bluuebunny/crossref_metadata_2025 huggingface dataset:
Single-Molecule Fluorescence Imaging in Living Cells
Authors: Xia Tie , Li Nan , Fang Xiaohong
Published: January 2013

The transition of single-molecule fluorescence detection and imaging from in vitro to living cells has greatly enriched our knowledge on the behavior of single biomolecules in their native environments and their roles in cellular processes. Here we review recent advances of single-molecule biophysical approaches to live-cell studies based on fluorescence imaging. We start by discussing the practical considerations in designing single-molecule fluorescence imaging in cells, including the choice of fluorescent probes, labeling methods, instrumentation, and imaging techniques. We then describe representative examples in detail to illustrate the physicochemical parameters that can be obtained by imaging individually labeled biomolecules in cells and what can be learned from such characterizations.
Structure and Dynamics of Interfacial Water Studied by Heterodyne-Detected Vibrational Sum-Frequency Generation
Authors: Nihonyanagi Satoshi , Mondal Jahur A. , Yamaguchi Shoichi , Tahara Tahei
Published: January 2013

Vibrational sum-frequency generation (VSFG) spectroscopy is a powerful tool to study interfaces. Recently, multiplex heterodyne-detected VSFG (HD-VSFG) has been developed, which enables the direct measurement of complex second-order nonlinear susceptibility [χ(2)]. HD-VSFG has remarkable advantages over conventional VSFG. For example, the imaginary part of χ(2) [Imχ(2)] obtained with this interferometric technique is the direct counterpart to the infrared [Imχ(1)] and Raman [Imχ(3)] spectra in the bulk, and it is free from the spectral deformation inevitable in conventional VSFG [|χ(2)|2] spectra. The Imχ(2) signal is obtained with a sign that contains unambiguous information about the up/down orientation of interfacial molecules. Furthermore, HD-VSFG can be straightforwardly extended to time-resolved measurements when combined with photoexcitation. In this review, we describe the present status of experiments and applications of multiplex HD-VSFG spectroscopy, in particular with regard to the orientation and structure of interfacial water at charged, neutral, and biorelevant water interfaces.
Shedding New Light on Retinal Protein Photochemistry
Authors: Wand Amir , Gdor Itay , Zhu Jingyi , Sheves Mordechai , Ruhman Sanford
Published: January 2013

The ultrafast spectroscopic investigation of novel retinal proteins challenges existing notions concerning the course of primary events in these natural photoreceptors. We review two illustrations here. The first demonstrates that changes in the initial retinal configuration can alter the duration of photochemistry by nearly an order of magnitude in Anabaena sensory rhodopsin, making it as rapid as the ballistic photoisomerization in visual pigments. This prompted a reinvestigation of the much studied bacteriorhodopsin, leading to a similar trend as well, contrary to earlier reports. The second involves the study of xanthorhodopsin, an archaeal proton pump that includes an attached light-harvesting carotenoid. Pump-probe experiments demonstrate the efficient transfer of energy from carotenoid to retinal, providing a first glimpse at a cooperative multichromophore function, which is probably characteristic of many other proteins as well. Finally, we discuss measures required to advance our knowledge from kinetics to mode-specific dynamics concerning this expanding family of biological photoreceptors.
Empowerment of the European Parliament
Authors: Hix Simon , Høyland Bjørn
Published: January 2013

One of the most remarkable democratic developments in Europe in recent decades has been the empowerment of the only directly elected supranational assembly in the world: the European Parliament (EP). We first review the development of the legislative powers of the EP vis-à-vis the other European Union (EU) institutions, discussing the theoretical models of the power of the EP and the main empirical methods that have been used to evaluate these models. We then turn to the impact of the growing power of the EP on political organization and behavior inside the legislature. We demonstrate that the “electoral connection” is weak and discuss what this means for understanding legislative politics in the EP. The concluding section demonstrates differences in behavior across policy areas, which have received scant attention, and suggests avenues for further research.
The Political Economy of the Euro
Authors: De Grauwe Paul
Published: January 2013

The Eurozone's present state of crisis originated from decisions made at its creation. The decision to create a monetary union was motivated by political objectives and completely disregarded the economics of a monetary union. Political leaders did not understand the necessary economic conditions for a successful monetary union and did not recognize the inherent fragility of the monetary union they established. They showed the same disturbing lack of understanding of the economics of the sovereign debt crisis in 2010. They misdiagnosed the problem, and their response included disastrous decisions that intensified the crisis. This review explains these errors and concludes with recommendations for saving the euro.
Energy and Human Health
Authors: Smith Kirk R. , Frumkin Howard , Balakrishnan Kalpana , Butler Colin D. , Chafe Zoë A. , Fairlie Ian , Kinney Patrick , Kjellstrom Tord , Mauzerall Denise L. , McKone Thomas E. , McMichael Anthony J. , Schneider Mycle
Published: January 2013

Energy use is central to human society and provides many health benefits. But each source of energy entails some health risks. This article reviews the health impacts of each major source of energy, focusing on those with major implications for the burden of disease globally. The biggest health impacts accrue to the harvesting and burning of solid fuels, coal and biomass, mainly in the form of occupational health risks and household and general ambient air pollution. Lack of access to clean fuels and electricity in the world's poor households is a particularly serious risk for health. Although energy efficiency brings many benefits, it also entails some health risks, as do renewable energy systems, if not managed carefully. We do not review health impacts of climate change itself, which are due mostly to climate-altering pollutants from energy systems, but do discuss the potential for achieving near-term health cobenefits by reducing certain climate-related emissions.
Links Among Human Health, Animal Health, and Ecosystem Health
Authors: Rabinowitz Peter , Conti Lisa
Published: January 2013

In the face of growing world human and animal populations and rapid environmental change, the linkages between human, animal, and environmental health are becoming more evident. Because animals and humans have shared risk to health from changing environments, it seems logical to expand the perspective of public health beyond a single species to detect and manage emerging public health threats. Mitigating the effects of climate change, emerging pathogens, toxicant releases, and changes in the built environment requires a retooling of global public health resources and capabilities across multiple species. Furthermore, human and animal health professionals must overcome specific barriers to interprofessional collaboration to implement needed health strategies. This review outlines the relationships between human, animal, and ecosystem health and the public health challenges and opportunities that these links present.
Reducing Hospital Errors: Interventions that Build Safety Culture
Authors: Singer Sara J. , Vogus Timothy J.
Published: January 2013

Hospital errors are a seemingly intractable problem and continuing threat to public health. Errors resist intervention because too often the interventions deployed fail to address the fundamental source of errors: weak organizational safety culture. This review applies and extends a theoretical model of safety culture that suggests it is a function of interrelated processes of enabling, enacting, and elaborating that can reduce hospital errors over time. In this model, enabling activities help shape perceptions of safety climate, which promotes enactment of safety culture. We then classify a broad array of interventions as enabling, enacting, or elaborating a culture of safety. Our analysis, which is intended to guide future attempts to both study and more effectively create and sustain a safety culture, emphasizes that isolated interventions are unlikely to reduce the underlying causes of hospital errors. Instead, reducing errors requires systemic interventions that address the interrelated processes of safety culture in a balanced manner.
Transportation and Public Health
Authors: Litman Todd
Published: January 2013

This article investigates various ways that transportation policy and planning decisions affect public health and better ways to incorporate public health objectives into transport planning. Conventional planning tends to consider some public health impacts, such as crash risk and pollution emissions measured per vehicle-kilometer, but generally ignores health problems resulting from less active transport (reduced walking and cycling activity) and the additional crashes and pollution caused by increased vehicle mileage. As a result, transport agencies tend to undervalue strategies that increase transport system diversity and reduce vehicle travel. This article identifies various win-win strategies that can help improve public health and other planning objectives.
Impaired contractile recovery after low-flow myocardial ischemia in a porcine model of metabolic syndrome
Authors: Huang Janice V. , Lu Li , Ye Shuyu , Bergman Bryan C. , Sparagna Genevieve C. , Sarraf Mohammad , Reusch Jane E. B. , Greyson Clifford R. , Schwartz Gregory G.
Published: January 2013

Clinical metabolic syndrome conveys a poor prognosis in patients with acute coronary syndrome, not fully accounted for by the extent of coronary atherosclerosis. To explain this observation, we determined whether postischemic myocardial contractile and metabolic function are impaired in a porcine dietary model of metabolic syndrome without atherosclerosis. Micropigs ( n = 28) were assigned to a control diet (low fat, no added sugars) or an intervention diet (high saturated fat and simple sugars, no added cholesterol) for 7 mo. The intervention diet produced obesity, hypertension, dyslipidemia, and impaired glucose tolerance, but not atherosclerosis. Under open-chest, anesthetized conditions, pigs underwent 45 min of low-flow myocardial ischemia and 120 min of reperfusion. In both diet groups, contractile function was similar at baseline and declined similarly during ischemia. However, after 120 min of reperfusion, regional work recovered to 21 ± 12% of baseline in metabolic syndrome pigs compared with 61 ± 13% in control pigs ( P = 0.01). Ischemia-reperfusion caused a progressive decline in mechanical/metabolic efficiency (regional work/O2consumption) in metabolic syndrome hearts, but not in control hearts. Metabolic syndrome hearts demonstrated altered fatty acyl composition of cardiolipin and increased Akt phosphorylation in both ischemic and nonischemic regions, suggesting tonic activation. Metabolic syndrome hearts used more fatty acid than control hearts ( P = 0.03). When fatty acid availability was restricted by prior insulin exposure, differences between groups in postischemic contractile recovery and mechanical/metabolic efficiency were eliminated. In conclusion, pigs with characteristics of metabolic syndrome demonstrate impaired contractile and metabolic recovery after low-flow myocardial ischemia. Contributory mechanisms may include remodeling of cardiolipin, abnormal activation of Akt, and excessive utilization of fatty acid substrates.
SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells
Authors: Dhar Kajari , Rakesh Kriti , Pankajakshan Divya , Agrawal Devendra K.
Published: January 2013

Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α-stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.
Simvastatin and GGTI-2133, a geranylgeranyl transferase inhibitor, increase erythrocyte deformability but reduce low O2tension-induced ATP release
Authors: Clapp K. M. , Ellsworth M. L. , Sprague R. S. , Stephenson A. H.
Published: January 2013

Statin drugs inhibit 3-hydroxy-3-methylglutaryl CoA reductase, which reduces the synthesis of both cholesterol and isoprenoids (geranylgeranyl pyrophosphate and farnesyl pyrophosphate), with the latter being lipid molecules responsible for the posttranslational modification of small GTP-binding proteins such as Rho. Effects of statins, independent of lowering blood cholesterol levels, are thought to occur by inhibition of Rho/Rho kinase. The Rho kinase inhibitor Y-27632 has been reported to increase both erythrocyte deformability and low O2tension-induced ATP release. Here, we tested the hypothesis that by inhibiting Rho/Rho kinase, simvastatin would increase both erythrocyte deformability and low O2tension-induced ATP release. Male Sprague-Dawley rats were divided into two groups, control or simvastatin treated [simvastatin-supplemented chow (0.02%)], for 4 wk. Simvastatin treatment increased rat erythrocyte deformability compared with controls ( n = 6, P < 0.05). However, erythrocytes of simvastatin-treated rats ( n = 9, P < 0.05) exhibited impaired low O2tension-induced ATP release. Similarly, the geranylgeranyl transferase inhibitor GGTI-2133 (10 μM) also increased deformability and impaired low O2tension-induced ATP release in healthy human erythrocytes ( P < 0.05). Interestingly, ATP release in response to mastoparan 7 ( n = 7, P < 0.05), which directly activates Gi, and isoproterenol ( n = 5, P < 0.05), which signals through Gs, was not altered by incubation with GGTI-2133. These results suggest that although statins increase erythrocyte deformability, likely by inhibiting geranylgeranylation, the finding that both statins and a geranylgeranyl transferase inhibitor attenuated low O2tension-induced ATP release demonstrates that factors in addition to erythrocyte deformability are critical for ATP release in response to this physiological stimulus.
Effect of stellate ganglia stimulation on global and regional left ventricular function as assessed by speckle tracking echocardiography
Authors: Zhou Wei , Yamakawa Kentaro , Benharash Peyman , Ajijola Olujimi , Ennis Daniel , Hadaya Joseph , Vaseghi Marmar , Shivkumar Kalyanam , Mahajan Aman
Published: January 2013

Left ventricular (LV) twist mechanics and regional strain during cardiac sympathetic efferent activation are poorly understood. The purpose of this study was to compare the effects of left stellate ganglia (LSG) and right stellate ganglia (RSG) stimulation on cardiac twist/untiwst mechanics and regional strain. In nine pigs, echocardiographic imaging and LV pressure-volume measurements were performed before and during unilateral and bilateral stellate ganglion stimulation. LSG and RSG stimulation significantly augmented LV end-systolic pressure by 24% and 22% ( P < 0.01), maximal rate of LV pressure change by 167% and 165% ( P < 0.01), and time constant of LV relaxation by 20% and 12% ( P < 0.01), respectively. RSG stimulation resulted in a greater chronotropic response than LSG stimulation (RSG: 68% vs. LSG: 12%, P < 0.01). Both LSG and RSG stimulation significantly increased global epicardial and endocardial LV rotation and diastolic untwisting rate and reduced the time to peak rotation ( P < 0.05). However, LSG stimulation predominantly increased radial and circumferential strain in the LV inferoseptal, inferior, posterior, and lateral regions, whereas RSG stimulation primarily increased radial and circumferential strain in the anteroseptal, anterior, and lateral LV regions. Stimulation of both stellate ganglia led to a uniform increase in all LV segments. Our data suggest that LSG and RSG stimulation lead to a global increase in LV twist, driven by distinct regional strain heterogeneity that may result from myocardial innervation from the LSG and RSG. These findings provide a better understanding of the global and regional functional consequences of regional myocardial innervation from the LSG and RSG.
L-type Ca2+ channel contributes to alterations in mitochondrial calcium handling in the mdx ventricular myocyte
Authors: Viola Helena M. , Davies Stefan M. K. , Filipovska Aleksandra , Hool Livia C.
Published: January 2013

The L-type Ca2+ channel is the main route for calcium entry into cardiac myocytes, and it is essential for contraction. Alterations in whole cell L-type Ca2+ channel current and Ca2+ homeostasis have been implicated in the development of cardiomyopathies. Cytoskeletal proteins can influence whole cell L-type Ca2+ current and mitochondrial function. Duchenne muscular dystrophy is a fatal X-linked disease that leads to progressive muscle weakness due to the absence of cytoskeletal protein dystrophin. This includes dilated cardiomyopathy, but the mechanisms are not well understood. We sought to identify the effect of alterations in whole cell L-type Ca2+ channel current on mitochondrial function in the murine model of Duchenne muscular dystrophy ( mdx). Activation of the L-type Ca2+ channel with the dihydropyridine agonist BayK(−) caused a significantly larger increase in cytosolic Ca2+ in mdx vs. wild-type ( wt) ventricular myocytes. Consistent with elevated cytosolic Ca2+, resting mitochondrial Ca2+, NADH, and mitochondrial superoxide were significantly greater in mdx vs. wt myocytes. Activation of the channel with BayK(−) caused a further increase in mitochondrial Ca2+, NADH, and superoxide in mdx myocytes. The ratios of the increases were similar to the ratios recorded in wt myocytes. In mitochondria isolated from 8-wk-old mdx hearts, respiration and mitochondrial electron transport chain complex activity were similar to mitochondria isolated from wt hearts. We conclude that mitochondria function at a higher level of resting calcium in the intact mdx myocyte and activation of the L-type Ca2+ channel contributes to alterations in calcium handling by the mitochondria. This perturbation may contribute to the development of cardiomyopathy.
Kruppel-like factor 2 protects against ischemic stroke by regulating endothelial blood brain barrier function
Authors: Shi Hong , Sheng Baiyang , Zhang Feng , Wu Chunying , Zhang Rongli , Zhu Junqing , Xu Kui , Kuang Youzhi , Jameson Stephen C. , Lin Zhiyong , Wang Yanming , Chen Jun , Jain Mukesh K. , Atkins G. Brandon
Published: January 2013

During an ischemic stroke normal brain endothelial function is perturbed, resulting in blood brain barrier (BBB) breakdown with subsequent infiltration of activated inflammatory blood cells, ultimately leading to neuronal cell death. Kruppel-like factor 2 (KLF2) is regulated by flow, is highly expressed in vascular endothelial cells (ECs), and serves as a key molecular switch regulating endothelial function and promoting vascular health. In this study we sought to determine the role of KLF2 in cerebrovascular function and the pathogenesis of ischemic stroke. Transient middle cerebral artery occlusion was performed in KLF2-deficient (KLF2−/−), KLF2 overexpressing (KLF2tg), and control mice, and stroke volume was analyzed. BBB function was assessed in vivo by real-time neuroimaging using positron emission tomography and Evan's blue dye assay. KLF2−/−mice exhibited significantly larger strokes and impairment in BBB function. In contrast, KLF2tgmice were protected against ischemic stroke and demonstrated preserved BBB function. In concordance, gain- and loss-of-function studies in primary brain microvascular ECs using transwell assays revealed KLF2 to be BBB protective. Mechanistically, KLF2 was demonstrated, both in vitro and in vivo, to regulate the critical BBB tight junction factor occludin. These data are first to identify endothelial KLF2 as a key regulator of the BBB and a novel neuroprotective factor in ischemic stroke.
Targets of vascular protection in acute ischemic stroke differ in type 2 diabetes
Authors: Kelly-Cobbs Aisha I. , Prakash Roshini , Li Weiguo , Pillai Bindu , Hafez Sherif , Coucha Maha , Johnson Maribeth H. , Ogbi Safia N. , Fagan Susan C. , Ergul Adviye
Published: January 2013

Hemorrhagic transformation is an important complication of acute ischemic stroke, particularly in diabetic patients receiving thrombolytic treatment with tissue plasminogen activator, the only approved drug for the treatment of acute ischemic stroke. The objective of the present study was to determine the effects of acute manipulation of potential targets for vascular protection [i.e., NF-κB, peroxynitrite, and matrix metalloproteinases (MMPs)] on vascular injury and functional outcome in a diabetic model of cerebral ischemia. Ischemia was induced by middle cerebral artery occlusion in control and type 2 diabetic Goto-Kakizaki rats. Treatment groups received a single dose of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III), the nonspecific NF-κB inhibitor curcumin, or the broad-spectrum MMP inhibitor minocycline at reperfusion. Poststroke infarct volume, edema, hemorrhage, neurological deficits, and MMP-9 activity were evaluated. All acute treatments reduced MMP-9 and hemorrhagic transformation in diabetic groups. In addition, acute curcumin and minocycline therapy reduced edema in these animals. Improved neurological function was observed in varying degrees with treatment, as indicated by beam-walk performance, modified Bederson scores, and grip strength; however, infarct size was similar to untreated diabetic animals. In control animals, all treatments reduced MMP-9 activity, yet bleeding was not improved. Neuroprotection was only conferred by curcumin and minocycline. Uncovering the underlying mechanisms contributing to the success of acute therapy in diabetes will advance tailored stroke therapies.
Antenatal glucocorticoids counteract LPS changes in TGF-β pathway and caveolin-1 in ovine fetal lung
Authors: Collins Jennifer J. P. , Kunzmann Steffen , Kuypers Elke , Kemp Matthew W. , Speer Christian P. , Newnham John P. , Kallapur Suhas G. , Jobe Alan H. , Kramer Boris W.
Published: January 2013

Inflammation and antenatal glucocorticoids, the latter given to mothers at risk for preterm birth, affect lung development and may contribute to the development of bronchopulmonary dysplasia (BPD). The effects of the combined exposures on inflammation and antenatal glucocorticoids on transforming growth factor (TGF)-β signaling are unknown. TGF-β and its downstream mediators are implicated in the etiology of BPD. Therefore, we asked whether glucocorticoids altered intra-amniotic lipopolysaccharide (LPS) effects on TGF-β expression, its signaling molecule phosphorylated sma and mothers against decapentaplegic homolog 2 (pSmad2), and the downstream mediators connective tissue growth factor (CTGF) and caveolin-1 (Cav-1). Ovine singleton fetuses were randomized to receive either an intra-amniotic injection of LPS and/or maternal betamethasone (BTM) intramuscularly 7 and/or 14 days before delivery at 120 days gestational age (GA; term = 150 days GA). Saline was used for controls. Protein levels of TGF-β1 and -β2 were measured by ELISA. Smad2 phosphorylation was assessed by immunohistochemistry and Western blot. CTGF and Cav-1 mRNA and protein levels were determined by RT-PCR and Western blot. Free TGF-β1 and -β2 and total TGF-β1 levels were unchanged after LPS and/or BTM exposure, although total TGF-β2 increased in animals exposed to BTM 7 days before LPS. pSmad2 immunostaining increased 7 days after LPS exposure although pSmad2 protein expression did not increase. Similarly, CTGF mRNA and protein levels increased 7 days after LPS exposure as Cav-1 mRNA and protein levels decreased. BTM exposure before LPS prevented CTGF induction and Cav-1 downregulation. This study demonstrated that the intrauterine inflammation-induced TGF-β signaling can be inhibited by antenatal glucocorticoids in fetal lungs.
LPS-mediated endothelial activation in pulmonary endothelial cells: role of Nox2-dependent IKK-β phosphorylation
Authors: Menden Heather , Tate Everett , Hogg Neil , Sampath Venkatesh
Published: January 2013

Lipopolysaccharide (LPS)-mediated endothelial activation contributes to lung inflammation and alveolar remodeling seen in premature infants with bronchopulmonary dysplasia (BPD). The mechanisms underlying LPS-mediated oxidative stress and proinflammatory signaling in human pulmonary microvascular endothelial cells (HPMEC) remain unclear. We hypothesized that NADPH oxidase (Nox) mediates LPS-induced endothelial activation in HPMEC by regulating phosphorylation of Toll-like receptor (TLR) pathway proteins. LPS-induced expression of intercellular adhesion molecule 1 (ICAM-1) was associated with increased 2-OH-E+(marker for superoxide formation) levels and was attenuated by apocynin and the Nox inhibitor, VAS2870. LPS triggered membrane translocation of p67 phox, suggesting activation of Nox2. Silencing Nox2, but not Nox4, suppressed LPS-induced ICAM-1 expression in HPMEC. Immunoprecipitation studies showed that inhibitor of κ-B kinase-β (IKK-β) serine phosphorylation induced by LPS was inhibited by Nox2 silencing. We examined whether Nox2-dependent, LPS-mediated IKK-β phosphorylation was regulated by protein phosphatase 2A (PP2A) or TGF-β associated kinase-1 (TAK1) in HPMEC. LPS increased PP2A activity in HPMEC, and inhibition of PP2A did not alter LPS-mediated ICAM-1 expression but attenuated IKK-β phosphorylation. TAK1 inhibition decreased LPS-induced ICAM-1 expression in HPMEC, and Nox2 silencing attenuated LPS-mediated TAK1 phosphorylation (Thr184/187). We demonstrate that Nox2 regulates LPS-mediated endothelial activation in pulmonary endothelial cells by modulating phosphorylation of key kinases in the TLR signaling cascade. Our data support a novel mechanism by which Nox-dependent signaling regulates proinflammatory signaling in pulmonary endothelial cells. Inhibition of vascular Nox may potentially limit lung injury and alveolar remodeling caused by infections in BPD.
A Multifunctional Chimeric Chaperone Serves as a Novel Immune Modulator Inducing Therapeutic Antitumor Immunity
Authors: Yu Xiaofei , Guo Chunqing , Yi Huanfa , Qian Jie , Fisher Paul B. , Subjeck John R. , Wang Xiang-Yang
Published: January 2013

Converting the immunosuppressive tumor environment into one that is favorable to the induction of antitumor immunity is indispensable for effective cancer immunotherapy. Here, we strategically incorporate a pathogen (i.e., flagellin)-derived, NF-κB–stimulating “danger” signal into the large stress protein or chaperone Grp170 (HYOU1/ORP150) that was previously shown to facilitate antigen crosspresentation. This engineered chimeric molecule (i.e., Flagrp170) is capable of transporting tumor antigens and concurrently inducing functional activation of dendritic cells (DC). Intratumoral administration of adenoviruses expressing Flagrp170 induces a superior antitumor response against B16 melanoma and its distant lung metastasis compared with unmodified Grp170 and flagellin. The enhanced tumor destruction is accompanied with significantly increased tumor infiltration by CD8+ cells as well as elevation of IFN-γ and interleukin (IL)-12 levels in the tumor sites. In situ Ad.Flagrp170 therapy provokes systemic activation of CTLs that recognize several antigens naturally expressing in melanoma (e.g., gp100/PMEL and TRP2/DCT). The mechanistic studies using CD11c-DTR transgenic mice and Batf3-deficient mice reveal that CD8α+ DCs are required for the improved T-cell crosspriming. Antibody neutralization assays show that IL-12 and IFN-γ are essential for the Flagrp170-elicited antitumor response, which also involves CD8+ T cells and natural killer cells. The therapeutic efficacy of Flagrp170 and its immunostimulating activity are also confirmed in mouse prostate cancer and colon carcinoma. Together, targeting the tumor microenvironment with this chimeric chaperone is highly effective in mobilizing or restoring antitumor immunity, supporting the potential therapeutic use of this novel immunomodulator in the treatment of metastatic diseases. Cancer Res; 73(7); 2093–103. ©2013 AACR.
Genetic Variation in Transforming Growth Factor Beta 1 and Mammographic Density in Singapore Chinese Women
Authors: Lee Eunjung , Van Den Berg David , Hsu Chris , Ursin Giske , Koh Woon-Puay , Yuan Jian-Min , Stram Daniel O. , Yu Mimi C. , Wu Anna H.
Published: January 2013

TGF-β plays a critical role in normal mammary development and morphogenesis. Decreased TGF-β signaling has been associated with increased mammographic density. Percent mammographic density (PMD) adjusted for age and body mass index (BMI) is a strong risk factor and predictor of breast cancer risk. PMD is highly heritable, but few genetic determinants have been identified. We investigated the association between genetic variation in TGFB1 and PMD using a cross-sectional study of 2,038 women who were members of the population-based Singapore Chinese Health Study cohort. We assessed PMD using a computer-assisted method. We used linear regression to examine the association between nine tagging single-nucleotide polymorphisms (SNP) of TGFB1 and PMD and their interaction with parity, adjusting for age, BMI, and dialect group. We calculated P values adjusted for correlated tests (PACT) to account for multiple testing. The strongest association was observed for rs2241716. Adjusted PMD was higher by 1.5% per minor allele (PACT = 0.04). When stratifying by parity, this association was limited to nulliparous women. For nulliparous women, adjusted PMD was higher by 8.6% per minor allele (PACT = 0.003; P for interaction with parity = 0.002). Three additional TGFB1 tagging SNPs, which were in linkage disequilibrium with rs2241716, were statistically significantly associated with adjusted PMD (PACT < 0.05) for nulliparous women. However, none of these three SNPs showed statistically significant association after adjusting for rs2241716. Our data support that TGFB1 genetic variation may be an important genetic determinant of mammographic density measure that predicts breast cancer risk, particularly in nulliparous women. Cancer Res; 73(6); 1876–82. ©2012 AACR.
Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity
Authors: Atkins Charity , Liu Qi , Minthorn Elisabeth , Zhang Shu-Yun , Figueroa David J. , Moss Katherine , Stanley Thomas B. , Sanders Brent , Goetz Aaron , Gaul Nathan , Choudhry Anthony E. , Alsaid Hasan , Jucker Beat M. , Axten Jeffrey M. , Kumar Rakesh
Published: January 2013

The unfolded protein response (UPR) is a signal transduction pathway that coordinates cellular adaptation to microenvironmental stresses that include hypoxia, nutrient deprivation, and change in redox status. These stress stimuli are common in many tumors and thus targeting components of the UPR signaling is an attractive therapeutic approach. We have identified a first-in-class, small molecule inhibitor of the eukaryotic initiation factor 2-alpha kinase 3 (EIF2AK3) or PERK, one of the three mediators of UPR signaling. GSK2656157 is an ATP-competitive inhibitor of PERK enzyme activity with an IC50 of 0.9 nmol/L. It is highly selective for PERK with IC50 values >100 nmol/L against a panel of 300 kinases. GSK2656157 inhibits PERK activity in cells with an IC50 in the range of 10–30 nmol/L as shown by inhibition of stress-induced PERK autophosphorylation, eIF2α substrate phosphorylation, together with corresponding decreases in ATF4 and CAAT/enhancer binding protein homologous protein (CHOP) in multiple cell lines. Oral administration of GSK2656157 to mice shows a dose- and time-dependent pharmacodynamic response in pancreas as measured by PERK autophosphorylation. Twice daily dosing of GSK2656157 results in dose-dependent inhibition of multiple human tumor xenografts growth in mice. Altered amino acid metabolism, decreased blood vessel density, and vascular perfusion are potential mechanisms for the observed antitumor effect. However, despite its antitumor activity, given the on-target pharmacologic effects of PERK inhibition on pancreatic function, development of any PERK inhibitor in human subjects would need to be cautiously pursued in cancer patients. Cancer Res; 73(6); 1993–2002. ©2012 AACR.
Enhanced Sonographic Imaging to Diagnose Lymph Node Metastasis: Importance of Blood Vessel Volume and Density
Authors: Li Li , Mori Shiro , Kodama Mizuho , Sakamoto Maya , Takahashi Shoki , Kodama Tetsuya
Published: January 2013

Lymph node size is an important variable in ultrasound diagnosis of lymph node metastasis. However, the size criterion often leads to oversight of tumor-positive lymph nodes within the range of “normal” size, such that more accurate diagnostic criteria for lymph node metastasis are required. In this study, we show how diagnosis of lymph node metastasis can be improved by evaluating changes in blood vessel volume and density using a novel contrast-enhanced high-frequency ultrasound (CE-HFUS) system with Sonazoid. An MRL/MpJ-lpr/lpr (MRL/lpr) mouse model of lymph node metastasis was used in which lymph nodes are similar in size to humans. Metastasis via lymphatic vessels to proper axillary lymph nodes (proper ALN) was induced by injection of tumor cells into the subiliac lymph nodes. Within 21 days of injection, significant increases in blood vessel volume and density, but no increases in the size of the proper ALNs, were observed. The increase in blood vessel density was confirmed with immunohistochemical analysis and was positively related to tumor cell proliferation as measured using bioluminescence imaging. Together, our results showed that alterations in blood vessel volume and density precede alterations in lymph node size in the early stages of lymph node metastasis. Detection of these changes by ultrasonography may offer new criteria for early diagnosis of lymph node metastasis. Cancer Res; 73(7); 2082–92. ©2013 AACR.
Viral Load of High-Risk Human Papillomaviruses as Reliable Clinical Predictor for the Presence of Cervical Lesions
Authors: Schmitt Markus , Depuydt Christophe , Benoy Ina , Bogers Johannes , Antoine Jerome , Pawlita Michael , Arbyn Marc
Published: January 2013

Background: Infections with high-risk human papillomaviruses (Hr-HPV) can cause malignant transformation of the human cervical epithelium. HPV DNA tests generally are very sensitive to detect cervical neoplastic lesions but also identify transient HPV infections. As a consequence, the specificity and positive predictive value are low.

Methods: We analyzed viral load of Hr- and possibly Hr-HPV types more than seven orders of magnitude (on a log10 scale) in 999 consecutive BD-SurePath liquid-based cervical cytology samples from routine cervical screening enriched with atypical squamous cells of undetermined significance (n = 100), low-grade squamous intraepithelial lesions (LSIL; n = 100), and high-grade squamous intraepithelial lesions (HSIL; n = 97) using type-specific multiplex quantitative real-time PCR and the BSGP5+/6+-PCR/MPG assay. In the 36-month follow-up, 79 histologically verified CIN2+ and 797 double-negative cytology cases were identified.

Results: Viral loads in LSIL and HSIL were significantly increased compared with no intraepithelial lesion or malignancy in both the quantitative PCR (qPCR) and BSGP5+/6+-PCR/MPG assay (P < 0.0001). The mean viral loads in LSIL and HSIL were not significantly different. Using a newly determined high viral load cut off for 14 Hr-HPV types, the sensitivity for prevalent CIN3+ remained at 100% for both assays compared with the minimal detection threshold. The specificity (corresponding to double-negative cytology at subsequent screening episodes) increased substantially (qPCR, from 91.1% to 95.7%; BSGP5+/6+-PCR/MPG, from 79.8% to 96.2%).

Conclusions: Compared with DNA positivity alone, high Hr-HPV viral loads could reduce the amount of false positive results detected by the BSGP5+/6+-PCR/MPG and qPCR by 81.4% and 52.1%, respectively.

Impact: Quantitative type-specific HPV DNA assays show high flexibility in defining thresholds that allow optimizing clinical accuracy for cervical cancer precursors. Cancer Epidemiol Biomarkers Prev; 22(3); 406–14. ©2013 AACR.
The Effect of Menthol on Cigarette Smoking Behaviors, Biomarkers and Subjective Responses
Authors: Strasser Andrew A. , Ashare Rebecca L. , Kaufman Madeline , Tang Kathy Z. , Mesaros A. Clementina , Blair Ian A.
Published: January 2013

Background: As part of the Family Smoking Prevention and Tobacco Control Act, the U.S. Food and Drug Administration charged the Tobacco Products Scientific Advisory Committee with developing a report and recommendations about the effect of menthol in cigarettes on the public health. The purpose of this study was to examine smoking behaviors, biomarkers of exposure, and subjective responses when switching from a novel menthol cigarette to a non-menthol cigarette to isolate the effect of menthol and to approximate the effect a menthol ban might have on smokers.

Methods: Thirty-two adult smokers completed this 35-day randomized, open-label, laboratory study. After a 5-day baseline period, participants were randomized to the experimental group (n = 22) where they would smoke menthol Camel crush for 15 days followed by 15 days of non-menthol Camel crush, or the control group (n = 10) where they smoked their own brand cigarette across all periods. Participants attended study visits every 5 days and completed measures of smoking rate, smoking topography, biomarkers of exposure, and subjective responses.

Results: Although total puff volume tended to increase when the experimental group switched from menthol to non-menthol (P = 0.06), there were no corresponding increases in cigarette consumption or biomarkers of exposure (P > 0.1). Subjective ratings related to taste and smell decreased during the non-menthol period (P < 0.01), compared with the menthol.

Conclusions: Results suggest menthol has minimal impact on smoking behaviors, biomarkers of exposure, and subjective ratings.

Impact: When controlling for all other cigarette design features, menthol in cigarettes had minimal effect on outcome measures. Cancer Epidemiol Biomarkers Prev; 22(3); 382–9. ©2013 AACR.
Targetome Profiling, Pathway Analysis and Genetic Association Study Implicate miR-202 in Lymphomagenesis
Authors: Hoffman Aaron E. , Liu Ran , Fu Alan , Zheng Tongzhang , Slack Frank , Zhu Yong
Published: January 2013

Background: miRNAs have been implicated in numerous tumorigenic pathways, and previous studies have associated miR-202 dysregulation with various cancer types, including follicular lymphoma.

Methods: The miR-202 targetome was identified by ribonucleoprotein immunoprecipitation-microarray (RIP-Chip), and functional interactions among identified targets were investigated using the Ingenuity Pathway Analysis tool. We also conducted a population-based genetic association study of a polymorphism within the miR-202 stem-loop sequence and risk of non-Hodgkin lymphoma. In vitro gain-of-function experiments were further conducted to elucidate the functional significance of the variant.

Results: A total of 141 potential members of the miR-202 targetome were identified by a transcriptome-wide RIP-Chip assay. Functional interactions among identified targets suggested that miR-202-regulated genes are involved in biologic pathways relevant for hematologic function and cancer. Consistent with this, a genetic association analysis using human blood samples revealed a significant association between a germline mutation (rs12355840) in the miR-202 precursor sequence and follicular lymphoma risk. An in vitro functional assay further showed that the variant allele resulted in diminished miR-202 levels, possibly by altering precursor-processing efficiency.

Conclusions: Taken together, our findings suggest that miR-202 is involved in follicular lymphomagenesis.

Impact: These findings implicate miR-202 as a potential tumor suppressor in follicular lymphoma and warrant the investigation of miR-202 as a novel biomarker of follicular lymphoma risk. Cancer Epidemiol Biomarkers Prev; 22(3); 327–36. ©2013 AACR.
Trials and Tribulations of Interrogating Biomarkers to Define Efficacy of Cancer Risk Reductive Interventions
Authors: Brenner Dean E. , Hawk Ernest
Published: January 2013

The challenges of clinical screening of cancer risk reductive interventions (“chemopreventive”) have slowed progress in deployment of therapeutics to reverse or delay the carcinogenesis process. The preoperative or window-of-opportunity design clinical trial design enrolls subjects rapidly, has short study periods, and quantifies tissue biomarkers that reflect both anti-carcinogenesis mechanism of the risk reductive intervention and key molecular events of the carcinogenesis process for a specific epithelial target. High subject screened to on study ratios reduce the efficiency and increase cost of this research strategy. Small-sized tissue samples obtained by minimally invasive endoscopic technologies limit the number of biomarkers that can be detected and quantified, forcing investigators into choosing either a broad-based but superficial multi-mechanism exploration of signaling intermediates or a more focused analysis of multiple molecular events in a linear signaling-specific pathway. More efficient strategies of the future might involve isolation and expansion of pluripotent cells from at-risk epithelium or intraepithelial neoplastic lesions. Such a strategy would allow interrogation of key carcinogenesis-associated pathways and mechanisms in representative primary single-cell cultures amenable to genomic, proteomics, or transfection-based technologies. Cancer Prev Res; 6(2); 71–3. ©2013 AACR.
Ultra-low-molecular-weight heparins: Precise structural features impacting specific anticoagulant activities
Authors: Lima Marcelo , Viskov Christian , Herman Frederic , Gray Angel , de Farias Eduardo , Cavalheiro Renan , Sassaki Guilherme , Hoppensteadt Debra , Fareed Fareed , Nader Helena
Published: January 2013

Ultra-low-molecular-weight heparins (ULMWHs) with better efficacy and safety ratios are under development; however, there are few structural data available. The main structural features and molecular weight of ULMWHs were studied and compared to enoxaparin. Their monosaccharide composition and average molecular weights were determined and preparations studied by nuclear magnetic resonance spectroscopy, scanning ultraviolet spectroscopy, circular dichroism and gel permeation chromatography. In general, ULMWHs presented higher 3-O-sulphated glucosamine and unsaturated uronic acid residues, the latter being comparable with their higher degree of depolymerisation. The analysis showed that ULMWHs are structurally related to LMWHs; however, their monosaccharide/oligosaccharide compositions and average molecular weights differed considerably explaining their different anticoagulant activities. The results relate structural features to activity, assisting the development of new and improved therapeutic agents, based on depolymerised heparin, for the prophylaxis and treatment of thrombotic disorders.
Role of Dynamin-Related Protein 1 (Drp1)-Mediated Mitochondrial Fission in Oxygen Sensing and Constriction of the Ductus Arteriosus
Authors: Hong Zhigang , Kutty Shelby , Toth Peter T. , Marsboom Glenn , Hammel James M. , Chamberlain Carolyn , Ryan John J. , Zhang Hannah J. , Sharp Willard W. , Morrow Erik , Trivedi Kalyani , Weir E. Kenneth , Archer Stephen L.
Published: January 2013

Rationale:

Closure of the ductus arteriosus (DA) is essential for the transition from fetal to neonatal patterns of circulation. Initial P
O
2
-dependent vasoconstriction causes functional DA closure within minutes. Within days a fibrogenic, proliferative mechanism causes anatomic closure. Though modulated by endothelial-derived vasodilators and constrictors, O
2
sensing is intrinsic to ductal smooth muscle cells and oxygen-induced DA constriction persists in the absence of endothelium, endothelin, and cyclooxygenase mediators. O
2
increases mitochondrial-derived H
2
O
2
, which constricts ductal smooth muscle cells by raising intracellular calcium and activating rho kinase. However, the mechanism by which oxygen changes mitochondrial function is unknown.

Objective:

The purpose of this study was to determine whether mitochondrial fission is crucial for O
2
-induced DA constriction and closure.

Methods and Results:

Using DA harvested from 30 term infants during correction of congenital heart disease, as well as DA from term rabbits, we demonstrate that mitochondrial fission is crucial for O
2
-induced constriction and closure. O
2
rapidly (<5 minutes) causes mitochondrial fission by a cyclin-dependent kinase- mediated phosphorylation of dynamin-related protein 1 (Drp1) at serine 616. Fission triggers a metabolic shift in the ductal smooth muscle cells that activates pyruvate dehydrogenase and increases mitochondrial H
2
O
2
production. Subsequently, fission increases complex I activity. Mitochondrial-targeted catalase overexpression eliminates P
O
2
-induced increases in mitochondrial-derived H
2
O
2
and cytosolic calcium. The small molecule Drp1 inhibitor, Mdivi-1, and siDRP1 yield concordant results, inhibiting O
2
-induced constriction (without altering the response to phenylephrine or KCl) and preventing O
2
-induced increases in oxidative metabolism, cytosolic calcium, and ductal smooth muscle cells proliferation. Prolonged Drp1 inhibition reduces DA closure in a tissue culture model.

Conclusions:

Mitochondrial fission is an obligatory, early step in mammalian O
2
sensing and offers a promising target for modulating DA patency.