 amino acids. The remaining MAb mapped to DENV NS1 region of 25-39 amino acids. These two NS1 regions were found to be highly conserved among all four DENV serotypes by sequences analysis and database comparison. These MAbs were used to develop an NS1 capture ELISA and tested using a small panel of clinical specimens. The results from the NS1 capture ELISA indicated at least a three-fold increase in NS1 antigen detection in heat-denatured samples compared to untreated specimens. Furthermore, artificial immunecomplexed results also demonstrated the binding efficiency of these MAbs to heat denatured NS1. Taken together, these MAbs allow for the incorporation of a heat dissociation step to improve the sensitivity of DENV NS1 antigen detection in secondary infections., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mitochondrial ADP transport may represent a convergence point unifying two prominent working models for the development of insulin resistance, as reactive lipids (specifically palmitoyl-CoA [P-CoA]) can inhibit ADP transport and subsequently increase mitochondrial reactive oxygen species emissions. In the current study, we aimed to determine if exercise training in humans diminished P-CoA attenuation of mitochondrial ADP respiratory sensitivity. Six weeks of exercise training increased whole-body glucose homeostasis and skeletal muscle Akt signaling and reduced markers of oxidative stress without reducing maximal mitochondrial H2O2 emissions. To ascertain if enhanced mitochondrial ADP transport contributed to the improvement in the in vivo oxidative state, we determined mitochondrial ADP sensitivity in the presence and absence of P-CoA. In the absence of P-CoA, exercise training reduced mitochondrial ADP sensitivity. In contrast, exercise training increased mitochondrial ADP sensitivity with P-CoA present. We further show that P-CoA noncompetitively inhibits mitochondrial ADP transport and the ability of ADP to attenuate mitochondrial H2O2 emission. Altogether, the current data provide a potential mechanism for how P-CoA contributes to insulin resistance and highlight the ability of exercise training to diminish P-CoA attenuation in mitochondrial ADP transport., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Objective: Skeletal muscle AMP-activated protein kinase (AMPK) is important for regulating glucose homeostasis, mitochondrial content and exercise capacity. R419 is a mitochondrial complex-I inhibitor that has recently been shown to acutely activate AMPK in myotubes. Our main objective was to examine whether R419 treatment improves insulin sensitivity and exercise capacity in obese insulin resistant mice and whether skeletal muscle AMPK was important for mediating potential effects., Methods: Glucose homeostasis, insulin sensitivity, exercise capacity, and electron transport chain content/activity were examined in wildtype (WT) and AMPK β1β2 muscle-specific null (AMPK-MKO) mice fed a high-fat diet (HFD) with or without R419 supplementation., Results: There was no change in weight gain, adiposity, glucose tolerance or insulin sensitivity between HFD-fed WT and AMPK-MKO mice. In both HFD-fed WT and AMPK-MKO mice, R419 enhanced insulin tolerance, insulin-stimulated glucose disposal, skeletal muscle 2-deoxyglucose uptake, Akt phosphorylation and glucose transporter 4 (GLUT4) content independently of alterations in body mass. In WT, but not AMPK-MKO mice, R419 improved treadmill running capacity. Treatment with R419 increased muscle electron transport chain content and activity in WT mice; effects which were blunted in AMPK-MKO mice., Conclusions: Treatment of obese mice with R419 improved skeletal muscle insulin sensitivity through a mechanism that is independent of skeletal muscle AMPK. R419 also increases exercise capacity and improves mitochondrial function in obese WT mice; effects that are diminished in the absence of skeletal muscle AMPK. These findings suggest that R419 may be a promising therapy for improving whole-body glucose homeostasis and exercise capacity.

Introduction: Pompe disease is a progressive disease that affects skeletal muscles and leads to loss of ambulation. We investigated the activation of the tibialis anterior (TA) in late-onset Pompe disease (LOPD) individuals during maximal voluntary contraction (MVC) and evoked involuntary responses., Methods: Four LOPD patients and matched control subjects performed MVC of the TA using dorsiflexion and TA evoked responses. Activation of the TA was recorded with surface electromyography., Results: The Pompe patients exhibited greater power at frequencies below 60 Hz and reduced power above 100 Hz. They also exhibited a reduced increase in M-wave and prolonged M-wave latency and duration in response to stimulation., Conclusions: These results provide evidence that LOPD individuals have an altered activation pattern of the TA during maximal contractions. The observed activation pattern may reflect impairments in voluntary command, neuromuscular junction pathology, or compensatory drive due to a reduced number of functional motoneurons., (© 2014 Wiley Periodicals, Inc.)

Metformin is the mainstay therapy for type 2 diabetes (T2D) and many patients also take salicylate-based drugs [i.e., aspirin (ASA)] for cardioprotection. Metformin and salicylate both increase AMP-activated protein kinase (AMPK) activity but by distinct mechanisms, with metformin altering cellular adenylate charge (increasing AMP) and salicylate interacting directly at the AMPK β1 drug-binding site. AMPK activation by both drugs results in phosphorylation of ACC (acetyl-CoA carboxylase; P-ACC) and inhibition of acetyl-CoA carboxylase (ACC), the rate limiting enzyme controlling fatty acid synthesis (lipogenesis). We find doses of metformin and salicylate used clinically synergistically activate AMPK in vitro and in vivo, resulting in reduced liver lipogenesis, lower liver lipid levels and improved insulin sensitivity in mice. Synergism occurs in cell-free assays and is specific for the AMPK β1 subunit. These effects are also observed in primary human hepatocytes and patients with dysglycaemia exhibit additional improvements in a marker of insulin resistance (proinsulin) when treated with ASA and metformin compared with either drug alone. These data indicate that metformin-salicylate combination therapy may be efficacious for the treatment of non-alcoholic fatty liver disease (NAFLD) and T2D.

Background: Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes., Methods: To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1-4 per os at viral titers of 5-6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution., Results: Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus., Conclusions: This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.

Background and Objectives: Identification of hospitalized patients who smoke has shown significant improvement in recent years, but provision of evidence-based tobacco cessation treatment remains a challenge. This study evaluated the utilization of an electronic health record (EHR) to facilitate implementation of evidence-based clinical practice guidelines for smoking cessation on a vascular surgery inpatient unit., Methods: A pre-and post-intervention cohort study was conducted over 6 months at a single academic medical center with a comprehensive EHR. All patients admitted to the vascular surgery service and documented as current smokers were included. A vascular surgery discharge order set with an evidence-based smoking cessation module was developed and implemented. The primary outcome was prescription of nicotine replacement therapy (NRT) at the time of discharge. The secondary outcome was referral for smoking cessation counseling at the time of discharge., Results: There were 52 and 42 smokers in the pre-and post-intervention cohorts, respectively. Over the 3 months following implementation of the EHR order set, prescription of NRT at the time of discharge did not change significantly (27% vs 19%, p =0.30). Referral for outpatient smoking cessation counseling increased in the post-intervention group, but did not reach significance (64% vs 72%, p =0.20)., Conclusions: Implementation of a brief tobacco dependence treatment order set in an existing EHR increased cessation counseling referrals on a vascular surgery inpatient unit. One potential limitation of the study was the modest sample size. Not being able to make smoking cessation treatment a mandatory component in discharge orders may also have contributed to the modest effect. Assessing the differential effect of EHR-based order implementation will be important in future research on this topic.

Background: Dietary supplementation with almonds has demonstrated dose-dependent decreases in low-density lipoprotein cholesterol (LDL-C), likely because of their composition of beneficial nutrients including mono- and polyunsaturated fatty acids, fiber, and protein., Objective: The primary objective of this study was to determine the changes in the lipid profile (LDL-C, high-density lipoprotein cholesterol [HDL-C], triglycerides, total cholesterol, non-HDL-C), LDL-C particle size, and lipoprotein (a) when 100 g of almonds daily were added to background statin therapy for 4 weeks., Methods: Subjects (N = 48) receiving a consistent statin dose were randomized to 100 g of almonds daily and to The National Cholesterol Education Program Adult Treatment Panel's third report Therapeutic Lifestyle Changes Diet counseling (almond group; n = 22) or solely Adult Treatment Panel's third report Therapeutic Lifestyle Changes Diet counseling (non-almond group; n = 26), for 4 weeks., Results: No significant changes in weight and weekly physical activity were noted between the 2 groups from baseline. However, the almond group consumed significantly more calories at 4 weeks compared with controls. The almond group experienced a 4.9% reduction in non-HDL-C compared with a 3.5% increase for the non-almond group (P = .02). Additionally, notable improvements were observed in LDL-C and triglycerides, but did not achieve statistical significance (P = .068 for both parameters). There was also a shift from LDL pattern A to pattern B particles (P = .003) in the almond group. No significant differences in total cholesterol (P = .1), HDL-C (P = .3), or lipoprotein (a) (P = .1) were observed., Conclusion: Adding 100 g of almonds daily to chronic statin therapy for 4 weeks significantly reduced non-HDL-C., Trial Registration: clinicaltrials.gov Identifier: NCT00603876., (Copyright © 2015 National Lipid Association. Published by Elsevier Inc. All rights reserved.)

Mass spectrometry imaging (MSI) is an emerging technology for the mapping of molecular distributions in tissues. In most of the existing studies, imaging is performed by sampling on a predefined rectangular grid that does not reflect the natural cellular pattern of the tissue. Delivering laser pulses by a sharpened optical fiber in laser ablation electrospray ionization (LAESI) mass spectrometry (MS) has enabled the direct analysis of single cells and subcellular compartments. Cell-by-cell imaging had been demonstrated using LAESI-MS, where individual cells were manually selected to serve as natural pixels for tissue imaging. Here we describe a protocol for a novel cell-by-cell LAESI imaging approach that automates cell recognition and addressing for systematic ablation of individual cells. Cell types with particular morphologies can also be selected for analysis. First, the cells are recognized as objects in a microscope image. The coordinates of their centroids are used by a stage-control program to sequentially position the cells under the optical fiber tip for laser ablation. This approach increases the image acquisition efficiency and stability, and enables the investigation of extended or selected tissue areas. In the LAESI process, the ablation events result in mass spectra that represent the metabolite levels in the ablated cells. Peak intensities of selected ions are used to represent the metabolite distributions in the tissue with single-cell resolution.

Half a million patients are hospitalized with severe dengue every year, many of whom would die without timely, appropriate clinical intervention. The majority of dengue cases are uncomplicated; however, 2-5% progress to severe dengue. Severe dengue cases have been reported with increasing frequency over the last 30 years. To discover biomarkers for severe dengue, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to analyze dengue virus positive serum samples from the acute phase of infection. Using this method, 16 proteins were identified as candidate biomarkers for severe dengue. From these 16 biomarkers, three candidates were selected for confirmation by enzyme-linked immunosorbent assay and Western blot: vitronectin (Vtn, 55.1 kDa), hemopexin (Hx, 52.4 kDa), and serotransferrin (Tf, 79.2 kDa). Vitronectin, Hx, and Tf best differentiated between dengue and severe dengue., (© The American Society of Tropical Medicine and Hygiene.)

Background: Since their introduction several years ago, integrated (0 + 5) vascular surgery residency programs are being increasingly developed across the country. To date, however, there is no defined "universal" curriculum for these programs and each program is responsible for creating its own curriculum. The aim of this study was to review the experiences of current 0 + 5 program directors (PDs) to determine what factors contributed to the curricular development within their institution., Methods: Semistructured interviews were conducted with 0 + 5 PDs to explore their experiences with program development, factors influencing the latter, and rationale for current curricula. The interview script was loosely structured to explore several factors including time of incoming residents' first exposure to the vascular surgical service, timing and rationale behind the timing of core surgical rotations throughout the 5 year program, educational value of nonsurgical rotations, opportunities for leadership and scholarly activity, and influence the general surgery program and institutional climate had on curricular structure. All interviews were conducted by a single interviewer. All interviews were qualitatively analyzed using emergent theme analysis., Results: Twenty-six 0 + 5 PDs participated in the study. A total of 69% believed establishing professional identity early reduces resident attrition and recommend starting incoming trainees on vascular surgical services. Sixty-two percent spread core surgical rotations over the first 3 years to optimize general surgical exposure and most of the programs have eliminated specific rotations, as they were not considered valuable to the goals of training. Factors considered most important by PDs in curricular development include building on existing institutional opportunities (96%), avoiding rotations considered unsuccessful by "experienced" programs (92%), and maintaining a good working relationship with general surgery (77%). Fifty-eight percent of PDs voiced concern over the lack of standardization among the differing programs and most of the PDs agree that some degree of programmatic standardization is critical for the continued success of the 0 + 5 training paradigm., Conclusions: Qualitative evaluation of PD experiences with the development of 0 + 5 vascular surgery residency programs reveals the key factors that commonly influence program design. Programs continue to evolve in both structure and content as PDs respond to these influences. Learning from the collective experience of PDs and some standardization of the curricula may help current and future programs avoid common pitfalls in curricular development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The successful management of captive animals requires attention to multiple interconnected factors. One critical aspect of the daily life of a captive animal is the recommended and/or provisioned diet. This study focuses on the diets of zoo-housed gorillas. A national survey of diets among zoo-housed gorillas was conducted to examine diets being offered to captive gorillas in the United States and Canada. This survey serves as a follow-up to a 1995 dietary survey of zoo-housed gorillas and goes further to quantify nutritional profiles at responding institutions. Results are encouraging, as zoos have made clear improvements in dietary nutrient profiles offered over the past 15 years. However, we suggest that zoological and sanctuary institutions follow Gorilla Species Survival Plan (SSP) recommendations and work to continuously improve diets provided, which could improve gorillas' health and well-being., (© 2014 Wiley Periodicals, Inc.)

The centronuclear myopathies (CNMs) are a group of inherited neuromuscular disorders classified as congenital myopathies. While several causative genes have been identified, some patients do not harbor any of the currently known mutations. These diverse disorders have common histological features, which include a high proportion of centrally nucleated muscle fibers, and clinical attributes of muscle weakness and respiratory insufficiency. Respiratory problems in CNMs may manifest initially during sleep, but daytime symptoms, ineffective airway clearance, and hypoventilation predominate as more severe respiratory muscle dysfunction evolves. Respiratory muscle capacity can be evaluated using a variety of clinical tests selected with consideration for the age and baseline motor function of the patient. Similar clinical tests of respiratory function can also be incorporated into preclinical CNM canine models to offer insight for clinical trials. Because respiratory problems account for significant morbidity in patients, routine assessments of respiratory muscle function are discussed., (© 2014 Wiley Periodicals, Inc.)

Androgens regulate body composition and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. Recently, we demonstrated that trenbolone (a potent synthetic testosterone analogue that is not a substrate for 5-alpha reductase or for aromatase) induces myotrophic effects in skeletal muscle without causing prostate enlargement, which is in contrast to the known prostate enlarging effects of testosterone. These previous results suggest that the 5α-reduction of testosterone is not required for myotrophic action. We now report differential gene expression in response to testosterone versus trenbolone in the highly androgen-sensitive levator ani/bulbocavernosus (LABC) muscle complex of the adult rat after 6weeks of orchiectomy (ORX), using real time PCR. The ORX-induced expression of atrogenes (Muscle RING-finger protein-1 [MuRF1] and atrogin-1) was suppressed by both androgens, with trenbolone producing a greater suppression of atrogin-1 mRNA compared to testosterone. Both androgens elevated expression of anabolic genes (insulin-like growth factor-1 and mechano-growth factor) after ORX. ORX-induced increases in expression of glucocorticoid receptor (GR) mRNA were suppressed by trenbolone treatment, but not testosterone. In ORX animals, testosterone promoted WNT1-inducible-signaling pathway protein 2 (WISP-2) gene expression while trenbolone did not. Testosterone and trenbolone equally enhanced muscle regeneration as shown by increases in LABC mass and in protein expression of embryonic myosin by western blotting. In addition, testosterone increased WISP-2 protein levels. Together, these findings identify specific mechanisms by which testosterone and trenbolone may regulate skeletal muscle maintenance and growth., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aims/hypothesis: Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK-ACC2-malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice., Methods: Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates., Results: ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance., Conclusions/interpretation: These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.

Objectives: Recent studies have shown that brief periods of mechanical ventilation in animals and humans can lead to ventilator-induced diaphragmatic dysfunction, which includes muscle atrophy, reduced force development, and impaired mitochondrial function. Studies in animal models have shown that short periods of increased diaphragm activity during mechanical ventilation support can attenuate ventilator-induced diaphragmatic dysfunction but corresponding human data are lacking. The purpose of this study was to examine the effect of intermittent diaphragm contractions during cardiothoracic surgery, including controlled mechanical ventilation, on mitochondrial respiration in the human diaphragm., Design: Within subjects repeated measures study., Setting: Operating room in an academic health center., Patients: Five subjects undergoing elective cardiothoracic surgery., Interventions: In patients (age 65.6 ± 6.3 yr) undergoing cardiothoracic surgery, one phrenic nerve was stimulated hourly (30 pulses/min, 1.5 msec duration, 17.0 ± 4.4 mA) during the surgery. Subjects received 3.4 ± 0.6 stimulation bouts during surgery. Thirty minutes following the last stimulation bout, samples of diaphragm muscle were obtained from the anterolateral costal regions of the stimulated and inactive hemidiaphragms., Measurements and Main Results: Mitochondrial respiration was measured in permeabilized muscle fibers with high-resolution respirometry. State III mitochondrial respiration rates (pmol O2/s/mg wet weight) were 15.05 ± 3.92 and 11.42 ± 2.66 for the stimulated and unstimulated samples, respectively (p < 0.05). State IV mitochondrial respiration rates were 3.59 ± 1.25 and 2.11 ± 0.97 in the stimulated samples and controls samples, respectively (p < 0.05)., Conclusion: These are the first data examining the effect of intermittent contractions on mitochondrial respiration rates in the human diaphragm following surgery/mechanical ventilation. Our results indicate that very brief periods (duty cycle ~1.7%) of activity can improve mitochondrial function in the human diaphragm following surgery/mechanical ventilation.

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors in response to several limitations inherent to the clinical design: 1) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; 2) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and 3) a possibly faster depletion of pathogenic myofibers in this patient population. Immune responses triggered by the first vector administration, and to subsequent ones, represent a major obstacle for successful gene transfer in young patient population. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or ERT treatment for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. This study presents a case of Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate the immune responses. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

It is recognized that diaphragm muscle plasticity occurs with mechanical overloads, yet less is known about synergistic parasternal intercostal muscle fiber remodeling. We conducted overload training with intrinsic transient tracheal occlusion (ITTO) exercises in conscious animals. We hypothesized that ITTO would yield significant fiber hypertrophy and myogenic activation that would parallel diaphragm fiber remodeling. Sprague-Dawley rats underwent placement of a tracheal cuff and were randomly assigned to receive daily 10 min sessions of conscious ITTO or observation (sham) over 2 wk. After training, fiber morphology, myosin heavy chain (MHC) isoform composition, cross-sectional area, proportion of Pax7-positive nuclei, and presence of embryonic MHC (eMHC) were quantified. Type IIx/b fibers were 20% larger after ITTO training than with sham training (ITTO: 4,431 +/– 676 μm2, sham: 3,689 +/– 400 μm2, p < 0.05), and type I fibers were more prevalent after ITTO (p < 0.01). Expression of Pax7 was increased in ITTO parasternals and diaphragm (p < 0.05). In contrast, the proportion of eMHC-positive fibers was increased only in ITTO parasternals (1.2% [3.4%–0.6%], sham: 0% [0.6%–0%], p < 0.05). Although diaphragm and parasternal type II fibers hypertrophy to a similar degree, myogenic remodeling appears to differ between the two muscles.

The prevalence of obesity is high resulting from chronic imbalances between energy intake and expenditure. On the expenditure side, regular exercise is associated with health benefits, including enhanced brain function. The benefits of exercise are not immediate and require persistence to be realized. Brain regions associated with health-related decisions, such as whether or not to exercise or controlling the impulse to engage in immediately rewarding activities (e.g., sedentary behavior), include reward processing and cognitive control regions. A 9 month aerobic exercise study will be conducted in 180 sedentary adults (n = 90 healthy weight [BMI = 18.5 to 26.0 kg/m(2)]; n = 90 obese [BMI = 29.0 to 41.0 kg/m(2)) to examine the brain processes underlying reward processing and impulse control that may affect adherence in a new exercise regimen. The primary aim is to use functional magnetic resonance imaging (fMRI) to examine reward processing and impulse control among participants that adhere (exercise >80% of sessions) and those that do not adhere to a nine-month exercise intervention with secondary analyses comparing sedentary obese and sedentary healthy weight participants. Our results will provide valuable information characterizing brain activation underlying reward processing and impulse control in sedentary obese and healthy weight individuals. In addition, our results may identify brain activation predictors of adherence and success in the exercise program along with measuring the effects of exercise and improved fitness on brain activation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: While inspiratory muscle weakness is common in prolonged mechanical ventilation, inspiratory muscle strength training (IMST) can facilitate strengthening and ventilator weaning. However, the inspiratory load compensation (ILC) responses to threshold loads are not well characterized in patients. We retrospectively compared ILC responses according to the clinical outcomes of IMST (ie, maximum inspiratory pressure [PImax], weaning outcome), in difficult-to-wean ICU patients., Methods: Sixteen tracheostomized subjects (10 weaned, 6 unweaned) from a previous clinical trial underwent IMST 5 days/week, at the highest tolerated load, in conjunction with daily, progressive spontaneous breathing trials. PImax and ILC with a 10 cm H2O load were compared in the subjects before and after IMST. Changes in ILC performance were further characterized (5, 10, 15 cm H2O loads) in the trained subjects who weaned., Results: Demographics, respiratory mechanics, and initial PImax (52 ± 26 cm H2O vs 42 ± 13 cm H2O) did not significantly differ between the groups. Upon enrollment, PImax significantly correlated with flow ILC responses with the 10 cm H2O load (r = 0.64, P = .008). After IMST, PImax significantly increased in the entire sample (P = .03). Both before and after IMST, subjects who weaned generated greater flow and volume ILC than subjects who failed to wean. Additionally, ILC flow, tidal volume, and duty cycle increased upon ventilator weaning, at loads of 5, 10, and 15 cm H2O., Conclusions: Flow ILC at a threshold load of 10 cm H2O in ventilated, tracheostomized subjects positively correlated with PImax. Although PImax improved in both groups, the flow and volume ILC responses of the weaned subjects were more robust, both before and after IMST. The results suggest that ILC response is different in weaned and unweaned subjects, reflecting dynamic inspiratory muscular efforts that could be influential in weaning.

Mitochondrial dysfunction and reactive oxygen species (ROS) have been implicated in the aetiology of skeletal muscle insulin resistance, although there is considerable controversy regarding these concepts. Mitochondrial function has been traditionally assessed in the presence of saturating ADP, but ATP turnover and the resultant ADP is thought to limit respiration in vivo. Therefore, we investigated the potential link between submaximal ADP-stimulated respiration rates, ROS generation and skeletal muscle insulin sensitivity in a model of type 2 diabetes mellitus, the ZDF rat. Utilizing permeabilized muscle fibres we observed that submaximal ADP-stimulated respiration rates (250-2000 μm ADP) were lower in ZDF rats than in lean controls, which coincided with decreased adenine nucleotide translocase 2 (ANT2) protein content. This decrease in submaximal ADP-stimulated respiration occurred in the absence of a decrease in electron transport chain function. Treating ZDF rats with resveratrol improved skeletal muscle insulin resistance and this was associated with elevated submaximal ADP-stimulated respiration rates as well as an increase in ANT2 protein content. These results coincided with a greater ability of ADP to attenuate mitochondrial ROS emission and an improvement in cellular redox balance. Together, these data suggest that mitochondrial dysfunction is present in skeletal muscle insulin resistance when assessed at submaximal ADP concentrations and that ADP dynamics may influence skeletal muscle insulin sensitivity through alterations in the propensity for mitochondrial ROS emission.