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Find video protocols related to scientific articles indexed in Pubmed.
Development and testing of a past year measure of sedentary behavior: the SIT-Q.
BMC Public Health
PUBLISHED: 09-01-2014
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Most sedentary behavior measures focus on occupational or leisure-time sitting. Our aim was to develop a comprehensive measure of adult sedentary behavior and establish its measurement properties.
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The Sedentary Time and Activity Reporting Questionnaire (STAR-Q): reliability and validity against doubly labeled water and 7-day activity diaries.
Am. J. Epidemiol.
PUBLISHED: 07-19-2014
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We determined measurement properties of the Sedentary Time and Activity Reporting Questionnaire (STAR-Q), which was designed to estimate past-month activity energy expenditure (AEE). STAR-Q validity and reliability were assessed in 102 adults in Alberta, Canada (2009-2011), who completed 14-day doubly labeled water (DLW) protocols, 7-day activity diaries on day 15, and the STAR-Q on day 14 and again at 3 and 6 months. Three-month reliability was substantial for total energy expenditure (TEE) and AEE (intraclass correlation coefficients of 0.84 and 0.73, respectively), while 6-month reliability was moderate. STAR-Q-derived TEE and AEE were moderately correlated with DLW estimates (Spearman's ?s of 0.53 and 0.40, respectively; P < 0.001), and on average, the STAR-Q overestimated TEE and AEE (median differences were 367 kcal/day and 293 kcal/day, respectively). Body mass index-, age-, sex-, and season-adjusted concordance correlation coefficients (CCCs) were 0.24 (95% confidence interval (CI): 0.07, 0.36) and 0.21 (95% CI: 0.11, 0.32) for STAR-Q-derived versus DLW-derived TEE and AEE, respectively. Agreement between the diaries and STAR-Q (metabolic equivalent-hours/day) was strongest for occupational sedentary time (adjusted CCC = 0.76, 95% CI: 0.64, 0.85) and overall strenuous activity (adjusted CCC = 0.64, 95% CI: 0.49, 0.76). The STAR-Q demonstrated substantial validity for estimating occupational sedentary time and strenuous activity and fair validity for ranking individuals by AEE.
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A high-density, high-channel count, multiplexed ?ECoG array for auditory-cortex recordings.
J. Neurophysiol.
PUBLISHED: 06-11-2014
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Our understanding of the large-scale population dynamics of neural activity is limited, in part, by our inability to record simultaneously from large regions of the cortex. Here, we validated the use of a large-scale active microelectrode array that simultaneously records 196 multiplexed micro-electrocortigraphical (?ECoG) signals from the cortical surface at a very high density (1,600 electrodes/cm(2)). We compared ?ECoG measurements in auditory cortex using a custom "active" electrode array to those recorded using a conventional "passive" ?ECoG array. Both of these array responses were also compared with data recorded via intrinsic optical imaging, which is a standard methodology for recording sound-evoked cortical activity. Custom active ?ECoG arrays generated more veridical representations of the tonotopic organization of the auditory cortex than current commercially available passive ?ECoG arrays. Furthermore, the cortical representation could be measured efficiently with the active arrays, requiring as little as 13.5 s of neural data acquisition. Next, we generated spectrotemporal receptive fields from the recorded neural activity on the active ?ECoG array and identified functional organizational principles comparable to those observed using intrinsic metabolic imaging and single-neuron recordings. This new electrode array technology has the potential for large-scale, temporally precise monitoring and mapping of the cortex, without the use of invasive penetrating electrodes.
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Machine learning helps identify CHRONO as a circadian clock component.
PLoS Biol.
PUBLISHED: 04-01-2014
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Over the last decades, researchers have characterized a set of "clock genes" that drive daily rhythms in physiology and behavior. This arduous work has yielded results with far-reaching consequences in metabolic, psychiatric, and neoplastic disorders. Recent attempts to expand our understanding of circadian regulation have moved beyond the mutagenesis screens that identified the first clock components, employing higher throughput genomic and proteomic techniques. In order to further accelerate clock gene discovery, we utilized a computer-assisted approach to identify and prioritize candidate clock components. We used a simple form of probabilistic machine learning to integrate biologically relevant, genome-scale data and ranked genes on their similarity to known clock components. We then used a secondary experimental screen to characterize the top candidates. We found that several physically interact with known clock components in a mammalian two-hybrid screen and modulate in vitro cellular rhythms in an immortalized mouse fibroblast line (NIH 3T3). One candidate, Gene Model 129, interacts with BMAL1 and functionally represses the key driver of molecular rhythms, the BMAL1/CLOCK transcriptional complex. Given these results, we have renamed the gene CHRONO (computationally highlighted repressor of the network oscillator). Bi-molecular fluorescence complementation and co-immunoprecipitation demonstrate that CHRONO represses by abrogating the binding of BMAL1 to its transcriptional co-activator CBP. Most importantly, CHRONO knockout mice display a prolonged free-running circadian period similar to, or more drastic than, six other clock components. We conclude that CHRONO is a functional clock component providing a new layer of control on circadian molecular dynamics.
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Cell type-specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models.
PLoS Genet.
PUBLISHED: 04-01-2014
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In animals, circadian rhythms in physiology and behavior result from coherent rhythmic interactions between clocks in the brain and those throughout the body. Despite the many tissue specific clocks, most understanding of the molecular core clock mechanism comes from studies of the suprachiasmatic nuclei (SCN) of the hypothalamus and a few other cell types. Here we report establishment and genetic characterization of three cell-autonomous mouse clock models: 3T3 fibroblasts, 3T3-L1 adipocytes, and MMH-D3 hepatocytes. Each model is genetically tractable and has an integrated luciferase reporter that allows for longitudinal luminescence recording of rhythmic clock gene expression using an inexpensive off-the-shelf microplate reader. To test these cellular models, we generated a library of short hairpin RNAs (shRNAs) against a panel of known clock genes and evaluated their impact on circadian rhythms. Knockdown of Bmal1, Clock, Cry1, and Cry2 each resulted in similar phenotypes in all three models, consistent with previous studies. However, we observed cell type-specific knockdown phenotypes for the Period and Rev-Erb families of clock genes. In particular, Per1 and Per2, which have strong behavioral effects in knockout mice, appear to play different roles in regulating period length and amplitude in these peripheral systems. Per3, which has relatively modest behavioral effects in knockout mice, substantially affects period length in the three cellular models and in dissociated SCN neurons. In summary, this study establishes new cell-autonomous clock models that are of particular relevance to metabolism and suitable for screening for clock modifiers, and reveals previously under-appreciated cell type-specific functions of clock genes.
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Behavioral flexibility is increased by optogenetic inhibition of neurons in the nucleus accumbens shell during specific time segments.
Learn. Mem.
PUBLISHED: 03-19-2014
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Behavioral flexibility is vital for survival in an environment of changing contingencies. The nucleus accumbens may play an important role in behavioral flexibility, representing learned stimulus-reward associations in neural activity during response selection and learning from results. To investigate the role of nucleus accumbens neural activity in behavioral flexibility, we used light-activated halorhodopsin to inhibit nucleus accumbens shell neurons during specific time segments of a bar-pressing task requiring a win-stay/lose-shift strategy. We found that optogenetic inhibition during action selection in the time segment preceding a lever press had no effect on performance. However, inhibition occurring in the time segment during feedback of results--whether rewards or nonrewards--reduced the errors that occurred after a change in contingency. Our results demonstrate critical time segments during which nucleus accumbens shell neurons integrate feedback into subsequent responses. Inhibiting nucleus accumbens shell neurons in these time segments, during reinforced performance or after a change in contingencies, increases lose-shift behavior. We propose that the activity of nucleus shell accumbens shell neurons in these time segments plays a key role in integrating knowledge of results into subsequent behavior, as well as in modulating lose-shift behavior when contingencies change.
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The natural history of egg allergy in an observational cohort.
J. Allergy Clin. Immunol.
PUBLISHED: 03-19-2014
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There are few studies on the natural history of egg allergy, and most are single-site and nonlongitudinal and have not identified early predictors of outcomes.
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Prevalence of allergic sensitization in the United States: results from the National Health and Nutrition Examination Survey (NHANES) 2005-2006.
J. Allergy Clin. Immunol.
PUBLISHED: 02-09-2014
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Allergic sensitization is an important risk factor for the development of atopic disease. The National Health and Nutrition Examination Survey (NHANES) 2005-2006 provides the most comprehensive information on IgE-mediated sensitization in the general US population.
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Dissecting childhood asthma with nasal transcriptomics distinguishes subphenotypes of disease.
J. Allergy Clin. Immunol.
PUBLISHED: 02-02-2014
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Bronchial airway expression profiling has identified inflammatory subphenotypes of asthma, but the invasiveness of this technique has limited its application to childhood asthma.
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Reduction in myocardial infarction admissions in Liverpool after the smoking ban: potential socioeconomic implications for policymaking.
BMJ Open
PUBLISHED: 11-28-2013
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To analyse the trends and trend changes in myocardial infraction (MI) and coronary heart disease (CHD) admissions, to investigate the effects of the 2007 smoke-free legislation on these trends, and to consider the policy implications of any findings.
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Translational control of entrainment and synchrony of the suprachiasmatic circadian clock by mTOR/4E-BP1 signaling.
Neuron
PUBLISHED: 06-17-2013
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Protein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor(+/-) mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock.
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Lack of a relation between serum 25-hydroxyvitamin D concentrations and asthma in adolescents.
Am. J. Clin. Nutr.
PUBLISHED: 04-17-2013
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Decreased 25-hydroxyvitamin D [25(OH)D] concentrations have been associated with an increased prevalence and severity of asthma and a lower response to inhaled corticosteroids.
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Circadian regulation of ATP release in astrocytes.
J. Neurosci.
PUBLISHED: 06-10-2011
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Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release.
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G?16 interacts with tetratricopeptide repeat 1 (TPR1) through its ?3 region to activate Ras independently of phospholipase C? signaling.
BMC Struct. Biol.
PUBLISHED: 04-13-2011
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G protein-coupled receptors constitute the largest family of cell surface receptors in the mammalian genome. As the core of the G protein signal transduction machinery, the G? subunits are required to interact with multiple partners. The GTP-bound active state of many G? subunits can bind a multitude of effectors and regulatory proteins. Yet it remains unclear if the different proteins utilize distinct or common structural motifs on the G? subunit for binding. Using G?16 as a model, we asked if its recently discovered adaptor protein tetratricopeptide repeat 1 (TPR1) binds to the same region as its canonical effector, phospholipase C? (PLC?).
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Food allergies and asthma.
Curr Opin Allergy Clin Immunol
PUBLISHED: 04-07-2011
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To consider the possible links between food allergy and asthma.
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Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children.
N. Engl. J. Med.
PUBLISHED: 03-18-2011
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Research has underscored the effects of exposure and sensitization to allergens on the severity of asthma in inner-city children. It has also revealed the limitations of environmental remediation and guidelines-based therapy in achieving greater disease control.
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Delay in feedback repression by cryptochrome 1 is required for circadian clock function.
Cell
PUBLISHED: 01-18-2011
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Direct evidence for the requirement of delay in feedback repression in the mammalian circadian clock has been elusive. Cryptochrome 1 (Cry1), an essential clock component, displays evening-time expression and serves as a strong repressor at morning-time elements (E box/E box). In this study, we reveal that a combination of day-time elements (D box) within the Cry1-proximal promoter and night-time elements (RREs) within its intronic enhancer gives rise to evening-time expression. A synthetic composite promoter produced evening-time expression, which was further recapitulated by a simple phase-vector model. Of note, coordination of day-time with night-time elements can modulate the extent of phase delay. A genetic complementation assay in Cry1(-/-):Cry2(-/-) cells revealed that substantial delay of Cry1 expression is required to restore circadian rhythmicity, and its prolonged delay slows circadian oscillation. Taken together, our data suggest that phase delay in Cry1 transcription is required for mammalian clock function.
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Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis.
Nat. Med.
PUBLISHED: 05-26-2010
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During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)?. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.
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The Childhood Asthma Control Test: retrospective determination and clinical validation of a cut point to identify children with very poorly controlled asthma.
J. Allergy Clin. Immunol.
PUBLISHED: 04-29-2010
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The Childhood Asthma Control Test (C-ACT) has demonstrated validity in classifying children aged 4 to 11 years as having either "well-controlled" or "not well-controlled" asthma. However, new asthma management guidelines distinguish 3 levels of asthma control.
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Galpha16 activates Ras by forming a complex with tetratricopeptide repeat 1 (TPR1) and Son of Sevenless (SOS).
Cell. Signal.
PUBLISHED: 04-23-2010
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Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.
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Emergence of noise-induced oscillations in the central circadian pacemaker.
PLoS Biol.
PUBLISHED: 02-23-2010
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Bmal1 is an essential transcriptional activator within the mammalian circadian clock. We report here that the suprachiasmatic nucleus (SCN) of Bmal1-null mutant mice, unexpectedly, generates stochastic oscillations with periods that overlap the circadian range. Dissociated SCN neurons expressed fluctuating levels of PER2 detected by bioluminescence imaging but could not generate circadian oscillations intrinsically. Inhibition of intercellular communication or cyclic-AMP signaling in SCN slices, which provide a positive feed-forward signal to drive the intracellular negative feedback loop, abolished the stochastic oscillations. Propagation of this feed-forward signal between SCN neurons then promotes quasi-circadian oscillations that arise as an emergent property of the SCN network. Experimental analysis and mathematical modeling argue that both intercellular coupling and molecular noise are required for the stochastic rhythms, providing a novel biological example of noise-induced oscillations. The emergence of stochastic circadian oscillations from the SCN network in the absence of cell-autonomous circadian oscillatory function highlights a previously unrecognized level of circadian organization.
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Predictors of remitting, periodic, and persistent childhood asthma.
J. Allergy Clin. Immunol.
PUBLISHED: 02-18-2010
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The course of mild to moderate persistent asthma in children is not clearly established.
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Status of asthma control in pediatric primary care: results from the pediatric Asthma Control Characteristics and Prevalence Survey Study (ACCESS).
J. Pediatr.
PUBLISHED: 02-15-2010
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To estimate the prevalence of uncontrolled asthma in pediatric patients with asthma visiting their primary care provider for any medical reason.
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A genome-wide RNAi screen for modifiers of the circadian clock in human cells.
Cell
PUBLISHED: 05-30-2009
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Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.
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Asthma morbidity among inner-city adolescents receiving guidelines-based therapy: role of predictors in the setting of high adherence.
J. Allergy Clin. Immunol.
PUBLISHED: 04-21-2009
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With the expanding effort to provide guidelines-based therapy to adolescents with asthma, attention must be directed to evaluating which factors predict future asthma control when guidelines-based management is applied.
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Association of obesity with IgE levels and allergy symptoms in children and adolescents: results from the National Health and Nutrition Examination Survey 2005-2006.
J. Allergy Clin. Immunol.
PUBLISHED: 02-23-2009
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The prevalence of both obesity and allergic disease has increased among children over the last several decades. Previous literature on the relationship between obesity and allergic disease has been inconsistent. It is not known whether systemic inflammation could be a factor in this relationship.
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Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 01-23-2009
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A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.
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Immunopathology of chronic rhinosinusitis in young children.
J. Pediatr.
PUBLISHED: 01-21-2009
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Previous investigation demonstrated predominantly lymphocytic inflammation in sinus mucosa of young children with chronic rhinosinusitis (CRS) rather than eosinophilic inflammation typical of adult CRS. Immunohistopathological study was undertaken to define further the cellular response in pediatric CRS.
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Mediator release assay for assessment of biological potency of German cockroach allergen extracts.
J. Allergy Clin. Immunol.
PUBLISHED: 01-12-2009
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Cockroach is an important allergen in inner-city asthma. The diagnosis and treatment of cockroach allergy has been impeded by the lack of standardized cockroach extracts.
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The natural history of milk allergy in an observational cohort.
J. Allergy Clin. Immunol.
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There are few studies on the natural history of milk allergy. Most are single-site and not longitudinal, and these have not identified a means for early prediction of outcomes.
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Cry1-/- circadian rhythmicity depends on SCN intercellular coupling.
J. Biol. Rhythms
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In mammals, the suprachiasmatic nucleus (SCN) is the central pacemaker organizing circadian rhythms of behavior and physiology. At the cellular level, the mammalian clock consists of autoregulatory feedback loops involving a set of "clock genes," including the Cryptochrome (Cry) genes, Cry1 and Cry2. Experimental evidence suggests that Cry1 and Cry2 play distinct roles in circadian clock function. In mice, Cry1 is required for sustained circadian rhythms in dissociated SCN neurons or fibroblasts but not in organotypic SCN slices or at the behavioral level, whereas Cry2 is not required at any of these levels. It has been argued that coupling among SCN cellular oscillators compensates for clock gene defects to preserve oscillatory function. Here we test this hypothesis in Cry1(-/-) mice by first disrupting intercellular coupling in vivo using constant light (resulting in behavioral arrhythmicity) and then examining circadian clock gene expression in SCN slices at the single cell level. In this manner, we were able to test the role of intercellular coupling without drugs and while preserving tissue organization, avoiding the confounding influences of more invasive manipulations. Cry1(-/-) mice (as well as control Cry2(-/-) mice) bearing the PER2::LUC knock-in reporter were transferred from a standard light:dark cycle to constant bright light (~650 lux) to induce arrhythmic locomotor patterns. In SCN slices from these animals, we used bioluminescence imaging to monitor PER2::LUC expression in single cells. We show that SCN slices from rhythmic Cry1(-/-) and Cry2(-/-) mice had similarly high percentages of functional single-cell oscillators. In contrast, SCN slices from arrhythmic Cry1(-/-) mice had significantly fewer rhythmic cells than SCN slices from arrhythmic Cry2(-/-) mice. Thus, constant light in vivo disrupted intercellular SCN coupling to reveal a cell-autonomous circadian defect in Cry1(-/-) cells that is normally compensated by intercellular coupling in vivo.
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Monitoring cell-autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters.
J Vis Exp
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In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere. Individual cells are the functional units for generation and maintenance of circadian rhythms, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms. Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection or stable transduction. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.
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Predictors for asthma at age 7 years for low-income children enrolled in the Childhood Asthma Prevention Study.
J. Pediatr.
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To identify the predictive factors of early childhood wheezing in children of low socioeconomic status.
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Oral immunotherapy for treatment of egg allergy in children.
N. Engl. J. Med.
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For egg allergy, dietary avoidance is the only currently approved treatment. We evaluated oral immunotherapy using egg-white powder for the treatment of children with egg allergy.
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Identification of a novel cryptochrome differentiating domain required for feedback repression in circadian clock function.
J. Biol. Chem.
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Circadian clocks in mammals are based on a negative feedback loop in which transcriptional repression by the cryptochromes, CRY1 and CRY2, lies at the heart of the mechanism. Despite similarities in sequence, domain structure, and biochemical activity, they play distinct roles in clock function. However, detailed biochemical studies have not been straightforward and Cry function has not been examined in real clock cells using kinetic measurements. In this study, we demonstrate, through cell-based genetic complementation and real-time molecular recording, that Cry1 alone is able to maintain cell-autonomous circadian rhythms, whereas Cry2 cannot. Using this novel functional assay, we identify a cryptochrome differentiating ?-helical domain within the photolyase homology region (PHR) of CRY1, designated as CRY1-PHR(313-426), that is required for clock function and distinguishes CRY1 from CRY2. Contrary to speculation, the divergent carboxyl-terminal tail domain (CTD) is dispensable, but serves to modulate rhythm amplitude and period length. Finally, we identify the biochemical basis of their distinct function; CRY1 is a much more potent transcriptional repressor than CRY2, and the strength of repression by various forms of CRY proteins significantly correlates with rhythm amplitude. Taken together, our results demonstrate that CRY1-PHR(313-426), not the divergent CTD, is critical for clock function. These findings provide novel insights into the evolution of the diverse functions of the photolyase/cryptochrome family of flavoproteins and offer new opportunities for mechanistic studies of CRY function.
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Asthma outcomes: biomarkers.
J. Allergy Clin. Immunol.
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Measurement of biomarkers has been incorporated within clinical research studies of asthma to characterize the population and associate the disease with environmental and therapeutic effects.
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Development and validation of the Composite Asthma Severity Index--an outcome measure for use in children and adolescents.
J. Allergy Clin. Immunol.
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Asthma severity is reflected in many aspects of the disease, including impairment and future risks, particularly for exacerbations. According to the Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma, however, to assess more comprehensively the severity of asthma the level of current treatment needed to maintain a level of control should be included.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.