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This paper introduces super absorbent polymer valves and colorimetric sensing reagents as enabling components of soft, skin-mounted microfluidic devices designed to capture, store, and chemically analyze sweat released from eccrine glands. The valving technology enables robust means for guiding the flow of sweat from an inlet location into a collection of isolated reservoirs, in a well-defined sequence. Analysis in these reservoirs involves a color responsive indicator of chloride concentration with a formulation tailored to offer stable operation with sensitivity optimized for the relevant physiological range. Evaluations on human subjects with comparisons against ex situ analysis illustrate the practical utility of these advances.
Assuntos
Colorimetria/métodos , Microfluídica/métodos , Polímeros/química , Suor/química , Humanos , Dispositivos Lab-On-A-Chip , Pele/metabolismoRESUMO
BACKGROUND: Chromatin is an extraordinarily complex structure. Much of this complexity results from the presence of numerous histone post-translational modifications and histone variants. Alterations in the patterns of histone post-translational modifications are emerging as a feature of many types of cancer and have been shown to have prognostic value. RESULTS: We have applied a liquid chromatography/mass spectrometry-based approach to comprehensively characterize the histone proteome in primary samples from chronic lymphocytic leukemia (CLL) patients, as well as bladder and breast cancer cell culture models. When compared to non-malignant CD19+ B cells from healthy donors, the CLL histone proteome showed a distinct signature of differentially expressed species, spanning all the histones studied and including both post-translationally modified species and unmodified, non-allelic replication-dependent histone isoforms. However, the large changes in histone H3 and H4 that are characteristic of many cancer types were not observed. One of species of H2A (mass = 14,063 Da) was the most strongly associated with time to treatment in CLL patients. CLL patient samples also demonstrated histone profiles that were distinct from those of the bladder and breast cancer cells. CONCLUSIONS: Signatures of histone profiles are complex and can distinguish between healthy individuals and CLL patients and may provide prognostic markers. In addition, histone profiles may define tissue specific malignancies.
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H1 and related linker histones are important both for maintenance of higher-order chromatin structure and for the regulation of gene expression. The biology of the linker histones is complex, as they are evolutionarily variable, exist in multiple isoforms and undergo a large variety of posttranslational modifications in their long, unstructured, NH2- and COOH-terminal tails. We review recent progress in understanding the structure, genetics and posttranslational modifications of linker histones, with an emphasis on the dynamic interactions of these proteins with DNA and transcriptional regulators. We also discuss various experimental challenges to the study of H1 and related proteins, including limitations of immunological reagents and practical difficulties in the analysis of posttranslational modifications by mass spectrometry.
Assuntos
Histonas/metabolismo , Sequência de Aminoácidos , Histonas/química , Histonas/genética , Dados de Sequência Molecular , Família Multigênica , Nucleossomos/química , Processamento de Proteína Pós-Traducional , Alinhamento de SequênciaRESUMO
Breast cancer is the second leading cause of cancer-related deaths in women. The need for new clinical biomarkers in breast cancer is necessary to further predict prognosis and therapeutic response. In this article, the LC-MS histone H1 phosphorylation profiles were established for three distinct breast cancer cell lines. The results show that the extent of H1 phosphorylation can distinguish between the different cell lines. The histone H1 from the metastatic cell line, MDA-MB-231, was subjected to chemical derivitization and LC-MS/MS analysis. The results suggest that the phosphorylation at threonine 146 is found on both histone H1.2 and histone H1.4. Cell lines were then treated with an extracellular stimulus, estradiol or kinase inhibitor LY294002, to monitor changes in histone H1 phosphorylation. The data show that histone H1 phosphorylation can increase and decrease in response to extracellular stimuli. Finally, primary breast tissues were stained for the histone H1 phosphorylation at threonine 146. Variable staining patterns across tumor grades and subtypes were observed with pT146 labeling correlating with tumor grade. These results establish the potential for histone H1 phosphorylation at threonine 146 as a clinical biomarker in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Histonas/metabolismo , Fosfoproteínas/metabolismo , Sequência de Aminoácidos , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Feminino , Humanos , Imuno-Histoquímica , Células MCF-7 , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , Proteômica/métodos , Treonina/metabolismoRESUMO
Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell-to-cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label-free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label-free spectral counting applied to determine relative protein abundances in EVs.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Mieloma Múltiplo/metabolismo , Coloração e Rotulagem/métodos , Extratos Celulares , Linhagem Celular Tumoral , Cromatografia Líquida , Vesículas Citoplasmáticas/ultraestrutura , Humanos , Espectrometria de Massas , Mieloma Múltiplo/ultraestrutura , Proteínas de Neoplasias/metabolismo , Proteômica , Reprodutibilidade dos TestesRESUMO
Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers harboring oncogenic mutant K-Ras. Here, we tested a novel ferrocene derivative on the growth of pancreatic ductal adenocarcinoma and non-small cell lung cancer. Our compound, which elevated cellular ROS levels, inhibited the growth of K-Ras-driven cancers, and abrogated the PM binding and signaling of K-Ras in an isoform-specific manner. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified that K-Ras His95 residue plays an important role in this process, and it is putatively oxidized by cellular ROS. Together, our study demonstrates that the redox system directly regulates K-Ras/PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced growth and metastasis of K-Ras-driven cancers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Neoplasias Pancreáticas , Humanos , Animais , Antioxidantes , Metalocenos/farmacologia , Espécies Reativas de Oxigênio , Oxirredução , Estresse Oxidativo , MamíferosRESUMO
Due to the overall low abundance of volatile compounds in exhaled breath, it is necessary to preconcentrate the sample prior to traditional thermal desorption (TD) gas chromatography mass spectrometry analysis. While certain aspects of TD tubes, such as volatile storage, have been evaluated, many aspects remain uncharacterized. Two common TD tubes, Tenax TA and Biomonitoring 5TD tubes, were evaluated for background content and flow rate variability. The data illustrate that the Biomonitoring 5TD tubes have the highest number (23) and abundance of background contamination greater than 3x the mean noise when compared to Tenax TA (13) and empty tubes (9). Tentative identifications of the compounds in the background contamination experiment show that greater than 59% (16/27) of the compounds identified have been reported in the breath literature. The data illustrate the TD tube background abundance could account for more than 70% of the chromatographic signal from exhaled breath for these select compounds. Flow rate measurements of 200 Tenax TA and 200 Biomonitoring 5TD tubes show a large range in measured flow rates among the TD tubes (Tenax: 252.9-284.0 ml min-1, 5TD: 220.6-255.1 ml min-1). Finally, TD tubes of each type, Tenax TA and Biomonitoring 5TD, previously established to have high, medium, and low flow rates, show insignificant differences (p> 0.05) among the tubes of different flow rates, using both gas standards and an exhaled breath from a peppermint experiment. Collectively, these results establish overall background compounds attributed to each TD tube type tested. Additionally, while measured flow rate variability is present and plausibly impacts exhaled breath results, the data demonstrate no statistically significant difference was observed between tubes showing high, medium, and low flow rates from two separate sample types.
Assuntos
Compostos Orgânicos Voláteis , Humanos , Compostos Orgânicos Voláteis/análise , Testes Respiratórios/métodos , Expiração , Cromatografia Gasosa-Espectrometria de Massas/métodos , Padrões de ReferênciaRESUMO
Exhaled breath research has been hindered by a lack of standardization in collection and analysis methodologies. Recently, the Respiration Collector forIn VitroAnalysis (ReCIVA) sampling device has illustrated the potential to provide a consistent and convenient method for exhaled breath collection onto adsorbent media. However, the significant costs, compared to exhaled breath bags, associated with the standardized collector is believed to be the reason for limited widespread use by researchers in the exhaled breath field. For example, in addition to the sampling hardware, a single-use disposable silicon mask affixed with a filter is required for each exhaled breath collection. To reduce the financial burden, streamline device upkeep, reduce waste material, and ease the logistical burden associated with the single use masks, it is hypothesized that the consumable masks and filters could be sterilized by autoclaving for reuse. The masks were contaminated, autoclaved, and then tested for any surviving pathogens with spore strip standards and by measuring the optical density of cultures. The compound background collected when using the ReCIVA with new masks was compared to that collected with repeatedly autoclaved masks via thermal desorption gas chromatography mass spectrometry (TD-GC-MS). The capacity to block particulate matter of new filters was tested against that of autoclaved filters by introducing an aerosol and comparing pre-filter and post-filter particle counts. Finally, breath samplings were conducted with new masks and autoclaved masks to test for changes in measurements by TD-GC-MS of exogenous and endogenous compounds. The data illustrate the autoclave cycle sterilizes masks spiked with saliva to background levels (p= 0.2527). The results indicate that background levels of siloxane compounds are increased as masks are repetitively autoclaved. The data show that mask filters have significant breakthrough of 1µm particles after five repetitive autoclaving cycles compared to new filters (p= 0.0219). Finally, exhaled breath results utilizing a peppermint ingestion protocol indicate two compounds associated with peppermint, menthone and 1-Methyl-4-(1-methylethyl)-cyclohexanol, and an endogenous exhaled breath compound, isoprene, show no significant difference if sampled with a new mask or a mask autoclaved five times (p> 0.1063). Collectively, the data indicate that ReCIVA masks and filters can be sterilized via autoclave and reused. The results suggest ReCIVA mask and filter reuse should be limited to three times to limit potentially problematic background contaminants and filter dysfunction.
Assuntos
Testes Respiratórios , Esterilização , Humanos , Testes Respiratórios/métodos , Cromatografia Gasosa-Espectrometria de Massas , Expiração , Padrões de ReferênciaRESUMO
Inhaled medications are commonplace for administering bronchodilators, anticholinergics, and corticosteroids. While they have a defined legitimate use, they are also used in sporting events as performance-enhancing drugs. These performance enhancers can be acquired via both legal (i.e., at a pharmacy through over-the-counter medications or through a prescription) and illicit (i.e., black market and foreign pharmacies) means, thus making monitoring procurement impossible. While urine tests can detect these pharmacological agents hours after they have been inhaled, there is a significant lag time before they are observed in urine. Direct detection of these inhaled agents is complicated and requires a multiplexed approach due to the sheer number of inhaled pharmacological agents. Therefore, detection of propellants, which carry the drug into the lungs, provides a simpler path forward toward detection of broad pharmacological agents. In this paper, we demonstrate the first use of terahertz spectroscopy (THz) to detect inhaled medications in human subjects. Notably, we were able to detect and quantitate the propellant, HFA-134a, in breath up to 30 min after using an asthma inhaler, enabling the use of a point-of-care device to monitor exhaled breath for the presence of propellants. We also demonstrate via simulations that the same approach can be leveraged to detect and identify next-generation propellants, specifically HFA-152a. As a result, we provide evidence that a single point-of-care THz sensor can detect when individuals have used pressure-mediated dose inhalers (pMDIs) without further modification of the hardware.
Assuntos
Asma , Espectroscopia Terahertz , Humanos , Propelentes de Aerossol/uso terapêutico , Asma/tratamento farmacológico , Nebulizadores e Vaporizadores , Broncodilatadores/química , Broncodilatadores/uso terapêuticoRESUMO
Isoprene is one of the most abundant and most frequently evaluated volatile organic compounds in exhaled breath. Recently, several individuals with background levels of exhaled isoprene have been identified. Here, case study data are provided for an individual, identified from a previous study, with this low prevalence phenotype. It is hypothesized that the individual will illustrate low levels of exhaled isoprene at rest and during exercise. At rest, the subject (7.1 ppb) shows background (µ= 14.2 ± 7.0 ppb) levels of exhaled isoprene while the control group illustrates significantly higher quantities (µ= 266.2 ± 72.3 ppb) via proton transfer reaction mass spectrometry (PTR-MS). The result, background levels of isoprene at rest, is verified by thermal desorption gas chromatography mass spectrometry (TD-GC-MS) collections with the individual showing -3.6 ppb exhaled isoprene while the room background containedµ= -4.1 ± 0.1 ppb isoprene. As isoprene has been shown previously to increase at the initiation of exercise, exercise bike experiments were performed with the individual identified with low isoprene, yielding low and invariant levels of exhaled isoprene (µ= 6.6 ± 0.1 ppb) during the exercise while control subjects illustrated an approximate 2.5-fold increase (preµ= 286.3 ± 43.8 ppb, exerciseµ= 573.0 ± 147.8 ppb) in exhaled isoprene upon exercise start. Additionally, exhaled breath bag data showed a significant decrease in isoprene (delta post/pre, p = 0.0078) of the control group following the exercise regimen. Finally, TD-GC-MS results for exhaled isoprene from the individual's family (mother, father, sister and maternal grandmother) illustrated that the mother and father exhibited isoprene values (28.5 ppb, 77.2 ppb) below control samples 95% confidence interval (µ= 166.8 ± 43.3 ppb) while the individual's sister (182.0 ppb) was within the control range. These data provide evidence for a large dynamic range in exhaled isoprene in this family. Collectively, these results provide additional data surrounding the existence of a small population of individuals with background levels of exhaled isoprene.
Assuntos
Testes Respiratórios , Butadienos , Testes Respiratórios/métodos , Espectrometria de Massas/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Butadienos/análise , Hemiterpenos/análise , ExpiraçãoRESUMO
Sweat is emerging as a prominent biosource for real-time human performance monitoring applications. Although promising, sources of variability must be identified to truly utilize sweat for biomarker applications. In this proof-of-concept study, a targeted metabolomics method was applied to sweat collected from the forearms of participants in a 12-week exercise program who ingested either low or high nutritional supplementation twice daily. The data establish the use of dried powder mass as a method for metabolomic data normalization from sweat samples. Additionally, the results support the hypothesis that ingestion of regular nutritional supplementation semi-quantitatively impact the sweat metabolome. For example, a receiver operating characteristic (ROC) curve of relative normalized metabolite quantities show an area under the curve of 0.82 suggesting the sweat metabolome can moderately predict if an individual is taking nutritional supplementation. Finally, a significant correlation between physical performance and the sweat metabolome are established. For instance, the data illustrate that by utilizing multiple linear regression modeling approaches, sweat metabolite quantities can predict VO2 max (p = 0.0346), peak lower body Windage (p = 0.0112), and abdominal circumference (p = 0.0425). The results illustrate the need to account for dietary nutrition in biomarker discovery applications involving sweat as a biosource.
RESUMO
As the demand for real-time exercise performance feedback increases, excreted sweat has become a biosource of interest for continuous human performance assessment. For sweat to truly fulfill this requirement, analyte concentrations must be normalized to adequately assess day-to-day differences within and among individuals. In this manuscript, data are presented highlighting the use of accurate localized sweat rate as a means for ion and global metabolomic data normalization. The results illustrate large sweat rate variability among individuals over the course of two distinct exercises protocols. Furthermore, the data show sweat rate is not symmetrical at similar locations among right and left forearms of individuals (p = 0.0007). Sweat ion conductivity analysis suggest overall sweat rate normalization reduces variability collectively among ion values and participants with principal component analysis showing 77.8% of variation in the data set attributable to sweat rate normalization. Global metabolomic analysis of sweat illustrated overall rate normalization increases the variability among test subjects with 72.7% of the variation explained by sweat rate normalization. Finally, overall rate normalized metabolomic features of sweat significantly correlated (ρ ≥ 0.7, ρ ≤ -0.7) with measured performance metrics of the individual, establishing the potential for sweat to be used as a biosource for performance monitoring. Collectively, these data illustrate the importance of accurate localized sweat rate determination, for analyte data normalization, in support for the use of sweat in biomarker discovery efforts to predict human performance.
Assuntos
Metabolômica , Suor , Biomarcadores , Exercício Físico , Humanos , Análise de Componente PrincipalRESUMO
Histone H1 is commonly used to assay kinase activity in vitro. As many promising targeted therapies affect kinase activity of specific enzymes involved in cancer transformation, H1 phosphorylation can serve as potential pharmacodynamic marker for drug activity within the cell. In this study we utilized a phosphoproteomic workflow to characterize histone H1 phosphorylation changes associated with two targeted therapies in the Kasumi-1 acute myeloid leukemia cell line. The phosphoproteomic workflow was first validated with standard casein phosphoproteins and then applied to the direct analysis of histone H1 from Kasumi-1 nuclear lysates. Ten H1 phosphorylation sites were identified on the H1 variants, H1.2, H1.3, H1.4, H1.5 and H1.x. LC MS profiling of intact H1s demonstrated global dephosphorylation of H1.5 associated with therapy by the cyclin-dependent kinase inhibitor, flavopiridol and the Heat Shock Protein 90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin. In contrast, independent treatments with a nucleotide analog, proteosome inhibitor and histone deacetylase inhibitor did not exhibit decreased H1.5 phosphorylation. The data presented herein demonstrate that potential of histones to assess the cellular response of reagents that have direct and indirect effects on kinase activity that alters histone phosphorylation. As such, this approach may be a highly informative marker for response to targeted therapies influencing histone phosphorylation.
Assuntos
Benzoquinonas/farmacologia , Biomarcadores Farmacológicos/análise , Flavonoides/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Histonas/metabolismo , Lactamas Macrocíclicas/farmacologia , Leucemia Mieloide Aguda/metabolismo , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sequência de Aminoácidos , Caseínas/química , Histonas/química , Humanos , Dados de Sequência Molecular , Terapia de Alvo Molecular , Fosfopeptídeos/química , Células Tumorais CultivadasRESUMO
The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Sistemas CRISPR-Cas , Proteínas do Citoesqueleto/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ligação Proteica , RNA Mensageiro/metabolismo , NucleolinaRESUMO
The Respiration Collector for In Vitro Analysis (ReCIVA) sampler, marketed by Owlstone Medical, provides a step forward in exhaled breath sampling through active sampling directly onto thermal desorption (TD) tubes. Although an improvement to the issues surrounding breath bag sampling, the ReCIVA device, first released in 2015, is a relatively new research and clinical tool that requires further exploration. Here, data are presented comparing two distinct ReCIVA devices. The results, comparing ReCIVA serial numbers #33 and #65, demonstrate that overall statistically insignificant results are obtained via targeted isoprene quantitation (p > 0.05). However, when the data are parsed by the TD tube type used to capture breath volatiles, either Tenax TA or the dual bed Tenax/Carbograph 5TD (5TD), a statistical difference (p < 0.05) among the two different TD tubes was present. These data, comparing the two ReCIVA devices with both Tenax TA and 5TD tubes, are further supported by a global metabolomics analysis yielding 85% of z-scores, comparing ReCIVA devices, below the limit for significance. Experiments to determine the effect of breathing rate on ReCIVA function, using guided breathing for low (7.5 breaths min-1) and high (15 breaths min-1) breathing rates, demonstrate the ReCIVA device shows no statistical difference among breathing rates for quantitated isoprene (p > 0.05). Global metabolomics analysis of the guided breathing rate data shows more than 87% of the z-scores, comparing high and low breathing rates using both the Tenax and the 5TD tubes, are below the level for significance. Finally, data are provided from a single participant who displayed background levels of isoprene while illustrating levels of acetone consistent with the remaining participants. Collectively, these data support the use of multiple ReCIVA devices for exhaled breath collection and provide evidence for an instance where exhaled isoprene is consistent with background levels.
Assuntos
Testes Respiratórios/instrumentação , Manejo de Espécimes/instrumentação , Temperatura , Butadienos/análise , Expiração , Hemiterpenos/análise , Humanos , Masculino , Padrões de Referência , Compostos Orgânicos Voláteis/análiseRESUMO
Due to increased interest in the use of excreted sweat for biomarker discovery, data must be generated supporting sample collection and handling methods to allow for controlled, large-scale biomarker discovery studies to be performed. In this manuscript, twelve amino acids were quantitated from exercise-induced excreted sweat held at room temperature or a simulated body temperature of 37⯰C for up to 90â¯min. The data illustrate a large dynamic range exists among amino acids in sweat. Additionally, the amino acid quantities vary across individuals and among the same individual under different storage conditions, with alanine, arginine, and threonine showing a significant statistical difference between sampling events (pâ¯<â¯0.05). Furthermore, the results establish amino acids are relatively invariant, at both storage temperatures tested, for up to 90â¯min illustrated by <10% (15/156) of the amino acids measurements demonstrating change greater than 10% from the time zero value. An untargeted metabolomics approach was also applied to the data set to evaluate global changes to the metabolome. The results show more than 88% of all data points fall within the established limits, regardless of temperature condition and ionization mode. Collectively, this study demonstrates that sweat is largely invariant at two distinct temperatures for up to 90â¯min. These results establish sweat collection and sample handling is possible for up to 90â¯min with minimal changes in metabolite abundances.
Assuntos
Metaboloma/fisiologia , Metabolômica/métodos , Suor/metabolismo , Aminoácidos/análise , Aminoácidos/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Cromatografia Líquida/métodos , Exercício Físico/fisiologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , MasculinoRESUMO
Due to several sources of potential variability associated with exhaled breath bag sampling procedures for off-line analysis, the Respiration Collector for in vitro Analysis (ReCIVA) sampler was developed. Although designed to improve upon several pitfalls of sampling with exhaled breath bags, the ReCIVA remains a minimally studied research tool. In this manuscript, several attributes of the ReCIVA sampler are investigated among three individual tests, such as background contamination, control software version, performance of different adsorbent tubes, duplicate sample production, and comparison to exhaled breath bags. The data shows greater than a 58% reduction in background siloxanes can be achieved with submersion of ReCIVA masks in ethyl alcohol or baking the masks at a high temperature (200 °C). The results illustrate the ReCIVA control software version plays a key role in the flow rates applied to thermal desorption (TD) tubes. Using exhaled isoprene as a representative analyte, the data suggest duplicate samples among ReCIVA pump banks can be achieved using two different thermal desorption tubes, Tenax TA and Tenax/Carbograph 5TD, when using an updated control software and manually calibrating the ReCIVA pumps to uniform flow rates (Tenax p = 0.3869, 5TD p = 0.3131). Additionally, using the updated control software and manual ReCIVA flow calibration, the data suggest the ReCIVA can produce statistically similar results among TD tube types (p = 0.3824) and compared to standard exhaled breath bags (p = 0.1534). Collectively, these results establish a method for manually calibrating the flow of the ReCIVA device to allow for the most consistent results. These data support further experimentation into the use of the ReCIVA sampler for exhaled breath research.
Assuntos
Testes Respiratórios/métodos , Butadienos/análise , Calibragem , Expiração , Hemiterpenos/análise , Humanos , Masculino , Padrões de Referência , Siloxanas/química , Manejo de EspécimesRESUMO
Sweat is a biofluid with several attractive attributes. However, investigation into sweat for biomarker discovery applications is still in its infancy. To add support for the use of sweat as a non-invasive media for human performance monitoring, volunteer participants were subjected to a physical exertion model using a treadmill. Following exercise, sweat was collected, aliquotted, and analyzed for metabolite and protein content via high-resolution mass spectrometry. Overall, the proteomic analysis illustrates significant enrichment steps will be required for proteomic biomarker discovery from single sweat samples as protein abundance is low in this medium. Furthermore, the results indicate a potential for protein degradation, or a large number of low molecular weight protein/peptides, in these samples. Metabolomic analysis shows a strong correlation in the overall abundance among sweat metabolites. Finally, hierarchical clustering of participant metabolite abundances show trends emerging, although no significant trends were observed (alpha = 0.8, lambda = 1 standard error via cross validation). However, these data suggest with a greater number of biological replicates, stronger, statistically significant results, can be obtained. Collectively, this study represents the first to simultaneously use both proteomic and metabolomic analysis to investigate sweat. These data highlight several pitfalls of sweat analysis for biomarker discovery applications.
Assuntos
Exercício Físico , Metabolômica , Proteômica , Suor/metabolismo , Adolescente , Adulto , Humanos , Metaboloma , Metabolômica/métodos , Pessoa de Meia-Idade , Militares , Resistência Física , Projetos Piloto , Proteoma/análise , Proteoma/metabolismo , Proteômica/métodos , Suor/química , Adulto JovemRESUMO
Hypoxia-like incidents in-flight have increased over the past decade causing severe safety concerns across the aviation community. As a result, the need to monitor flight crews in real-time for the onset of hypoxic conditions is paramount for continued aeronautical safety. Here, hypoxic events were simulated in the laboratory via a reduced oxygen-breathing device to determine the effect of recovery gas oxygen concentration (21% and 100%) on exhaled breath volatile organic compound composition. Data from samples collected both serially (throughout the exposure), prior to, and following exposures yielded 326 statistically significant features, 203 of which were unique. Of those, 72 features were tentatively identified while 51 were verified with authentic standards. A comparison of samples collected serially between recovery and hypoxia time points shows a statistically significant reduction in exhaled breath isoprene (2-methyl-1,3-butadiene, log2 FC -0.399, p = 0.005, FDR = 0.034, q = 0.033), however no significant difference in isoprene abundance was observed when comparing recovery gases (21% or 100% O2, p = 0.152). Furthermore, examination of pre-/post-exposure 1 l bag breath samples illustrate an overall increase in exhaled isoprene abundance post-exposure (log2 FC 0.393, p = 0.005, FDR = 0.094, q = 0.033) but again no significant difference between recovery gas (21% and 100%, p = 0.798) was observed. A statistically significant difference in trend was observed between isoprene abundance and recovery gases O2 concentration when plotted against minimum oxygen saturation (p = 0.0419 100% O2, p = 0.7034 21% O2). Collectively, these results suggest exhaled isoprene is dynamic in the laboratory ROBD setup and additional experimentation will be required to fully understand the dynamics of isoprene in response to acute hypoxic stress.
Assuntos
Testes Respiratórios/métodos , Butadienos/análise , Expiração , Hemiterpenos/análise , Hipóxia/diagnóstico , Pentanos/análise , Estresse Fisiológico , Adulto , Humanos , Hipóxia/sangue , Masculino , Oxigênio/sangue , Padrões de Referência , Fatores de Tempo , Adulto JovemRESUMO
Multiple myeloma (MM) is a hematologic malignancy of plasma cells (PCs). In the United States, MM accounts for approximately 1% of all diagnoses and 2% of all cancer-related deaths. Although MM is a treatable disease, most patients eventually relapse, and despite the development of numerous treatment options it is still considered incurable. Mechanisms of communication between MM-PCs and bone marrow microenvironment, including cell-cell contacts and release of pro-survival factors, promote cancer cell survival and drug resistance. Recently, the importance of extracellular vesicles (EVs) as mechanisms of communication between MM cells and other cells in the microenvironment has been reported. In this review, the authors provide the update on the biology and clinical aspects of EVs in MM.