Other articles by Nichole Reisdorph on PubMed

• 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.
• Human Rhinovirus Capsid Dynamics is Controlled by Canyon Flexibility Virology. Sep, 2003  |   Pubmed ID: 14517058 Quantitative enzyme accessibility experiments using nano liquid chromatography electrospray mass spectrometry combined with limited proteolysis and isotope-labeling was used to examine the dynamic nature of the human rhinovirus (HRV) capsid in the presence of three antiviral compounds, a neutralizing Fab, and drug binding cavity mutations. Using these methods, it was found that the antivirals WIN 52084 and picovir (pleconaril) stabilized the capsid, while dansylaziridine caused destabilization. Site-directed mutations in the drug-binding cavity were found to stabilize the HRV14 capsid against proteolytic digestion in a manner similar to WIN 52084 and pleconaril. Antibodies that bind to the NIm-IA antigenic site and penetrate the canyon were also observed to protect the virion against proteolytic cleavage. These results demonstrate that quantifying the effects of antiviral ligands on protein "breathing" can be used to compare their mode of action and efficacy. In this case, it is apparent that hydrophobic antiviral agents, antibodies, or mutations in the canyon region block viral breathing. Therefore, these studies demonstrate that mobility in the canyon region is a major determinant in capsid breathing.
• The CPH1 Gene of Chlamydomonas Reinhardtii Encodes Two Forms of Cryptochrome Whose Levels Are Controlled by Light-induced Proteolysis Plant Physiology. Apr, 2004  |   Pubmed ID: 15064387 Cryptochromes are proteins related to DNA photolyases and have been shown to function as blue-light photoreceptors and to play important roles in circadian rhythms in both plants and animals. The CPH1 gene from Chlamydomonas reinhardtii was originally predicted to encode a putative cryptochrome protein of 867 amino acids with a predicted molecular mass of 91 kD (Small et al., 1995). However, western blotting with antibodies specific to the CPH1 protein revealed the presence of two proteins that migrate at apparent molecular mass of approximately 126 and 143 kD. A reexamination of the assigned intron-exon boundaries has shown that the previously assigned intron 7 is in fact part of exon 7 which leads to a predicted protein of 1,007 amino acids corresponding to a size of 104.6 kD. The two forms of CPH1 that migrate slower on SDS-PAGE presumably result from unknown posttranslational modifications. In C. reinhardtii cells synchronized by light to dark cycles, the two slow migrating forms of CPH1 protein accumulate in the dark and disappear rapidly in the light. Both red and blue light are effective at inducing the degradation of the CPH1 proteins. Proteasomes are implicated because degradation is inhibited by MG132, a proteasome inhibitor. Studies with deletion mutants indicate that the C-terminal region is important for both the posttranslational modification and the protein's stability under both light and dark conditions.
• Proteomics in Pulmonary Medicine Chest. Aug, 2006  |   Pubmed ID: 16899860 Proteomics is the study of the entire protein complement of the genome (the proteome) in a biological system. Proteomic studies require a multidisciplinary approach and have only been practical with the convergence of technical and methodologic improvements including the following: advances in mass spectrometry and genomic sequencing that now permit the identification and relative quantization of small amounts (femtomole) of nearly any single protein; new methods in gel electrophoresis that allow the detection of subtle changes in protein expression, including posttranslational modifications; automation and miniaturization that permit high-throughput analysis of clinical samples; and new bioinformatics and computational methods that facilitate analysis and interpretation of the abundant data that are generated by proteomics experiments. This convergence makes proteomics studies practical for pulmonary researchers using BAL fluid, lung tissue, blood, and exhaled breath condensates, and will facilitate the research of complex, multifactorial lung diseases such as acute lung injury and COPD. This review describes how proteomics experiments are conducted and interpreted, their limitations, and how proteomics has been used in clinical pulmonary medicine.
• Analysis of 25 Underivatized Amino Acids in Human Plasma Using Ion-pairing Reversed-phase Liquid Chromatography/time-of-flight Mass Spectrometry Rapid Communications in Mass Spectrometry : RCM. 2007  |   Pubmed ID: 17659659 Amino acids in biological fluids have previously been shown to be detectable using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application of a recently described liquid chromatographic separation method was coupled with TOFMS to employ accurate mass for qualitative identification; resulting in additional qualitative data not available with standard single quadrupole data. In the current study, we evaluated 25 physiological amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids; in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves were linear over a range from 1.56 to 400 microM. The dynamic range was found to be within physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino acids was between 80-120% spike recovery and
• Mining the Liver Proteome for Drug Targets for Sepsis Critical Care Medicine. Oct, 2007  |   Pubmed ID: 17885383
• MPYS, a Novel Membrane Tetraspanner, is Associated with Major Histocompatibility Complex Class II and Mediates Transduction of Apoptotic Signals Molecular and Cellular Biology. Aug, 2008  |   Pubmed ID: 18559423 Although the best-defined function of type II major histocompatibility complex (MHC-II) is presentation of antigenic peptides to T lymphocytes, these molecules can also transduce signals leading alternatively to cell activation or apoptotic death. MHC-II is a heterodimer of two transmembrane proteins, each containing a short cytoplasmic tail that is dispensable for transduction of death signals. This suggests the function of an undefined MHC-II-associated transducer in signaling the death response. Here we describe a novel plasma membrane tetraspanner (MPYS) that is associated with MHC-II and mediates its transduction of death signals. MPYS is unusual among tetraspanners in containing an extended C-terminal cytoplasmic tail (approximately 140 amino acids) with multiple embedded signaling motifs. MPYS is tyrosine phosphorylated upon MHC-II aggregation and associates with inositol lipid and tyrosine phosphatases. Finally, MHC class II-mediated cell death signaling requires MPYS-dependent activation of the extracellular signal-regulated kinase signaling pathway.
• 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.
• Alterations in the Human Lung Proteome with Lipopolysaccharide BMC Pulmonary Medicine. 2009  |   Pubmed ID: 19432985 Recombinant human activated protein C (rhAPC) is associated with improved survival in high-risk patients with severe sepsis; however, the effects of both lipopolysaccharide (LPS) and rhAPC on the bronchoalveolar lavage fluid (BALF) proteome are unknown.
• Proteomics Methods and Applications for the Practicing Clinician Annals of Allergy, Asthma & Immunology : Official Publication of the American College of Allergy, Asthma, & Immunology. Jun, 2009  |   Pubmed ID: 19558013 To describe clinical proteomics from discovery techniques and their limitations, to applications in allergy, asthma, and immunology, and finally to how proteomics can be integrated into clinical practice.
• Leukotriene-E4 in Human Urine: Comparison of On-line Purification and Liquid Chromatography-tandem Mass Spectrometry to Affinity Purification Followed by Enzyme Immunoassay Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences. Oct, 2009  |   Pubmed ID: 19726242 A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.
• Peeling off the Layers: Skin Taping and a Novel Proteomics Approach to Study Atopic Dermatitis The Journal of Allergy and Clinical Immunology. Nov, 2009  |   Pubmed ID: 19748658
• Regulation of Vascular Contractility and Blood Pressure by the E2F2 Transcription Factor Circulation. Sep, 2009  |   Pubmed ID: 19752322 Recent studies have identified a polymorphism in the endothelin-converting enzyme (ECE)-1b promoter (-338C/A) that is strongly associated with hypertension in women. The polymorphism is located in a consensus binding sequence for the E2F family of transcription factors. E2F proteins are crucially involved in cell-cycle regulation, but their roles in cardiovascular function are poorly understood. Here, we investigated the potential role of E2F2 in blood pressure regulation.
• Chromogranin A is an Autoantigen in Type 1 Diabetes Nature Immunology. Mar, 2010  |   Pubmed ID: 20139986 Autoreactive CD4(+) T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4(+) T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-A(g7) in an atypical manner, occupying only the carboxy-terminal half of the I-A(g7) peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.
• 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.
• 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.
• 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.
• SPLUNC1 Regulation in Airway Epithelial Cells: Role of Toll-like Receptor 2 Signaling Respiratory Research. 2010  |   Pubmed ID: 21054862 Respiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling.
• Comparative Proteomic Profiling of Patients with Atopic Dermatitis Based on History of Eczema Herpeticum Infection and Staphylococcus Aureus Colonization The Journal of Allergy and Clinical Immunology. Jan, 2011  |   Pubmed ID: 21211653 Atopic dermatitis (AD) is the most common inflammatory skin disorder in the general population worldwide, and the majority of patients are colonized with Staphylococcus aureus. Eczema herpeticum is a disseminated herpes simplex virus infection that occurs in a small subset of patients.
• Increased Cell Surface Fas Expression is Necessary and Sufficient to Sensitize Lung Fibroblasts to Fas Ligation-induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis Journal of Immunology (Baltimore, Md. : 1950). Jul, 2011  |   Pubmed ID: 21632719 Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-α and IFN-γ reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-α- and IFN-γ-stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-κB- and p38(mapk)-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-α and IFN-γ to sensitize fibroblasts to Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF.
• Islet Amyloid Polypeptide is a Target Antigen for Diabetogenic CD4+ T Cells Diabetes. Sep, 2011  |   Pubmed ID: 21734016 To investigate autoantigens in β-cells, we have used a panel of pathogenic T-cell clones that were derived from the NOD mouse. Our particular focus in this study was on the identification of the target antigen for the highly diabetogenic T-cell clone BDC-5.2.9.
• Urinary Leukotriene E₄ Levels Identify Children with Tobacco Smoke Exposure at Risk for Asthma Exacerbation The Journal of Allergy and Clinical Immunology. Aug, 2011  |   Pubmed ID: 21807251 Children with asthma exposed to secondhand smoke (SHS) might be at higher risk for severe exacerbations, but biomarkers of susceptibility to SHS exposure have not been previously reported.
• 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.
• 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.
• 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.
• 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.
• The Association of Adiponectin with Computed Tomography Phenotypes in Chronic Obstructive Pulmonary Disease American Journal of Respiratory and Critical Care Medicine. Sep, 2013  |   Pubmed ID: 23777323 Chronic obstructive pulmonary disease (COPD) is a heterogeneous disorder associated with systemic manifestations that contribute to its morbidity and mortality. Recent work suggests that biomarker signatures in the blood may be useful in evaluating COPD phenotypes and may provide insight into the pathophysiology of systemic manifestations. Adiponectin, primarily produced by fat cells, has been implicated in the pathophysiology of emphysema.
• Integrative Omics Approach Identifies Interleukin-16 As a Biomarker of Emphysema Omics : a Journal of Integrative Biology. Dec, 2013  |   Pubmed ID: 24138069 Interleukin-16 (IL-16) is a multifunctional cytokine that has been associated with autoimmune and allergic diseases. To investigate comprehensively whether IL-16 is also associated with chronic obstructive pulmonary disease (COPD) and emphysema, we performed an integrated analysis of multiple "omics" data. Over 500 subjects participating in the COPDGene® study donated blood and were clinically characterized and genetically profiled. IL-16 mRNA levels were measured in peripheral blood mononuclear cells (PBMC), and protein levels were measured in fresh frozen plasma. A multivariate analysis found plasma IL-16 positively associated with age and body mass index, and negatively associated with current smoking and emphysema in the upper lobes. PBMC IL-16 expression was positively associated with gender and a composite score for airflow obstruction, emphysema, and gas trapping. Whole-genome expression quantitative trait locus (eQTL) analysis identified a novel IL-16 missense SNP (rs11556218) associated with lower IL-16 in plasma. In summary, an integrated "omics" analysis in a very large cohort identified an association between decreased IL-16 and emphysema and discovered a novel IL-16 cis-eQTL. Thus IL-16 plasma levels and IL-16 genotyping may be useful in a personalized medicine approach for lung disease.
• 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.
• Predictors of Asthma Control and Lung Function Responsiveness to Step 3 Therapy in Children with Uncontrolled Asthma The Journal of Allergy and Clinical Immunology. Feb, 2014  |   Pubmed ID: 24084071 Predictors of improvement in asthma control and lung function to step 3 therapy in children with persistent asthma have not been identified despite reported heterogeneity in responsiveness.
• MSPrep--summarization, Normalization and Diagnostics for Processing of Mass Spectrometry-based Metabolomic Data Bioinformatics (Oxford, England). Jan, 2014  |   Pubmed ID: 24174567 Although R packages exist for the pre-processing of metabolomic data, they currently do not incorporate additional analysis steps of summarization, filtering and normalization of aligned data. We developed the MSPrep R package to complement other packages by providing these additional steps, implementing a selection of popular normalization algorithms and generating diagnostics to help guide investigators in their analyses.
• Transient and Persistent Metabolomic Changes in Plasma Following Chronic Cigarette Smoke Exposure in a Mouse Model PloS One. 2014  |   Pubmed ID: 25007263 Cigarette smoke exposure is linked to the development of a variety of chronic lung and systemic diseases in susceptible individuals. Metabolomics approaches may aid in defining disease phenotypes, may help predict responses to treatment, and could identify biomarkers of risk for developing disease. Using a mouse model of chronic cigarette smoke exposure sufficient to cause mild emphysema, we investigated whether cigarette smoke induces distinct metabolic profiles and determined their persistence following smoking cessation. Metabolites were extracted from plasma and fractionated based on chemical class using liquid-liquid and solid-phase extraction prior to performing liquid chromatography mass spectrometry-based metabolomics. Metabolites were evaluated for statistically significant differences among group means (p-value≤0.05) and fold change ≥1.5). Cigarette smoke exposure was associated with significant differences in amino acid, purine, lipid, fatty acid, and steroid metabolite levels compared to air exposed animals. Whereas 60% of the metabolite changes were reversible, 40% of metabolites remained persistently altered even following 2 months of smoking cessation, including nicotine metabolites. Validation of metabolite species and translation of these findings to human plasma metabolite signatures induced by cigarette smoking may lead to the discovery of biomarkers or pathogenic pathways of smoking-induced disease.