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Articles by Nichole Reisdorph in JoVE
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में गहराई और जानकारीपूर्ण metabolomic विश्लेषण के लिए एकाधिक metabolite यौगिक श्रेणी ठीक करने के लिए कई कदम तैयारी तकनीक
Charmion Cruickshank-Quinn1, Kevin D. Quinn1, Roger Powell1, Yanhui Yang1, Michael Armstrong1, Spencer Mahaffey2, Richard Reisdorph1, Nichole Reisdorph1
1Department of Immunology, National Jewish Health, 2Department of Pharmacology, School of Medicine, University of Colorado Denver
metabolomics प्रयोगों में परिणामों की विश्वसनीयता नमूना तैयार करने की प्रभावशीलता और reproducibility पर निर्भर करता है. बाद में यौगिकों के हजारों को विश्लेषण करने, या ब्याज की बस यौगिक वर्गों के विकल्प के साथ जैविक तरल पदार्थ से मेटाबोलाइट्स की निकासी के लिए सक्षम बनाता है कि एक कठोर और गहन विधि है वर्णित है.
Other articles by Nichole Reisdorph on PubMed
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Changing Desiccation Tolerance of Pea Embryo Protoplasts During Germination
Journal of Experimental Botany.
Jun, 2003 |
Pubmed ID: 12730264 Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.
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Metabolic Markers of Hypoxia: Systems Biology Application in Biomedicine
Toxicology Mechanisms and Methods.
2008 |
Pubmed ID: 20020894 ABSTRACT The overall goal of this review is to provide insight into methodologies for 'omic investigations and hypoxic biomarkers that have been identified using 'omic techniques. First, a detailed description of current metabolomic, proteomic, and genomic technologies is provided, followed by a basic introduction to biostatistics and how to interpret 'omic data. Metabolomic biomarkers of diseases in which hypoxia plays a role are then reviewed by those that involved chronic (pulmonary disease, cardiovascular disease, cancer) and acute (stroke, myocardial infarction, ischemia) hypoxia. Data are presented with consideration for the source of hypoxia, the severity of hypoxia, the length of hypoxia, and the cell or organ affected, all of which can have significant effects on biomarker profiles. Drugs that promote and antagonize hypoxia are discussed and important points to consider during tissue collection in hypoxia 'omic studies are then reviewed.
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Proteomics and Metabolomics and Their Application to Analgesia Research
Methods in Molecular Biology (Clifton, N.J.).
2010 |
Pubmed ID: 20336441 Technological innovations have increased our potential to evaluate global changes in protein and small molecule levels in a rapid and comprehensive manner. This is especially true in mass spectrometry-based research where improvements, including ease-of-use, in high performance liquid chromatography (HPLC), column chemistries, instruments, software, and molecular databases have advanced the fields of proteomics and metabolomics considerably. Applications of these technologies in clinical research include biomarker discovery, drug targeting, and elucidating molecular networks, and a systems-based approach, utilizing multiple "omics," can also be taken. While the exact choice of workflow can dramatically impact the results of a study, the basic steps are similar, both within and between metabolomics and proteomics experiments. Although gel-based methods of quantitation are still widely used, our laboratory focuses on mass spectrometry-based methods, specifically protein and small molecule profiling.
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A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain
Molecular & Cellular Proteomics : MCP.
Sep, 2010 |
Pubmed ID: 20445002 The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.
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Aluminum Adjuvants Elicit Fibrin-dependent Extracellular Traps in Vivo
Blood.
Dec, 2010 |
Pubmed ID: 20876456 It has been recognized for nearly 80 years that insoluble aluminum salts are good immunologic adjuvants and that they form long-lived nodules in vivo. Nodule formation has long been presumed to be central for adjuvant activity by providing an antigen depot, but the composition and function of these nodules is poorly understood. We show here that aluminum salt nodules formed within hours of injection and contained the clotting protein fibrinogen. Fibrinogen was critical for nodule formation and required processing to insoluble fibrin by thrombin. DNase treatment partially disrupted the nodules, and the nodules contained histone H3 and citrullinated H3, features consistent with extracellular traps. Although neutrophils were not essential for nodule formation, CD11b(+) cells were implicated. Vaccination of fibrinogen-deficient mice resulted in normal CD4 T-cell and antibody responses and enhanced CD8 T-cell responses, indicating that nodules are not required for aluminum's adjuvant effect. Moreover, the ability of aluminum salts to retain antigen in the body, the well-known depot effect, was unaffected by the absence of nodules. We conclude that aluminum adjuvants form fibrin-dependent nodules in vivo, that these nodules have properties of extracellular traps, and the nodules are not required for aluminum salts to act as adjuvants.
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Changes in Ovarian Protein Expression During Primordial Follicle Formation in the Hamster
Molecular and Cellular Endocrinology.
Jan, 2012 |
Pubmed ID: 21821096 Although many proteins have been shown to affect the transition of primordial follicles to the primary stage, factors regulating the formation of primordial follicles remains sketchy at best. Differentiation of somatic cells into early granulosa cells during ovarian morphogenesis is the hallmark of primordial follicle formation; hence, critical changes are expected in protein expression. We wanted to identify proteins, the expression of which would correlate with the formation of primordial follicles as a first step to determine their biological function in folliculogenesis. Proteins were extracted from embryonic (E15) and 8-day-old (P8) hamster ovaries and fractionated by two-dimensional gel electrophoresis. Gels were stained with Proteosilver, and images of protein profiles corresponding to E15 and P8 ovaries were overlayed to identify protein spots showing altered expression. Some of the protein spots were extracted from SyproRuby-stained preparative gels, digested with trypsin, and analyzed by mass spectrometry. Both E15 and P8 ovaries had high molecular weight proteins at acidic, basic, and neutral ranges; however, we focused on small molecular weight proteins at 4-7 pH range. Many of those spots might represent post-translational modification. Mass spectrometric analysis revealed the identity of these proteins. The formation of primordial follicles on P8 correlated with many differentially and newly expressed proteins. Whereas Ebp1 expression was downregulated in ovarian somatic cells, Sfrs3 expression was specifically upregulated in newly formed granulosa cells of primordial follicles on P8. The results show for the first time that the morphogenesis of primordial follicles in the hamster coincides with altered and novel expression of proteins involved in cell proliferation, transcriptional regulation, and metabolism. Therefore, formation of primordial follicles is an active process requiring differentiation of somatic cells into early granulosa cells and their interaction with the oocytes.
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Peripheral Blood Mononuclear Cell Gene Expression in Chronic Obstructive Pulmonary Disease
American Journal of Respiratory Cell and Molecular Biology.
Aug, 2013 |
Pubmed ID: 23590301 Although most cases of chronic obstructive pulmonary disease (COPD) occur in smokers, only a fraction of smokers develop the disease. We hypothesized distinct molecular signatures for COPD and emphysema in the peripheral blood mononuclear cells (PBMCs) of current and former smokers. To test this hypothesis, we identified and validated PBMC gene expression profiles in smokers with and without COPD. We generated expression data on 136 subjects from the COPDGene study, using Affymetrix U133 2.0 microarrays (Affymetrix, Santa Clara, CA). Multiple linear regression with adjustment for covariates (gender, age, body mass index, family history, smoking status, and pack-years) was used to identify candidate genes, and ingenuity pathway analysis was used to identify candidate pathways. Candidate genes were validated in 149 subjects according to multiplex quantitative real-time polymerase chain reaction, which included 75 subjects not previously profiled. Pathways that were differentially expressed in subjects with COPD and emphysema included those that play a role in the immune system, inflammatory responses, and sphingolipid (ceramide) metabolism. Twenty-six of the 46 candidate genes (e.g., FOXP1, TCF7, and ASAH1) were validated in the independent cohort. Plasma metabolomics was used to identify a novel glycoceramide (galabiosylceramide) as a biomarker of emphysema, supporting the genomic association between acid ceramidase (ASAH1) and emphysema. COPD is a systemic disease whose gene expression signatures in PBMCs could serve as novel diagnostic or therapeutic targets.
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Steroidogenic Enzyme Cyp11a1 Regulates Type 2 CD8+ T Cell Skewing in Allergic Lung Disease
Proceedings of the National Academy of Sciences of the United States of America.
May, 2013 |
Pubmed ID: 23630275 Allergic asthma is a heterogeneous inflammatory disorder of the airways characterized by chronic airway inflammation and airway hyperresponsiveness. Numbers of CD8(+)IL-13(+) T cells are increased in asthmatics and during the development of experimental asthma in mice. In an atopic environment rich in IL-4, these CD8(+) T cells mediate asthmatic responses, but the mechanisms regulating the conversion of CD8(+) effector T cells from IFN-γ- to pathogenic IL-13-producing effector cells that contribute to an asthma phenotype have not been defined. Here, we show that cholesterol side-chain cleavage P450 enzyme, Cyp11a1, is a key regulator of CD8(+) T-cell conversion. Expression of the gene, protein, and enzymatic activity of Cyp11a1 were markedly increased in CD8(+) T cells differentiated in the presence of IL-2 plus IL-4 compared with cells differentiated in IL-2 alone. Inhibition of Cyp11a1 enzymatic activity with aminoglutethimide or reduction in the expression of Cyp11a1 using short hairpin RNA prevented the IL-4-induced conversion of IFN-γ- to IL-13-producing cells without affecting expression of the lineage-specific transcription factors T-box expressed in T cells (T-bet) or GATA binding protein 3 (GATA3). Adoptive transfer of aminoglutethimide-treated CD8(+) T cells into sensitized and challenged CD8-deficient recipients failed to restore airway hyperresponsiveness and inflammation. We demonstrate that Cyp11a1 controls the phenotypic conversion of CD8(+) T cells from IFN-γ to IL-13 production, linking steroidogenesis in CD8(+) T cells, a nonclassical steroidogenic tissue, to a proallergic differentiation pathway.
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New Sample Preparation Approach for Mass Spectrometry-based Profiling of Plasma Results in Improved Coverage of Metabolome
Journal of Chromatography. A.
Jul, 2013 |
Pubmed ID: 23672979 Sample preparation remains a challenge in untargeted metabolomics studies and no method currently results in complete extraction of all metabolite classes in human plasma. Because a large variety of molecules, with vast differences in dynamic range, could be involved in human disease, there is an urgent need to develop analytical techniques that result in comprehensive coverage of metabolites. Furthermore, analysis of more focused molecular classes could be necessary to more fully interrogate markers of human disease. However, such techniques, which generally involve multiple steps, often result in high variability. We have optimized a combined liquid-liquid and solid phase extraction method for plasma and have compared that to traditional methanol precipitation using spiked internal standards as controls. This method, based largely on previously published methods, results in 5 separate fractions enriched for aqueous species, phospholipids, fatty acids, neutral lipids, and hydrophobic lipids. Using liquid chromatography mass spectrometry as the analytical method, we detect over 3806 metabolites using the new method versus 1851 metabolites using methanol alone. Qualitative analysis and quantitative analysis of both internal standards (ISTDs) and endogenous metabolites demonstrate excellent reproducibility with CV's below 15% for the combined method compared to 30% using the methanol method. While both methods have applications for clinical metabolomics, fractionation resulted in greater overall coverage and can be used for initial classification of molecular species.
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Hands-on Workshops As an Effective Means of Learning Advanced Technologies Including Genomics, Proteomics and Bioinformatics
Genomics, Proteomics & Bioinformatics.
Dec, 2013 |
Pubmed ID: 24316330 Genomics and proteomics have emerged as key technologies in biomedical research, resulting in a surge of interest in training by investigators keen to incorporate these technologies into their research. At least two types of training can be envisioned in order to produce meaningful results, quality publications and successful grant applications: (1) immediate short-term training workshops and (2) long-term graduate education or visiting scientist programs. We aimed to fill the former need by providing a comprehensive hands-on training course in genomics, proteomics and informatics in a coherent, experimentally-based framework. This was accomplished through a National Heart, Lung, and Blood Institute (NHLBI)-sponsored 10-day Genomics and Proteomics Hands-on Workshop held at National Jewish Health (NJH) and the University of Colorado School of Medicine (UCD). The course content included comprehensive lectures and laboratories in mass spectrometry and genomics technologies, extensive hands-on experience with instrumentation and software, video demonstrations, optional workshops, online sessions, invited keynote speakers, and local and national guest faculty. Here we describe the detailed curriculum and present the results of short- and long-term evaluations from course attendees. Our educational program consistently received positive reviews from participants and had a substantial impact on grant writing and review, manuscript submissions and publications.
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