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BACKGROUND: The use of biomarkers in skin is a novel diagnostic tool. Interstitial fluid (ISF) from skin provides a snapshot of proteins secreted at the time of sampling giving insights into the patient's health status. METHODS: A minimally invasive technique for the transdermal collection of human ISF proteins. A low frequency ultrasonic skin permeation device (SonoPrep ultrasonic skin permeation system) was used to produce micropores in the stratum corneum through which ISF was extracted using a portable pulsed vacuum ISF collection device. RESULTS: On average, protein concentrations recovered ranged between 0.064 and 4.792 µg/µL (mean 1.258 µg/µL). Two-dimensional gel electrophoresis revealed that this sample type was amenable to this type of analysis. Gel images indicated that both highly abundant proteins and lower abundance proteins were isolated from the skin. Western blot analysis confirmed the presence of proteins commonly found in plasma and the epidermis. CONCLUSION: A minimally invasive method for the transdermal recovery of ISF proteins has been developed. We have demonstrated that ISF samples obtained using this approach can be analysed with proteomic techniques, such as two-dimensional gel electrophoresis and western blots, providing another tool for the identification of disease specific protein biomarkers.
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Dermatología/métodos , Epidermis/metabolismo , Líquido Extracelular/metabolismo , Proteómica/métodos , Ultrasonido/métodos , Adulto , Biomarcadores/metabolismo , Western Blotting/métodos , Dermatología/instrumentación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas/metabolismo , Proteómica/instrumentación , Valores de Referencia , Manejo de Especímenes/métodos , Electroforesis Bidimensional Diferencial en Gel/métodos , Ultrasonido/instrumentación , VacioRESUMEN
Two dimensional polyacrylamide gel electrophoresis (2D PAGE) is used to identify differentially expressed proteins and may be applied to biomarker discovery. A limitation of this approach is the inability to detect a protein when its concentration falls below the limit of detection. Consequently, differential expression of proteins may be missed when the level of a protein in the cases or controls is below the limit of detection for 2D PAGE. Standard statistical techniques have difficulty dealing with undetected proteins. To address this issue, we propose a mixture model that takes into account both detected and non-detected proteins. Non-detected proteins are classified either as (a) proteins that are not expressed in at least one replicate, or (b) proteins that are expressed but are below the limit of detection. We obtain maximum likelihood estimates of the parameters of the mixture model, including the group-specific probability of expression and mean expression intensities. Differentially expressed proteins can be detected by using a Likelihood Ratio Test (LRT). Our simulation results, using data generated from biological experiments, show that the likelihood model has higher statistical power than standard statistical approaches to detect differentially expressed proteins. An R package, Slider (Statistical Likelihood model for Identifying Differential Expression in R), is freely available at http://www.cebl.auckland.ac.nz/slider.php.
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Electroforesis en Gel Bidimensional/métodos , Modelos Biológicos , Modelos Estadísticos , Proteínas/metabolismo , Proteómica/métodos , Algoritmos , Análisis de Varianza , Simulación por Computador , Femenino , Humanos , Funciones de Verosimilitud , Preeclampsia , Embarazo , Sensibilidad y EspecificidadRESUMEN
INTRODUCTION: Point-of-care (POC) measurements using saliva samples have immense potential to assess systemic health and wellbeing, but sample viscosity and contaminants can affect analyses. We sought a portable clean-up method for whole saliva appropriate for use with POC measurement techniques such as biosensors. METHODS: Whole saliva from each of 13 male subjects was split into 5 fractions. Each fraction was treated with a different clean-up process: a freeze-thaw-centrifuge (FTC) step; centrifugation alone; or passage through a Mini-UniPrep polyethersulfone filter, cotton Salivette, or foam Oracol device. Following clean-up, each subject's treated saliva fractions were assayed for cortisol, testosterone, dehydroepiandrosterone (DHEA), and protein concentrations. The effects of clean-up methods on nonspecific binding (NSB) in a biosensor were also assessed. RESULTS: Compared with FTC, no analytes were affected by centrifugation alone. Cotton Salivettes significantly altered all analytes, with increases in cortisol (+64%), testosterone (+126%), and DHEA (off-scale) levels, and decreased protein (-21%) and biosensor NSB (-75%). Oracol foam devices decreased DHEA levels by 28%. Mini-UniPrep filtration decreased testosterone (-45%) and DHEA (-66%) concentrations while increasing cortisol (+40%). CONCLUSION: No method was optimal for all analytes, highlighting the need for validation of saliva treatment methods before their adoption in rapid POC analyses.
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Saliva/química , Manejo de Especímenes/métodos , Adulto , Centrifugación , Deshidroepiandrosterona/análisis , Filtración , Congelación , Humanos , Hidrocortisona/análisis , Masculino , Persona de Mediana Edad , Sistemas de Atención de Punto , Radioinmunoensayo , Proteínas y Péptidos Salivales/análisis , Resonancia por Plasmón de Superficie , Testosterona/análisisRESUMEN
Humans vary in acuity to many odors [1-4], with variation within olfactory receptor (OR) genes contributing to these differences [5-9]. How such variation also affects odor experience and food selection remains uncertain [10], given that such effects occur for taste [11-15]. Here we investigate ß-ionone, which shows extreme sensitivity differences [4, 16, 17]. ß-ionone is a key aroma in foods and beverages [18-21] and is added to products in order to give a pleasant floral note [22, 23]. Genome-wide and in vitro assays demonstrate rs6591536 as the causal variant for ß-ionone odor sensitivity. rs6591536 encodes a N183D substitution in the second extracellular loop of OR5A1 and explains >96% of the observed phenotypic variation, resembling a monogenic Mendelian trait. Individuals carrying genotypes for ß-ionone sensitivity can more easily differentiate between food and beverage stimuli with and without added ß-ionone. Sensitive individuals typically describe ß-ionone in foods and beverages as "fragrant" and "floral," whereas less-sensitive individuals describe these stimuli differently. rs6591536 genotype also influences emotional associations and explains differences in food and product choices. These studies demonstrate that an OR variant that influences olfactory sensitivity can affect how people experience and respond to foods, beverages, and other products.
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Preferencias Alimentarias , Variación Genética , Norisoprenoides/metabolismo , Odorantes/análisis , Receptores Odorantes/genética , Olfato , Adulto , Femenino , Alimentos , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Receptores Odorantes/metabolismo , Adulto JovenRESUMEN
Humans vary in their ability to smell numerous odors [1-3], including those associated with food [4-6]. Odor sensitivity is heritable [7-11], with examples linking genetic variation for sensitivity to specific odors typically located near olfactory receptor (OR) genes [12-16]. However, with thousands of aromas and few deorphaned ORs [17, 18], there has been little progress toward linking variation at OR loci to odor sensitivity [19, 20]. We hypothesized that OR genes contain the variation that explains much of the differences in sensitivity for odors, paralleling the genetics of taste [21, 22], which affect the flavor experience of foods [23-25]. We employed a genome-wide association approach for ten food-related odors and identified genetic associations to sensitivity for 2-heptanone (p = 5.1 × 10(-8)), isobutyraldehyde (p = 6.4 × 10(-10)), ß-damascenone (p = 1.6 × 10(-7)), and ß-ionone (p = 1.4 × 10(-31)). Each locus is located in/near distinct clusters of OR genes. These findings increase the number of olfactory sensitivity loci to nine and demonstrate the importance of OR-associated variation in sensory acuity for food-related odors. Analysis of genotype frequencies across human populations implies that variation in sensitivity for these odors is widespread. Furthermore, each participant possessed one of many possible combinations of sensitivities for these odors, supporting the notion that everyone experiences their own unique "flavor world."
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Variación Genética , Odorantes/análisis , Receptores Odorantes/genética , Olfato , Adulto , Femenino , Alimentos , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Persona de Mediana Edad , Receptores Odorantes/metabolismo , Adulto JovenRESUMEN
Creatine kinase (CK) is a marker of muscle damage and pathology present as multiple tissue-specific circulating isoforms. CK is often measured using enzyme activity assays that are unable to distinguish these isoforms. We have developed an immunoassay specific for the MM isoform of CK, found predominantly in skeletal muscle, which uses very small volumes of plasma (1-2 microL). A sandwich enzyme-linked immunosorbent assay (ELISA) for CK-MM was developed using isoform-specific antibodies. Cross-reactivity with CK-BB and MB isoforms was also assessed. The ELISA was validated using plasma samples from a group of athletes, and the measured CK-MM concentrations were correlated with CK enzyme activity assays measured by a contractor using the same samples. The CK-MM ELISA has a limit of detection of 0.02 ng/mL, an IC(50) of 2.3 ng/mL, and 5.8% cross-reactivity with CK-MB. CK-MM concentrations measured using this assay correlate well (p<0.0001, Spearman r=0.89) with enzyme activity assays. The CK-MM-specific ELISA can be used to help assess skeletal muscle damage independent of enzyme activity or interference from other CK isoforms, leading to more precise studies of muscle biology.
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Traumatismos en Atletas/sangre , Forma MM de la Creatina-Quinasa/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Músculo Esquelético/fisiopatología , Adolescente , Adulto , Anticuerpos Monoclonales , Forma MM de la Creatina-Quinasa/inmunología , Femenino , Humanos , Inmunoensayo , Masculino , Deportes , Adulto JovenRESUMEN
Preeclampsia is a common pregnancy complication that is an important cause of preterm birth and fetal growth restriction. Because there is no diagnostic test yet available for preeclampsia, we used a proteomic approach to identify novel serum/plasma biomarkers for this condition. We conducted case control studies comparing nulliparous women who developed preeclampsia at 36-38 weeks of gestation with healthy nulliparous women matched by gestational age at sampling. Serum/plasma was depleted of six abundant proteins and analyzed by two-dimensional gel electrophoresis (n = 12 per group) and difference gel electrophoresis (n = 12 per group). Differences in abundance of protein spots were detected by univariate and multivariate statistical analyses. Proteins were identified by mass spectrometry and expression of selected proteins was validated by immunoblotting. Proteins whose concentrations were selectively associated with preeclampsia included apolipoprotein E (apoE), apoC-II, complement factor C3c, fibrinogen, transthyretin, and complement factor H-related protein 2. An increase in a deglycosylated isoform of apoE3 and concomitantly decreased amounts of one apoE3 glycoisoform were identified in preeclamptic plasma and confirmed by immunoblotting. Altered production of these preeclampsia-related apoE3 isoforms might impair reverse cholesterol transport, contributing to arterial damage. These findings point to a novel mechanistic link between preeclampsia and subsequent cardiovascular disease.
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Apolipoproteína E3/sangre , Apolipoproteína E3/química , Regulación de la Expresión Génica , Preeclampsia/sangre , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/complicaciones , Cromatografía Liquida/métodos , Electroforesis en Gel Bidimensional , Femenino , Glicosilación , Humanos , Inmunoensayo , Metabolismo de los Lípidos , Espectrometría de Masas/métodos , Análisis Multivariante , Embarazo , Isoformas de ProteínasRESUMEN
The homodimeric lambda Cro protein has a "ball-and-socket" interface that includes insertion of an aromatic side chain, Phe 58, from each subunit into a cavity in the hydrophobic core of the other subunit. This overlap between the subunit core and dimer interface hypothetically explains the strong dimerization and weak monomer stability of lambda Cro in comparison to homologues. According to a model developed here and in a previous study [LeFevre, K. R., and Cordes, M. H. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2345-2350], the socket cavity evolved in part by replacement of a buried tryptophan in an ancestral stable monomer with a smaller side chain (Ala 33 in lambda Cro). The resulting core defect was in effect repaired by insertion of a different side chain (Phe 58) from a second subunit, generating the ball and socket. Consistent with such an evolutionary trade between intrasubunit and intersubunit interactions, we showed in the previous study that restoration of the ancestral Trp 33 in lambda Cro stabilized the monomer and reduced the extent of dimerization. Here, we report the solution structure of a stable lambda Cro monomer containing the Ala33Trp mutation, which confirms that the restored tryptophan fulfills its ancestral role as a core side chain, filling part of the socket cavity occupied by Phe 58 in the wild-type dimer. The structure also reveals, however, that the cavity is not completely filled by Trp 33, suggesting that its formation could have involved multiple mutations that reduced side chain volume. We offer suggestive evidence of a role of mutations at a second position.