Association Between Body Fat Response to Exercise Training and Multilocus ADR Genotypes

Objectives : To examine the contribution of adrenergic receptor (ADR) gene polymorphisms and their gene‐gene interactions to the variability of exercise training‐induced body fat response. Research Methods and Procedures : This was an intervention study that used a volunteer sample of 70 healthy, sedentary men (n = 29) and postmenopausal women (n = 41) 50 to 75 years of age, with a BMI ≤37 kg/m², from the Washington, DC, metropolitan area. Participants completed 6 weeks of dietary stabilization (American Heart Association diet) before 24 weeks of supervised aerobic exercise training. Diet was maintained throughout the intervention. Change in percent total body fat, percent trunk fat, and fat mass by DXA in ADR genotype groups (Glu¹²/Glu⁹ α2b‐ADR, Trp64Arg β3‐ADR, and Gln27Glu β2‐ADR) at baseline and after 24 weeks of aerobic exercise training was measured. Results : In multivariate analysis (covariates: age, gender, and baseline value of phenotype), best fit models for percent total body and trunk fat response to exercise training retained main effects of all three ADR gene loci and the effects of each gene‐gene interaction (p = 0.009 and 0.003, respectively). Similarly, there was a trend for the fat mass response model (p = 0.03). The combined genetic factors explained 17.5% of the overall model variability for percent total body fat, 22% for percent trunk fat, and 10% for fat mass. Discussion : The body fat response to exercise training in older adults is associated with the combined effects of the Glu¹²/Glu⁹ α2b‐, Trp64Arg β3‐, and Gln27Glu β2‐ADR gene variants and their gene‐gene interactions.     

Scalable GMP compliant suspension culture system for human ES cells

Suspension bioreactors are an attractive alternative to static culture of human embryonic stem cells (hESCs) for the generation of clinically relevant cell numbers in a controlled system. In this study, we have developed a scalable suspension culture system using serum-free defined media with spinner flasks for hESC expansion as cell aggregates. With optimized cell seeding density and splitting interval, we demonstrate prolonged passaging and expansion of several hESC lines with overall expansion, yield, viability and maintenance of pluripotency equivalent to adherent culture. Human ESCs maintained in suspension as aggregates can be passaged at least 20 times to achieve over 1×10(13) fold calculated expansion with high undifferentiation rate and normal karyotype. Furthermore, the aggregates are able to differentiate to cardiomyocytes in a directed fashion. Finally, we show that the cells can be cryopreserved in serum-free medium and thawed into adherent or suspension cultures to continue passaging and expansion. We have successfully used this method under cGMP or cGMP-equivalent conditions to generate cell banks of several hESC lines. Taken together, our suspension culture system provides a powerful approach for scale-up expansion of hESCs under defined and serum-free conditions for clinical and research applications.     

Future Impact of Various Interventions on the Burden of COPD in Canada: A Dynamic Population Model

Background

Chronic obstructive pulmonary disease (COPD) is a growing economic burden worldwide. Smoking cessation is thought to be the single most effective way of reducing the economic burden of COPD. The impact of other strategies such as interventions that predict risk of disease, reduce progression of disease, or reduce exacerbations has not been systematically studied.

Objectives

We estimated the economic and clinical burden of COPD over the next 25 years in Canada and the impact of three potential interventions (screening test for predisposition to COPD, new drugs to avoid progression into more severe disease stages, and predictive test for exacerbations) on COPD burden.

Methods

Using a dynamic simulation model, we projected the total burden of COPD (cost, morbidity, and mortality) from 2011 to 2035 using the population of Canada as a case study. The model stratified population based on sex, age, smoking status, respiratory symptoms, and their COPD stage. The cost and quality adjusted life years (QALYs) associated with each intervention were estimated.

Results

The model indicates that annual societal cost of COPD is $4.52 billion (B) Canadian dollars in 2011 and will reach $3.61B ($7.33B undiscounted) per year in 2035. Over the next 25 years, COPD will be responsible for approximately $101.4B in societal costs ($147.5B undiscounted) and 12.9 million QALYs lost (19.0 million undiscounted). Our results suggested that the best strategy to reduce the financial burden of COPD is by reducing exacerbations. Smoking cessation, while it is the cornerstone of COPD prevention, has only a modest effect in attenuating the financial burden of COPD over the next 25 years in Western countries such as Canada.

Conclusion

Our data suggest that any intervention that can reduce the number of exacerbations has a substantial impact on morbidity and costs of COPD and should be considered in conjunction with the ongoing efforts to reduce smoking rates.     

Targeted Ablation of Nesprin 1 and Nesprin 2 from Murine Myocardium Results in Cardiomyopathy,Altered Nuclear Morphology and Inhibition of the Biomechanical Gene Response

Recent interest has focused on the importance of the nucleus and associated nucleoskeleton in regulating changes in cardiac gene expression in response to biomechanical load. Mutations in genes encoding proteins of the inner nuclear membrane and nucleoskeleton, which cause cardiomyopathy, also disrupt expression of a biomechanically responsive gene program. Furthermore, mutations in the outer nuclear membrane protein Nesprin 1 and 2 have been implicated in cardiomyopathy. Here, we identify for the first time a role for the outer nuclear membrane proteins, Nesprin 1 and Nesprin 2, in regulating gene expression in response to biomechanical load. Ablation of both Nesprin 1 and 2 in cardiomyocytes, but neither alone, resulted in early onset cardiomyopathy. Mutant cardiomyocytes exhibited altered nuclear positioning, shape, and chromatin positioning. Loss of Nesprin 1 or 2, or both, led to impairment of gene expression changes in response to biomechanical stimuli. These data suggest a model whereby biomechanical signals are communicated from proteins of the outer nuclear membrane, to the inner nuclear membrane and nucleoskeleton, to result in changes in gene expression required for adaptation of the cardiomyocyte to changes in biomechanical load, and give insights into etiologies underlying cardiomyopathy consequent to mutations in Nesprin 1 and 2.     

Metabolic Reprogramming during Purine Stress in the Protozoan Pathogen Leishmania donovani

The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over three months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6–48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and/or post-translational mechanisms.     

Year-long presence of Eimeria echidnae and absence of Eimeria tachyglossi in captive short-beaked echidnas ( Tachyglossus aculeatus )

The short-beaked echidna ( Tachyglossus aculeatus ) is 1 of 5 extant species of monotreme, found only in Australia and Papua New Guinea. The aim of this study was to identify the species of coccidia present and establish a range of subclinical Eimeria spp. (Coccidia: Apicomplexa) oocyst shedding in echidnas from eastern Australia over 18 mo. The coccidia were detected in 89% (49/55) of fecal samples from 12 long-term monitored and healthy captive echidnas, 75% (3/4) of 4 healthy long-term captive echidnas, 83% (5/6) of 6 short-term captive echidnas, and 60% (6/10) of 10 wild echidnas. Echidnas captive for 4 to 23 yr shed 100-46,000 oocysts g(-1) of E. echidnae and remained clinically healthy during this study. Sub-adult and adult wild, and short-term captive, echidnas shed oocysts of both E. echidnae and E. tachyglossi . The lack of coccidia in juvenile short-beaked echidnas suggests these animals are probably non-immune and should not be placed in environments heavily contaminated with oocysts. In addition, no oocysts were found in captive long-beaked echidnas ( Zaglossus bartoni bartoni , n = 2) housed at Taronga Zoo. This study represents an important step in understanding the host-parasite interaction between coccidia and short-beaked echidnas.     

Identification of a truncated form of methionine sulfoxide reductase a expressed in mouse embryonic stem cells

Background  

Methionine Sulfoxide Reductase A (MsrA), an enzyme in the Msr gene family, is important in the cellular anti-oxidative stress defense mechanism. It acts by reducing the oxidized methionine sulfoxide in proteins back to sulfide and by reducing the cellular level of reactive oxygen species. MsrA, the only enzyme in the Msr gene family that can reduce the S-form epimers of methionine sulfoxide, has been located in different cellular compartments including mitochondria, cytosol and nuclei of various cell lines.     

Activation of NAD(P)H oxidase by lipid hydroperoxides: mechanism of oxidant-mediated smooth muscle cytotoxicity

Oxidized lipids, such as 13-hydroperoxyoctadecadienoic acid (13-HPODE), have been implicated in the pathogenesis of atherosclerosis. 13-HPODE, a constituent of oxidized low-density lipoproteins, can induce cytotoxicity of vascular smooth muscle cells (SMC), which may facilitate plaque destabilization and/or rupture. 13-HPODE-induced cytotoxicity has been linked to oxidative stress, although the mechanisms by which this occurs are unknown. In the present study, we show that 13-HPODE and 9-HPODE (10-30 microM) increased superoxide (O2*-) production and induced cytotoxicity in SMC. The 13-HPODE-induced increase in O2*- was blocked by transfecting the cells with antisense oligonucleotides against p22phox, suggesting that the O2*- was produced by NAD(P)H oxidase. Similar concentrations of the corresponding HPODE reduction products, 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE, neither increased O2*- production nor induced cytotoxicity, while 4-hydroxy nonenal (4-HNE), an unsaturated aldehyde lipid peroxidation product, induced cytotoxicity without increasing O2*- production. Treatment with superoxide dismutase or Tiron to scavenge O2*-, or transfection with p22phox antisense oligonucleotides to inhibit O2*- production, attenuated 13-HPODE-induced cytotoxicity, but not that induced by 4-HNE. These findings suggest that activation of NAD(P)H oxidase, and production of O2*-, play an important role in lipid hydroperoxide-induced smooth muscle cytotoxicity.     

Decrease in Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice

Respiratory syncytial virus (RSV) is a high priority target for vaccine development. One concern in RSV vaccine development is that a non-live virus vaccine would predispose for enhanced disease similar to that seen with the formalin inactivated RSV (FI-RSV) vaccine. Since a mAb specific to RSV G protein can reduce pulmonary inflammation and eosinophilia seen after RSV infection of FI-RSV vaccinated mice, we hypothesized that RSV G peptides that induce antibodies with similar reactivity may limit enhanced disease after subunit or other non-live RSV vaccines. In support of this hypothesis, we show that FI-RSV vaccinated mice administered RSV G peptide vaccines had a significant reduction in enhanced disease after RSV challenge. These data support the importance of RSV G during infection to RSV disease pathogenesis and suggest that use of appropriately designed G peptide vaccines to reduce the risk of enhanced disease with non-live RSV vaccines merits further study.     

Synthesis and anticancer activity of novel bisindolylhydroxymaleimide derivatives with potent GSK-3 kinase inhibition

Synthesis and biological evaluation of a series of novel indole derivatives as anticancer agents is described. A bisindolylmaleimide template has been derived as a versatile pharmacophore with which to pursue chemical diversification. Starting from maleimide, the introduction of an oxygen to the headgroup (hydroxymaleimide) was initially investigated and the bioactivity assessed by screening of kinase inhibitory activity, identifying substituent derived selectivity. Extension of the hydroxymaleimide template to incorporate substitution of the indole nitrogens was next completed and assessed again by kinase inhibition identifying unique selectivity patterns with respect to GSK-3 and CDK kinases. Subsequently, the anticancer activity of bisindolylmaleimides were assessed using the NCI-60 cell screen, disclosing the discovery of growth inhibitory profiles towards a number of cell lines, such as SNB-75 CNS cancer, A498 and UO-31 renal, MDA MB435 melanoma and a panel of leukemia cell lines. The potential for selective kinase inhibition by modulation of this template is evident and will inform future selective clinical candidates.