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1.  Evidence for metabolic aberrations in asymptomatic persons with type 2 diabetes after initiation of simvastatin therapy 
HMG-CoA reductase inhibitors (statins) prevent vascular events and are widely prescribed, particularly in persons with type 2 diabetes. However, intolerability due to myopathic symptoms often limits their use. We investigated the effects of simvastatin on parameters of mitochondrial function and muscle gene expression in 11 subjects with type 2 diabetes; none of whom had statin intolerance. After withdrawal of statins for two months, we obtained blood samples, performed vastus lateralis muscle biopsies, and assessed whole body resting energy expenditure (REE). We then re-initiated therapy using simvastatin, 20 mg/day, for one month before repeating these studies. As expected, simvastatin lowered LDL, but did not induce myalgias or significant elevations in serum creatine kinase. However, we found subtle but significant reductions in muscle citrate synthase activity and REE. In addition, quantitative PCR and gene set enrichment analysis of muscle samples revealed significantly repressed gene sets involved in mitochondrial function and induced gene sets involved in remodeling of the extracellular matrix. Further, the effects of simvastatin on muscle gene sets showed some similarities to previously described changes that occur in Duchenne muscular dystrophy, polymyositis, and dermatomyositis. Although statins inhibit an early step in coenzyme Q (CoQ) biosynthesis, we observed no differences in CoQ content within skeletal muscle mitochondria, muscle tissue, or circulating platelets. In summary, we report subtle changes in whole body energetics, mitochondrial citrate synthase activity, and microarray data consistent with subclinical myopathy. Although the benefits of statin therapy are clear, further understanding of muscular perturbations should help guide safety and tolerability.
doi:10.1016/j.trsl.2015.01.007
PMCID: PMC4509977  PMID: 25683525
2.  Quantitative secondary electron imaging for work function extraction at atomic level and layer identification of graphene 
Scientific Reports  2016;6:21045.
Two-dimensional (2D) materials usually have a layer-dependent work function, which require fast and accurate detection for the evaluation of their device performance. A detection technique with high throughput and high spatial resolution has not yet been explored. Using a scanning electron microscope, we have developed and implemented a quantitative analytical technique which allows effective extraction of the work function of graphene. This technique uses the secondary electron contrast and has nanometre-resolved layer information. The measurement of few-layer graphene flakes shows the variation of work function between graphene layers with a precision of less than 10 meV. It is expected that this technique will prove extremely useful for researchers in a broad range of fields due to its revolutionary throughput and accuracy.
doi:10.1038/srep21045
PMCID: PMC4754635  PMID: 26878907
3.  Modeling Linear and Cyclic PKS Intermediates through Atom Replacement 
Journal of the American Chemical Society  2014;136(48):16792-16799.
The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs.
doi:10.1021/ja5064857
PMCID: PMC4277753  PMID: 25406716
4.  Identification and Small Molecule Inhibition of an Activating Transcription Factor 4 (ATF4)-dependent Pathway to Age-related Skeletal Muscle Weakness and Atrophy* 
The Journal of Biological Chemistry  2015;290(42):25497-25511.
Background: Aging reduces skeletal muscle strength and mass.
Results: The transcription factor ATF4 is required for age-related muscle weakness and atrophy, and the small molecules ursolic acid and tomatidine reduce ATF4 activity, weakness, and atrophy in aged skeletal muscle.
Conclusion: ATF4 is an essential mediator of muscle aging.
Significance: These results identify new strategies for reducing weakness and muscle loss during aging.
Aging reduces skeletal muscle mass and strength, but the underlying molecular mechanisms remain elusive. Here, we used mouse models to investigate molecular mechanisms of age-related skeletal muscle weakness and atrophy as well as new potential interventions for these conditions. We identified two small molecules that significantly reduce age-related deficits in skeletal muscle strength, quality, and mass: ursolic acid (a pentacyclic triterpenoid found in apples) and tomatidine (a steroidal alkaloid derived from green tomatoes). Because small molecule inhibitors can sometimes provide mechanistic insight into disease processes, we used ursolic acid and tomatidine to investigate the pathogenesis of age-related muscle weakness and atrophy. We found that ursolic acid and tomatidine generate hundreds of small positive and negative changes in mRNA levels in aged skeletal muscle, and the mRNA expression signatures of the two compounds are remarkably similar. Interestingly, a subset of the mRNAs repressed by ursolic acid and tomatidine in aged muscle are positively regulated by activating transcription factor 4 (ATF4). Based on this finding, we investigated ATF4 as a potential mediator of age-related muscle weakness and atrophy. We found that a targeted reduction in skeletal muscle ATF4 expression reduces age-related deficits in skeletal muscle strength, quality, and mass, similar to ursolic acid and tomatidine. These results elucidate ATF4 as a critical mediator of age-related muscle weakness and atrophy. In addition, these results identify ursolic acid and tomatidine as potential agents and/or lead compounds for reducing ATF4 activity, weakness, and atrophy in aged skeletal muscle.
doi:10.1074/jbc.M115.681445
PMCID: PMC4646196  PMID: 26338703
aging; muscle atrophy; protein synthesis; skeletal muscle; skeletal muscle metabolism; ATF4; sarcopenia; skeletal muscle atrophy; tomatidine; ursolic acid
5.  Structure of the Neisserial Outer Membrane Protein Opa60: Loop Flexibility Essential to Receptor Recognition and Bacterial Engulfment 
The structure and dynamics of Opa proteins, which we report herein, are responsible for the receptor-mediated engulfment of Neisseria gonorrheae or Neisseria meningitidis by human cells and can offer deep understanding into the molecular recognition of pathogen–host receptor interactions. Such interactions are vital to understanding bacterial pathogenesis as well as the mechanism of foreign body entry to a human cell, which may provide insights for the development of targeted pharmaceutical delivery systems. The size and dynamics of the extracellular loops of Opa60 required a hybrid refinement approach wherein membrane and distance restraints were used to generate an initial NMR structural ensemble, which was then further refined using molecular dynamics in a DMPC bilayer. The resulting ensemble revealed that the extracellular loops, which bind host receptors, occupy compact conformations, interact with each other weakly, and are dynamic on the nanosecond time scale. We predict that this conformational sampling is critical for enabling diverse Opa loop sequences to engage a common set of receptors.
doi:10.1021/ja503093y
PMCID: PMC4105060  PMID: 24813921
6.  Systematic Reviews: Perhaps “The Answer to Policy Makers’ Prayers”? 
Environmental Health Perspectives  2014;122(10):A262-A263.
doi:10.1289/ehp.1408599
PMCID: PMC4181934  PMID: 25272205
7.  Systems-based Discovery of Tomatidine as a Natural Small Molecule Inhibitor of Skeletal Muscle Atrophy* 
The Journal of Biological Chemistry  2014;289(21):14913-14924.
Background: Skeletal muscle atrophy is a common and serious condition that lacks a pharmacologic therapy.
Results: We used a systems-based strategy to identify tomatidine, a natural compound from tomato plants, as a novel small molecule inhibitor of muscle atrophy.
Conclusion: Tomatidine may have utility as a therapeutic agent or lead compound for muscle atrophy.
Significance: These results suggest new therapeutic strategies for muscle atrophy.
Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. To address this problem, we used a systems-based discovery strategy to search for a small molecule whose mRNA expression signature negatively correlates to mRNA expression signatures of human skeletal muscle atrophy. This strategy identified a natural small molecule from tomato plants, tomatidine. Using cultured skeletal myotubes from both humans and mice, we found that tomatidine stimulated mTORC1 signaling and anabolism, leading to accumulation of protein and mitochondria, and ultimately, cell growth. Furthermore, in mice, tomatidine increased skeletal muscle mTORC1 signaling, reduced skeletal muscle atrophy, enhanced recovery from skeletal muscle atrophy, stimulated skeletal muscle hypertrophy, and increased strength and exercise capacity. Collectively, these results identify tomatidine as a novel small molecule inhibitor of muscle atrophy. Tomatidine may have utility as a therapeutic agent or lead compound for skeletal muscle atrophy.
doi:10.1074/jbc.M114.556241
PMCID: PMC4031541  PMID: 24719321
mTOR complex (mTORC); Muscle Atrophy; Skeletal Muscle; Skeletal Muscle Metabolism; Systems Biology; Tomatidine
8.  Dependence of Micelle Size and Shape on Detergent Alkyl Chain Length and Head Group 
PLoS ONE  2013;8(5):e62488.
Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.
doi:10.1371/journal.pone.0062488
PMCID: PMC3648574  PMID: 23667481
9.  Nano-structuring, surface and bulk modification with a focused helium ion beam 
Summary
We investigate the ability of a focused helium ion beam to selectively modify and mill materials. The sub nanometer probe size of the helium ion microscope used provides lateral control not previously available for helium ion irradiation experiments. At high incidence angles the helium ions were found to remove surface material from a silicon lamella leaving the subsurface structure intact for further analysis. Surface roughness and contaminants were both reduced by the irradiation process. Fabrication is also realized with a high level of patterning acuity. Implantation of helium beneath the surface of the sample is visualized in cross section allowing direct observation of the extended effects of high dose irradiation. The effect of the irradiation on the crystal structure of the material is presented. Applications of the sample modification process are presented and further prospects discussed.
doi:10.3762/bjnano.3.67
PMCID: PMC3458604  PMID: 23019554
EELS; EFTEM; helium ion microscopy; nanofabrication; TEM
10.  Ursolic Acid Increases Skeletal Muscle and Brown Fat and Decreases Diet-Induced Obesity, Glucose Intolerance and Fatty Liver Disease 
PLoS ONE  2012;7(6):e39332.
Skeletal muscle Akt activity stimulates muscle growth and imparts resistance to obesity, glucose intolerance and fatty liver disease. We recently found that ursolic acid increases skeletal muscle Akt activity and stimulates muscle growth in non-obese mice. Here, we tested the hypothesis that ursolic acid might increase skeletal muscle Akt activity in a mouse model of diet-induced obesity. We studied mice that consumed a high fat diet lacking or containing ursolic acid. In skeletal muscle, ursolic acid increased Akt activity, as well as downstream mRNAs that promote glucose utilization (hexokinase-II), blood vessel recruitment (Vegfa) and autocrine/paracrine IGF-I signaling (Igf1). As a result, ursolic acid increased skeletal muscle mass, fast and slow muscle fiber size, grip strength and exercise capacity. Interestingly, ursolic acid also increased brown fat, a tissue that shares developmental origins with skeletal muscle. Consistent with increased skeletal muscle and brown fat, ursolic acid increased energy expenditure, leading to reduced obesity, improved glucose tolerance and decreased hepatic steatosis. These data support a model in which ursolic acid reduces obesity, glucose intolerance and fatty liver disease by increasing skeletal muscle and brown fat, and suggest ursolic acid as a potential therapeutic approach for obesity and obesity-related illness.
doi:10.1371/journal.pone.0039332
PMCID: PMC3379974  PMID: 22745735
11.  mRNA Expression Signatures of Human Skeletal Muscle Atrophy Identify a Natural Compound that Increases Muscle Mass 
Cell metabolism  2011;13(6):627-638.
SUMMARY
Skeletal muscle atrophy is a common and debilitating condition that lacks a pharmacologic therapy. To develop a potential therapy, we identified 63 mRNAs that were regulated by fasting in both human and mouse muscle, and 29 mRNAs that were regulated by both fasting and spinal cord injury in human muscle. We used these two unbiased mRNA expression signatures of muscle atrophy to query the Connectivity Map, which singled out ursolic acid as a compound whose signature was opposite to those of atrophy-inducing stresses. A natural compound enriched in apples, ursolic acid reduced muscle atrophy and stimulated muscle hypertrophy in mice. It did so by enhancing skeletal muscle insulin/IGF-I signaling, and inhibiting atrophy-associated skeletal muscle mRNA expression. Importantly, ursolic acid’s effects on muscle were accompanied by reductions in adiposity, fasting blood glucose and plasma cholesterol and triglycerides. These findings identify a potential therapy for muscle atrophy and perhaps other metabolic diseases.
doi:10.1016/j.cmet.2011.03.020
PMCID: PMC3120768  PMID: 21641545
12.  The use of quartz crystal microbalance with dissipation (QCM-D) for studying nanoparticle-induced platelet aggregation 
Interactions between blood platelets and nanoparticles have both pharmacological and toxicological significance and may lead to platelet activation and aggregation. Platelet aggregation is usually studied using light aggregometer that neither mimics the conditions found in human microvasculature nor detects microaggregates. A new method for the measurement of platelet microaggregation under flow conditions using a commercially available quartz crystal microbalance with dissipation (QCM-D) has recently been developed. The aim of the current study was to investigate if QCM-D could be used for the measurement of nanoparticle-platelet interactions. Silica, polystyrene, and gold nanoparticles were tested. The interactions were also studied using light aggregometry and flow cytometry, which measured surface abundance of platelet receptors. Platelet activation was imaged using phase contrast and scanning helium ion microscopy. QCM-D was able to measure nanoparticle-induced platelet microaggregation for all nanoparticles tested at concentrations that were undetectable by light aggregometry and flow cytometry. Microaggregates were measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast and scanning helium ion microscopy.
doi:10.2147/IJN.S26679
PMCID: PMC3263416  PMID: 22275839
platelet aggregation; nanoparticles; light aggregometer; quartz crystal microbalance with dissipation; scanning helium ion microscopy
13.  The Politics of Achievable Mortality 
Public Health Reports  2010;125(2):168-170.
PMCID: PMC2821843  PMID: 20297742
14.  The Governance of Standard-Setting to Improve Health 
Preventing Chronic Disease  2010;7(6):A123.
This article describes recent events in the governance of standard-setting for 2 areas of US health policy — states' decisions about which prescription drugs to cover under Medicaid and other public programs and making health an aspect of foreign policy — and whether these events offer lessons for policy making. In prescription drug coverage, methodologic advances in research that evaluates health services and the politics of restraining the rate of growth in health expenditures enabled policy makers in most states to establish new public processes for assessing and applying evidence about the effectiveness of competing drugs. Their counterparts in foreign policy, in contrast, made few changes in existing processes for choosing which interventions to support. The history of governance in each area of policy making for health explains the selection of standards to evaluate evidence about interventions and whether and how to use this evidence to guide policy.
PMCID: PMC2995609  PMID: 20950530
15.  Realizing and Allocating Savings From Improving Health Care Quality and Efficiency 
Preventing Chronic Disease  2010;7(5):A99.
International efforts to increase the quality and efficiency of health care services may be creating financial savings that can be used to improve population health. This article examines evidence that such savings (ie, a quality/efficiency or value dividend) are accruing and how they have been allocated and assesses the prospects for reallocating future savings to improve population health. Savings have resulted mainly from reducing the number of inappropriate or harmful interventions, managing care of people with chronic disease more effectively, and implementing health information technology. Savings to date have accrued to the revenues of public and private collective purchasers of care and large provider organizations, but none seem to have been reallocated to address other determinants of health. Furthermore, improved quality sometimes increases spending.
PMCID: PMC2938415  PMID: 20712947
16.  HOIL-1L Interacting Protein (HOIP) as an NF-κB Regulating Component of the CD40 Signaling Complex 
PLoS ONE  2010;5(6):e11380.
The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-κB activation through the ubiquitin-dependent activation of IKKγ. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-κB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.
doi:10.1371/journal.pone.0011380
PMCID: PMC2894856  PMID: 20614026
17.  Mixing and Matching Detergents for Membrane Protein NMR Structure Determination 
One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. In this study, the structure and dynamics of an integral membrane protein in different detergents is investigated by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, and small angle X-ray scattering (SAXS). The results suggest that matching of the micelle dimensions to the protein’s hydrophobic surface avoids exchange processes that reduce the completeness of the NMR observations. Based on these dimensions, several mixed micelles were designed that improved the completeness of NMR observations. These findings provide a basis for the rational design of mixed micelles that may advance membrane protein structure determination by NMR.
doi:10.1021/ja808776j
PMCID: PMC2751809  PMID: 19425578
18.  Formation, Reactivity, and Properties of Nondative Late Transition Metal–Oxygen and–Nitrogen Bonds 
Accounts of chemical research  2002;35(1):44-56.
Complexes containing bonds between heteroatoms such as nitrogen and oxygen and “late” transition metals (i.e., those located on the right side of the transition series) have been implicated as reactive intermediates in numerous important catalytic systems. Despite this, our understanding of such M–X linkages still lags behind that of their M–H and M–C analogues. New synthetic strategies have now made possible the isolation and study of a variety of monomeric late-metal alkoxide, aryloxide, and amide complexes, including parent hydroxide and amide species. The heteroatoms in these materials form surprisingly strong bonds to their metal centers, and their bond energies do not necessarily correlate with the energies of the corresponding H–X bonds. The M–X complexes typically exhibit nucleophilic reactivity, in some cases form strong hydrogen bonds to proton donors, and even deprotonate relatively weak acids. These observations, as well as thermodynamic investigations, suggest that late metal–heteroatom bonds are strongly polarized and possess significant ionic character, properties that play an important role in their interactions with organic compounds.
doi:10.1021/ar000132x
PMCID: PMC1473979  PMID: 11790088
19.  What New Knowledge Would Help Policymakers Better Balance Investments for Optimal Health Outcomes? 
Health Services Research  2003;38(6 Pt 2):1923-1938.
Objective
Review the limitations in cross-sectoral health outcomes research and suggest a future research agenda.
Data Sources, Study Design, Data Collection
Literature review and workshop discussion.
Principal Findings
The research evidence that would aid public and private policy makers in answering the question the title poses is quite limited.
Conclusions
Much more evidence from diverse disciplines is needed, and key areas are suggested. Criteria for progress by 2010 are proposed.
doi:10.1111/j.1475-6773.2003.00209.x
PMCID: PMC1360980  PMID: 14727804
Health outcomes; population health; health determinants; cross-sectoral; cost-effectiveness

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