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Previous studies have primarily focused on predicting stress arousal, encompassing physiological, behavioral, and psychological responses to stressors, while neglecting the examination of stress appraisal. Stress appraisal involves the cognitive evaluation of a situation as stressful or non-stressful, and as a threat/pressure or a challenge/opportunity. In this study, we investigated several research questions related to the association between states of stress appraisal (i.e., boredom, eustress, coexisting eustress-distress, distress) and various factors such as stress levels, mood, productivity, physiological and behavioral responses, as well as the most effective ML algorithms and data signals for predicting stress appraisal. The results support the Yerkes-Dodson law, showing that a moderate stress level is associated with increased productivity and positive mood, while low and high levels of stress are related to decreased productivity and negative mood, with distress overpowering eustress when they coexist. Changes in stress appraisal relative to physiological and behavioral features were examined through the lenses of stress arousal, activity engagement, and performance. An XGBOOST model achieved the best prediction accuracies of stress appraisal, reaching 82.78% when combining physiological and behavioral features and 79.55% using only the physiological dataset. The small accuracy difference of 3% indicates that physiological data alone may be adequate to accurately predict stress appraisal, and the feature importance results identified electrodermal activity, skin temperature, and blood volume pulse as the most useful physiologic features. Implementing these models within work environments can serve as a foundation for designing workplace policies, practices, and stress management strategies that prioritize the promotion of eustress while reducing distress and boredom. Such efforts can foster a supportive work environment to enhance employee well-being and productivity.
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Estrés Psicológico , Lugar de Trabajo , Lugar de Trabajo/psicología , Afecto , Recolección de Datos , Condiciones de TrabajoRESUMEN
INTRODUCTION: Several blood inflammatory markers, such as high-sensitivity C-reactive protein (hs-CRP), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR), are linked to increased risk for cardiovascular diseases. This study aimed to evaluate these inflammatory markers after cardiac rehabilitation (CR) in patients with unstable ischemic heart disease (UIHD) who underwent successful percutaneous coronary intervention (PCI). METHODS: A cohort of 115 patients with successful PCI due to UIHD enrolled in the study from January 2018 to March 2021. We used a permuted block stratified randomization technique (2â :â 1 ratio). Seventy-seven patients were randomized to the CR group and 38 patients to the control group. The CR group underwent a 12-week pre-specified CR regimen. Blood samples were taken at baseline and follow-up at 12 weeks for both groups. RESULT: Among the 115 patients, 33 patients were female. The mean age was (53â ±â 5.55 years) in the control and (53â ±â 6.09 years) in the CR group. The two groups were comparable regarding their baseline characteristics and the values of the inflammatory markers. By contrast, at 12 weeks, the inflammatory marker values were significantly lower in the CR group compared to the control group; hs-CRP: 0.11 [0.08-0.14] vs. 0.21 [0.19-0.21], P -value <0.001; NLR: 2.17 [1.42-2.43] vs. 2.26 [2.07-2.6], P -value: 0.016; PLR: 91.2821 [63.3333-103.2000] vs. 92.600 [84.6154-110.0000], P -value: 0.027. CONCLUSION: CR after PCI in UIHD patients may attenuate some inflammatory markers, which might benefit cardiovascular health. Further studies are required to evaluate these findings with longer follow-up and the powered to measure major cardiovascular event rates.
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Rehabilitación Cardiaca , Isquemia Miocárdica , Intervención Coronaria Percutánea , Humanos , Femenino , Persona de Mediana Edad , Masculino , Proteína C-Reactiva/metabolismo , Intervención Coronaria Percutánea/efectos adversos , Biomarcadores , Linfocitos , Isquemia Miocárdica/diagnóstico , Isquemia Miocárdica/etiología , Neutrófilos/metabolismoRESUMEN
This research pioneers the application of a machine learning framework to predict the perceived productivity of office workers using physiological, behavioral, and psychological features. Two approaches were compared: the baseline model, predicting productivity based on physiological and behavioral characteristics, and the extended model, incorporating predictions of psychological states such as stress, eustress, distress, and mood. Various machine learning models were utilized and compared to assess their predictive accuracy for psychological states and productivity, with XGBoost emerging as the top performer. The extended model outperformed the baseline model, achieving an R2 of 0.60 and a lower MAE of 10.52, compared to the baseline model's R2 of 0.48 and MAE of 16.62. The extended model's feature importance analysis revealed valuable insights into the key predictors of productivity, shedding light on the role of psychological states in the prediction process. Notably, mood and eustress emerged as significant predictors of productivity. Physiological and behavioral features, including skin temperature, electrodermal activity, facial movements, and wrist acceleration, were also identified. Lastly, a comparative analysis revealed that wearable devices (Empatica E4 and H10 Polar) outperformed workstation addons (Kinect camera and computer-usage monitoring application) in predicting productivity, emphasizing the potential utility of wearable devices as an independent tool for assessment of productivity. Implementing the model within smart workstations allows for adaptable environments that boost productivity and overall well-being among office workers.
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Dispositivos Electrónicos Vestibles , Humanos , Muñeca/fisiología , Programas Informáticos , Aprendizaje Automático , Extremidad SuperiorRESUMEN
Human-Building Interaction (HBI) is a convergent field that represents the growing complexities of the dynamic interplay between human experience and intelligence within built environments. This paper provides core definitions, research dimensions, and an overall vision for the future of HBI as developed through consensus among 25 interdisciplinary experts in a series of facilitated workshops. Three primary areas contribute to and require attention in HBI research: humans (human experiences, performance, and well-being), buildings (building design and operations), and technologies (sensing, inference, and awareness). Three critical interdisciplinary research domains intersect these areas: control systems and decision making, trust and collaboration, and modeling and simulation. Finally, at the core, it is vital for HBI research to center on and support equity, privacy, and sustainability. Compelling research questions are posed for each primary area, research domain, and core principle. State-of-the-art methods used in HBI studies are discussed, and examples of original research are offered to illustrate opportunities for the advancement of HBI research.
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Entorno Construido , Humanos , Consenso , PredicciónRESUMEN
Noise is often considered a distractor; however recent studies suggest that sub-attentive individuals or individuals diagnosed with attention deficit hyperactivity disorder can benefit from white noise to enhance their cognitive performance. Research regarding the effect of white noise on neurotypical adults presents mixed results, thus the implications of white noise on the neurotypical population remain unclear. Thus, this study investigates the effect of 2 white noise conditions, white noise level at 45 dB and white noise level at 65 dB, on the cognitive performance, creativity, and stress levels of neurotypical young adults in a private office space. These conditions are compared to a baseline condition where participants are exposed to the office ambient noise. Our findings showed that the white noise level at 45 dB resulted in better cognitive performance in terms of sustained attention, accuracy, and speed of performance as well as enhanced creativity and lower stress levels. On the other hand, the 65 dB white noise condition led to improved working memory but higher stress levels, which leads to the conclusion that different tasks might require different noise levels for optimal performance. These results lay the foundation for the integration of white noise into office workspaces as a tool to enhance office workers' performance.
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Trastorno por Déficit de Atención con Hiperactividad , Memoria a Corto Plazo , Cognición , Humanos , Ruido , Adulto JovenRESUMEN
The objectives of this study are to investigate building professionals' experience, awareness, and interest in occupant health in buildings, and to assess the impact of the COVID-19 pandemic on their opinions, as well as to compare the research on occupant health in buildings to professionals' opinions. To address these objectives, a mixed research methodology, including a thorough review of the literature (NL = 190) and an online survey (NS = 274), was utilized. In general, there is an increasing research interest in occupant health and a heightened interest in health-related projects, among professionals, following the COVID-19 pandemic. Specifically, among the nine different building attributes examined, indoor air quality was the most researched building attribute with a focus on occupant health and was also presumed to be the most important by the professionals. Professionals considered fatigue and musculoskeletal pain to be the most important physical well-being issues, and stress, anxiety, and depression to be the most important mental well-being issues that need to be the focus of design, construction, and operation of buildings to support and promote occupant health, while eye-related symptoms and loss of concentration were the most researched physical and mental well-being symptoms in the literature, respectively. Finally, professionals indicated that COVID-19 pandemic had significant effect on their perspectives regarding buildings' impact on occupant health and they believed future building design, construction and operation will focus more on occupant health because of the pandemic experience.
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BACKGROUND: With the COVID-19 pandemic, organizations embraced Work From Home (WFH). An important component of transitioning to WFH is the effect on workers, particularly related to their productivity and work experience. OBJECTIVES: The objective of this study is to examine how worker-, workspace-, and work-related factors affected productivity and time spent at a workstation on a typical WFH day during the pandemic. METHODS: An online questionnaire was designed and administered to collect the necessary information. Data from 988 respondents were included in the analyses. RESULTS: Overall perception of productivity level among workers did not change relative to their in-office productivity before the pandemic. Female, older, and high-income workers were likely to report increased productivity. Productivity was positively influenced by better mental and physical health statuses, having a teenager, increased communication with coworkers and having a dedicated room for work. Number of hours spent at a workstation increased by approximately 1.5 hours during a typical WFH day. Longer hours were reported by individuals who had school age children, owned an office desk or an adjustable chair, and had adjusted their work hours. CONCLUSION: The findings highlight key factors for employers and employees to consider for improving the WFH experience.
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COVID-19 , Pandemias , Adolescente , Niño , Eficiencia , Femenino , Humanos , SARS-CoV-2 , TeletrabajoRESUMEN
Even before the COVID-19 pandemic, people spent on average around 90% of their time indoors. Now more than ever, with work-from-home orders in place, it is crucial that we radically rethink the design and operation of buildings. Indoor Environmental Quality (IEQ) directly affects the comfort and well-being of occupants. When IEQ is compromised, occupants are at increased risk for many diseases that are exacerbated by both social and economic forces. In the U.S. alone, the annual cost attributed to sick building syndrome in commercial workplaces is estimated to be between $10 billion to $70 billion. It is imperative to understand how parameters that drive IEQ can be designed properly and how buildings can be operated to provide ideal IEQ to safeguard health. While IEQ is a fertile area of scholarship, there is a pressing need for a systematic understanding of how IEQ factors impact occupant health. During extreme events, such as a global pandemic, designers, facility managers, and occupants need pragmatic guidance on reducing health risks in buildings. This paper answers ten questions that explore the effects of buildings on the health of occupants. The study establishes a foundation for future work and provides insights for new research directions and discoveries.
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BACKGROUND: Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC. METHODS AND RESULTS: A constant infusion of [2,3-(13)C(2)]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester (13)C-enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,3-(13)C(2)]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or â8 g/d. Red blood cell-derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was â28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,3-(13)C(2)]-cholesterol in fecal bile acids and neutral sterols, respectively. CONCLUSIONS: Three components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. (J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826.).
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DNA replication occurs almost exclusively during S-phase of the cell cycle and represents a simple biochemical metric of cell division. Previous methods for measuring cell proliferation rates have important limitations. Here, we describe experimental protocols for measuring cell proliferation and death rates based on the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells. Label incorporation is measured by gas chromatography/mass spectrometry. Modifications of the basic protocol permit analysis of small cell samples (down to 2,000 cells). The theoretical basis and operational requirements for effective use of these methods to measure proliferation and death rates of cells in vivo are described. These methods are safe for use in humans, have technical and interpretation advantages over alternative techniques and can be used on small numbers of cells. The protocols enable definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans.
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Proliferación Celular , Óxido de Deuterio/análisis , Animales , Agua Corporal , Muerte Celular , Colon/citología , Replicación del ADN , Óxido de Deuterio/metabolismo , Células Endoteliales/citología , Células Epiteliales/citología , Humanos , Marcaje Isotópico , Queratinocitos/citología , Ratones , Células Musculares/citología , Neoplasias , Neuronas/citología , Ratas , Técnicas de Cultivo de TejidosRESUMEN
DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.
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Proliferación Celular , Cromatografía de Gases/métodos , ADN/metabolismo , Óxido de Deuterio/análisis , Espectrometría de Masas/métodos , Animales , ADN/biosíntesis , Replicación del ADN , Desoxirribosa/análogos & derivados , Desoxirribosa/química , Desoxirribosa/metabolismo , Óxido de Deuterio/metabolismo , Humanos , Marcaje Isotópico , Conformación MolecularRESUMEN
Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders.
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Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Microtúbulos/química , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Animales , Axones/metabolismo , Progresión de la Enfermedad , Humanos , Cinética , Ratones , Ratones Transgénicos , Modelos Biológicos , Oxígeno/metabolismo , Superóxido Dismutasa/genética , Agua/química , Agua/metabolismoRESUMEN
Microglial activation is emerging as an important etiologic factor and therapeutic target in neurodegenerative and neuroinflammatory diseases. Techniques have been lacking, however, for measuring the different components of microglial activation independently in vivo. We describe a method for measuring microglial proliferation rates in vivo using heavy water (2H2O) labeling, and its application in screening for drugs that suppress neuro-inflammation. Brain microglia were isolated by flow cytometry as F4/80+, CD11b+, CD45(low) cells, and 2H enrichment in DNA was analyzed by gas chromatography/mass spectrometry. Basal proliferation rate was approximately 1%/week and systemic administration of bacterial lipopolysaccharide (LPS) markedly increased this rate in a dose-dependent manner. Induction of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice by MOG(35-55) peptide stimulated proliferation of CD45(low) microglia, which could be distinguished from the proliferation of CD45(high) infiltrating monocytes. Minocycline (45 mg/kg/day, i.p.) inhibited resident microglial proliferation in both the LPS and EAE models. Thirteen drugs were then screened for their ability to inhibit LPS-stimulated microglia proliferation. Female C57BL/6 mice were given LPS (1 mg/kg), and concomitant drug treatment while receiving 2H2O label for 7 days. Among the drugs screened, treatment with isotretinoin dose-dependently reduced LPS-induced microglial proliferation, representing an action of retinoids unknown previously. Follow-up studies in the EAE model confirmed that isotretinoin not only inhibited proliferation of microglia but also delayed the onset of clinical symptoms. In conclusion, 2H2O labeling represents a relatively high-throughput, quantitative, and highly reproducible technique for measuring microglial proliferation, and is useful for screening and discovering novel anti-neuroinflammatory drugs.
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Antiinflamatorios/farmacología , Encéfalo/efectos de los fármacos , Óxido de Deuterio , Evaluación Preclínica de Medicamentos/métodos , Inflamación/tratamiento farmacológico , Microglía/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Citometría de Flujo , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Enhanced production of collagen is central to fibrotic disorders such as hepatic cirrhosis and pulmonary fibrosis. We describe a sensitive, quantitative, and high-throughput technique for measuring hepatic collagen synthesis in vivo through metabolic labeling with heavy water ((2)H(2)O). Rats and mice received (2)H(2)O in drinking water for up to 35 days. Deuterium incorporation into collagen-bound amino acids (AA) alanine and hydroxyproline (OHP) was measured by gas chromatography-mass spectrometry. A threefold stimulation of collagen fractional synthesis was observed under the maximum dosage of carbon tetrachloride (CCl(4); 1.67 ml/kg). Deuterium enrichment was systematically 20% higher in AA from monomeric collagen relative to dimeric collagen, consistent with slower turnover of the latter. Administration of 1% griseofulvin to mice resulted in a significant, threefold increase in liver collagen synthesis, observable within 12 days and consistent with predicted interstrain differences (C57/Bl6J > BALB/c). Deuterium enrichments of OHP from total liver proteins correlated well with alanine or OHP from isolated collagen. Fibrogenesis subsided after withdrawal of CCl(4) exposure and was reduced to various degrees by coadministration of interferon-gamma, rosiglitazone, atorvastatin, or enalapril. Changes in isotopically measured collagen synthesis correlated with, but were more sensitive and reproducible than, standard histological staining (trichrome) for fibrosis. In summary, liver collagen synthesis can be measured sensitively and with high precision over a short time period, without radioactivity, thereby providing a relatively high-throughput in vivo strategy for rapidly measuring profibrotic activities of suspected hepatotoxicants and antifibrotic activities of drug candidates.
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Colágeno/biosíntesis , Óxido de Deuterio/administración & dosificación , Evaluación Preclínica de Medicamentos/métodos , Cromatografía de Gases y Espectrometría de Masas , Cirrosis Hepática Experimental/metabolismo , Hígado/metabolismo , Administración Oral , Alanina/metabolismo , Animales , Atorvastatina , Tetracloruro de Carbono , Enalapril/farmacología , Enalapril/uso terapéutico , Griseofulvina , Ácidos Heptanoicos/farmacología , Ácidos Heptanoicos/uso terapéutico , Hidroxiprolina/metabolismo , Interferón gamma/farmacología , Interferón gamma/uso terapéutico , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Cirrosis Hepática Experimental/prevención & control , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pirroles/farmacología , Pirroles/uso terapéutico , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Rosiglitazona , Especificidad de la Especie , Tiazolidinedionas/farmacología , Tiazolidinedionas/uso terapéutico , Factores de TiempoRESUMEN
OBJECTIVE: The purpose of this study was to compare an in vivo test of whole-body glycolysis, the deuterated-glucose disposal test (2H-GDT), with insulin sensitivity measured by the euglycemic-hyperinsulinemic glucose clamp and the steady-state plasma glucose (SSPG) test. RESEARCH DESIGN AND METHODS: The 2H-GDT consists of an oral glucose challenge containing deuterated glucose, followed by measurement of heavy water (2H2O) production, which represents whole-body glycolytic disposal of the glucose load. 2H2O production is corrected for ambient insulin concentration as an index of tissue insulin sensitivity. The 2H-GDT was compared with euglycemic-hyperinsulinemic glucose clamps in healthy lean subjects (n = 8) and subjects with the metabolic syndrome (n = 9) and with the SSPG test in overweight (n = 12) and obese (n = 6) subjects. RESULTS: A strong correlation with the clamp was observed for the 75-g and 30-g 2H-GDT (r = 0.95, P < 0.0001 and r = 0.88, P < 0.0001, respectively). The 2H-GDT and clamp studies revealed marked insulin resistance in subjects with metabolic syndrome compared with lean control subjects. The correlation with the clamp was maintained in each group (lean, r = 0.86, P < 0.01; metabolic syndrome, r = 0.81, P < 0.01) for the 75-g test. The 2H-GDT also correlated strongly with the SSPG test (r = -0.87, P < 0.0001) in overweight and obese subjects. CONCLUSIONS: The 2H-GDT, which measures whole-body glycolysis in humans in a quantitative manner, correlates highly with the euglycemic-hyperinsulinemic glucose clamp and the SSPG test. Impaired insulin-mediated whole-body glycolysis is a feature of insulin resistance, which provides a means of assessing insulin sensitivity in vivo.
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Glucosa/metabolismo , Resistencia a la Insulina , Síndrome Metabólico/metabolismo , Administración Oral , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Deuterio , Glucosa/administración & dosificación , Técnica de Clampeo de la Glucosa , Glucólisis , Homeostasis , Humanos , Hiperinsulinismo/sangre , Insulina/farmacología , Insulina/fisiología , Marcaje Isotópico/métodos , Síndrome Metabólico/fisiopatología , Modelos Biológicos , Sensibilidad y EspecificidadRESUMEN
In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water 2H enrichments for weeks or months. 2H is metabolically incorporated into C-H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so 2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of 2H2O-labeled rodent tissue proteins that metabolic 2H flux into C-H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By 2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, 2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.
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Aminoácidos/metabolismo , Óxido de Deuterio/administración & dosificación , Marcaje Isotópico/métodos , Biosíntesis de Proteínas , Proteínas/análisis , Animales , Proteínas Sanguíneas/análisis , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Química Encefálica , Tetracloruro de Carbono/toxicidad , Colágeno/análisis , Colágeno/metabolismo , Óxido de Deuterio/farmacocinética , Femenino , Fibrosis , Cromatografía de Gases y Espectrometría de Masas , Humanos , Cinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculos/química , Músculos/metabolismo , Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Microtubules are dynamic polymers with central roles in the mitotic checkpoint, mitotic spindle assembly, and chromosome segregation. Agents that block mitotic progression and cell proliferation by interfering with microtubule dynamics (microtubule-targeted tubulin-polymerizing agents (MTPAs)) are powerful antitumor agents. Effects of MTPAs (e.g. paclitaxel) on microtubule dynamics have not yet been directly demonstrated in intact animals, however. Here we describe a method that measures microtubule dynamics as an exchange of tubulin dimers into microtubules in vivo. The incorporation of deuterium ((2)H(2)) from heavy water ((2)H(2)O) into tubulin dimers and polymers is measured by gas chromatography/mass spectrometry. In cultured human lung and breast cancer cell lines, or in tumors implanted into nude mice, tubulin dimers and polymerized microtubules exhibited nearly identical label incorporation rates, reflecting their rapid exchange. Administration of paclitaxel during 24 h of (2)H(2)O labeling in vivo reduced (2)H labeling in polymers while increasing (2)H in dimers, indicating diminished flux of dimers into polymers (i.e. inhibition of microtubule dynamic equilibrium). In vivo inhibition of microtubule dynamics was dose-dependent and correlated with inhibition of DNA replication, a stable isotopic measure of tumor cell growth. In contrast, microtubule polymers from sciatic nerve of untreated mice were not in dynamic equilibrium with tubulin dimers, and paclitaxel increased label incorporation into polymers. Our results directly demonstrate altered microtubule dynamics as an important action of MTPAs in vivo. This sensitive and quantitative in vivo assay of microtubule dynamics may prove useful for pre-clinical and clinical development of the next generation of MTPAs as anticancer drugs.
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Óxido de Deuterio/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Dimerización , Humanos , Ratones , Ratones Desnudos , Microtúbulos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/cirugía , Paclitaxel/farmacología , Tubulina (Proteína)/efectos de los fármacosRESUMEN
Deoxyribose oxidation in DNA represents a biologically important facet of oxidative DNA damage that gives rise to protein-DNA cross-links and base adducts. Toward the goal of quantifying deoxyribose oxidation chemistry in cells, we report a method for the quantification of 3'-phosphoglycolaldehyde (PGA) residues, which likely arise from 3'-oxidation of deoxyribose in DNA. The method exploits the aldehyde moiety in PGA by derivatization as a stable oxime with pentafluorobenzylhydroxylamine, followed by solvent extraction and gas chromatography/negative chemical ionization/mass spectrometry. A stable isotopically labeled [(13)C(2)]PGA was synthesized and used as an internal standard. The assay showed a linear response over the range of 30 fmol to 300 pmol, and its precision was verified by analysis of a synthetic, PGA-containing oligodeoxynucleotide. The limit of detection in the presence of DNA was 30 fmol per sample, corresponding to two molecules of PGA in 10(6) nucleotides for 170 microg of DNA. Samples were exposed to 0-100 Gy of (60)Co gamma-radiation, which resulted in a linear dose-response of 1.5 PGA residues per 10(6) nucleotides per Gy and a radiation chemical yield (G-value) of 0.0016 micromol/J. When compared to the total quantity of deoxyribose oxidation occurring under the same conditions (141 oxidation events per 10(6) nucleotides per Gy; determined by plasmid topoisomer analysis), PGA formation occurs in 1% of deoxyribose oxidation events. This small fraction is consistent with current models of limited solvent accessibility of the 3'-position of deoxyribose, although partitioning of 3'-chemistry could lead to other damage products that would increase the fraction of oxidation at this site in deoxyribose.