multi-step-preparation-technique-to-recover-multiple-metabolite

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital ...

National Jewish Health

Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of approximately 20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin alpha1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis.

Genome-scale functional profiling of the mammalian AP-1 signaling pathway.

Expression of cyclin dependent kinase (Cdk) inhibitor p27(Kip1), which blocks cell cycle progression from G(1) to S phase, can be regulated via multiple mechanisms including transcription, protein degradation, and translation. Recently, it was shown that p27(Kip1) plays an important role in the cellular response to hypoxia. However, the mechanisms involved in the hypoxia-induced regulation of p27(Kip1) expression are still not clear. In this study, we compare the expression of p27(Kip1) in two related murine hepatoma cell lines, Hepa-1 and c4. Hepa-1 produces functional aryl hydrocarbon receptor nuclear translocator (ARNT). c4 cells are derived from Hepa-1, but are ARNT deficient. Interestingly, we observed cell line-dependent effects of hypoxia on the expression of p27(Kip1). The level of p27(Kip1) protein in Hepa-1 cells is enhanced by hypoxia, but is reduced by hypoxia in c4 cells. Further investigation demonstrated that hypoxia-induced, ARNT-mediated, transactivation of the p27(Kip1) gene in Hepa-1 cells is responsible for the increase in p27(Kip1) protein. Once c4 cells were stably transfected with the wild type ARNT gene, a hypoxia-induced increase in p27(Kip1) mRNA was observed and reduction of p27(Kip1) protein caused by hypoxia was blocked. Hence, our data indicate that ARNT is involved in transcriptional upregulation of the p27(Kip1) gene under hypoxic conditions.

Cyclin dependent kinase inhibitor p27(Kip1) is upregulated by hypoxia via an ARNT dependent pathway.

The rat class 3 aldehyde dehydrogenase gene (ALDH3A1) is expressed constitutively or by xenobiotic induction depending on the tissue in which it occurs. Although the mechanism that mediates inducible expression has been well characterized, relatively little is known about constitutive regulatory mechanisms. Previous ALDH3A1 promoter analyses have indicated that primary regulatory regions within the ALDH3A1 5' flanking region exert similar effects on both constitutive and inducible ALDH3A1 expression. However, promoter gene analyses that served as the basis of early work were limited by the lack of sufficient 5' flanking region sequence. To gain a more complete picture of how the 5' flanking region regulates both modes of expression, we have subcloned an 8.0-kilobase (kb) fragment from the 5' flanking region of the ALDH3A1 gene and subjected it to reporter gene analyses. We found a region located between 4.8 and 7.8 kb upstream of the noncoding first exon that drives strong ALDH3A1 reporter activity. This region contains xenobiotic response element consensus sequences that mediate constitutive and inducible ALDH3A1 reporter gene expression. Using the new generation of ALDH3A1 reporter constructs, we were unable to confirm the presence of a negative regulatory region that was apparent in previous studies using a shorter fragment of the 5' flanking region. We also demonstrate that 3-methylcholanthrene induces ALDH3A1 expression above high constitutive background in corneal epithelial cells.

Constitutive and 3-methylcholanthrene-induced rat ALDH3A1 expression is mediated by multiple xenobiotic response elements.

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.

Alterations in the human lung proteome with lipopolysaccharide.

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.

Proteomics methods and applications for the practicing clinician.

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.

Chromogranin A is an autoantigen in type 1 diabetes.

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.

Proteomics and metabolomics and their application to analgesia research.

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.

Islet amyloid polypeptide is a target antigen for diabetogenic CD4+ T cells.

Galectin-9 is a pleiotropic immune modulator affecting numerous cell types of innate and adaptive immunity. Patients with chronic infection with either hepatitis C virus (HCV) or HIV have elevated circulating levels. Limited data exist on the regulation of natural killer (NK) cell function through interaction with galectin-9. We found that galectin-9 ligation downregulates multiple immune-activating genes, including eight involved in the NK cell-mediated cytotoxicity pathway, impairs lymphokine-activated killing, and decreases the proportion of gamma interferon (IFN-γ)-producing NK cells that had been stimulated with interleukin-12 (IL-12)/IL-15. We demonstrate that the transcriptional and functional changes induced by galectin-9 are independent of Tim-3. Consistent with these results for humans, we find that the genetic absence of galectin-9 in mice is associated with greater IFN-γ production by NK cells and enhanced degranulation. We also show that in the setting of a short-term (4-day) murine cytomegalovirus infection, terminally differentiated NKs accumulate in the livers of galectin-9 knockout mice, and that hepatic NKs spontaneously produce significantly more IFN-γ in this setting. Taken together, our results indicate that galectin-9 engagement impairs the function of NK cells, including cytotoxicity and cytokine production.

Galectin-9 functionally impairs natural killer cells in humans and mice.

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.

MSPrep--summarization, normalization and diagnostics for processing of mass spectrometry-based metabolomic data.

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.

Hands-on workshops as an effective means of learning advanced technologies including genomics, proteomics and bioinformatics.

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.

Richard Reisdorph

Department of Immunology

National Jewish Health

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Richard Reisdorph

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Immunology

National Jewish Health

Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Department of Immunology