Project description:Isotope labeling measurements using mass spectrometry can provide informative insights on the metabolic systems of various organisms. The detailed identification of carbon positions included in the fragment ions of dicarboxylic and tricarboxylic acids in central carbon metabolism is needed for precise interpretation of the metabolic states. In this study, fragment ions containing the carbon backbone cleavage of dicarboxylic and tricarboxylic in the Krebs cycle were investigated by using gas chromatography (GC)-electron ionization (EI)-MS and GC-EI-MS/MS. The positions of decarboxylation in the dicarboxylic and tricarboxylic acids were successfully identified by analyses using position-specific 13C-labeled standards prepared by in vitro enzymatic reactions. For example, carboxyl groups of C1 and C6 of trimethylsilyl (TMS)- and tert-butyldimethylsilyl (TBDMS)-derivatized malic and citric acids were primarily cleaved by EI. MS/MS analyses were also performed, and fragment ions of TBDMS-citric and ?-ketoglutaric acids (?KG) with the loss of two carboxyl groups in collision-induced dissociation (CID) were observed.
Project description:Axon regeneration in the central nervous system (CNS) requires reactivating injured neurons’ intrinsic growth state and enabling growth in an inhibitory environment. Using an inbred mouse neuronal phenotypic screen, we find that CAST/Ei mouse adult dorsal root ganglion neurons extend axons more on CNS myelin than the other eight strains tested, especially when pre-injured. Injury-primed CAST/Ei neurons also regenerate markedly in the spinal cord and optic nerve more than those from C57BL/6 mice and show greater spouting following ischemic stroke. Heritability estimates indicate that extended growth in CAST/Ei neurons on myelin is genetically determined, and two whole-genome expression screens yield the Activin transcript Inhba as most correlated with this ability. These screens are presented here.
Project description:Rationale: Metabolomics analyses using gas chromatography mass spectrometry (GC-MS) - based metabolomics are heavily impeded by the lack of high-resolution mass spectrometers and limited spectral libraries to complement the excellent chromatography that GC platforms offer, a challenge that is being addressed with the implementation of high resolution (HR) platforms such as GC-Orbitrap-MS. Methods: We used serum samples from a non-human primate (NHP), a baboon (Papio hamadryas), with suitable quality controls to quantify the chemical space using an advanced HR MS platform for confident metabolite identification and robust quantification to assess the suitability of the platform for routine clinical metabolomics research. In a comparative approach, we also analyzed the same serum samples using a two-dimensional gas chromatography time-of-flight mass-spectrometer (2D GC-ToF-MS) for metabolite identification and quantification following established standard protocols. Results: Overall, the 2D GC-ToF-MS and GC-Orbitrap-MS analyses enabled identification and quantification of 555 total metabolites from the NHP serum with a spectral similarity score Rsim ≥ 900 and S/R ratio of > 25. A common set of 30 metabolites with HMDB and KEGG IDs were quantified in the serum samples by both platforms where the 2D GC-ToF-MS enabled quantification of a total 384 metabolites (118 HMDB IDs) and the GC-Orbitrap-MS analysis quantification of a total 200 metabolites (47 HMDB IDs). Conclusions: Our study provides insights into the benefits and limitations of the use of a higher mass accuracy instrument for untargeted GC-MS-based metabolomics with multi-dimensional chromatography in future studies addressing clinical conditions or exposome studies.
Project description:BACKGROUND: New strategies for high-throughput sequencing are constantly appearing, leading to a great increase in the number of completely sequenced genomes. Unfortunately, computational genome annotation is out of step with this progress. Thus, the accurate annotation of these genomes has become a bottleneck of knowledge acquisition. RESULTS: We exploited a proteogenomic approach to improve conventional genome annotation by integrating proteomic data with genomic information. Using Shigella flexneri 2a as a model, we identified total 823 proteins, including 187 hypothetical proteins. Among them, three annotated ORFs were extended upstream through comprehensive analysis against an in-house N-terminal extension database. Two genes, which could not be translated to their full length because of stop codon 'mutations' induced by genome sequencing errors, were revised and annotated as fully functional genes. Above all, seven new ORFs were discovered, which were not predicted in S. flexneri 2a str.301 by any other annotation approaches. The transcripts of four novel ORFs were confirmed by RT-PCR assay. Additionally, most of these novel ORFs were overlapping genes, some even nested within the coding region of other known genes. CONCLUSIONS: Our findings demonstrate that current Shigella genome annotation methods are not perfect and need to be improved. Apart from the validation of predicted genes at the protein level, the additional features of proteogenomic tools include revision of annotation errors and discovery of novel ORFs. The complementary dataset could provide more targets for those interested in Shigella to perform functional studies.
Project description:Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart defect with a world-wide prevalence of 3 to 4 cases per 10,000 life births. TOF is a congenital heart disease with four major cardiac defects, i.e., ventricular septal defect, overriding aortic root, infundibular stenosis of the pulmonary artery, and right ventricular hypertrophy. Treatment relies on correction surgery in early infancy. This study performed whole genome microarray gene expression profiling of cardiac specimens of the right ventricular outflow tract (RVOT), which were recovered during correction surgery of TOF from 11 pediatric patients diagnosed with TOF cardiac defects.
Project description:Mouse genetic crosses were established between the PyMT model of metastatic breast cancer and MOLF/Ei strain. Tumors were harvested from the animals for gene expression analysis to identify genes associated with progression to distant metastatic disease.
Project description:Tetralogy of Fallot (ToF) and Atrial Septal Defects (ASD) are the most common types of congenital heart disease and a major cause of childhood morbidity and mortality. Cardiopulmonary bypass (CPB) is used during corrective cardiac surgery to support circulation and heart stabilization. However, this technique triggers systemic inflammatory and stress response and consequent increased risk of postoperative complications. The aim of this study was to define the molecular bases of ToF and ASD pathogenesis and response to CPB and identify new specific biomarkers. We conducted comparative transcriptome analysis of right atrial biopsies from 10 ToF and 10 ASD patients before (Pre-CPB) and after (Post-CPB) surgery. Pre-CPB samples showed significant differential expression of 72 genes (DEGs), 28 of which were overexpressed in ToF and 44 in ASD. According to Gene Ontology annotation, the mostly enriched biological processes were represented by matrix organization and cell adhesion in ToF and by muscle development and contractility in ASD specimens. GSEA highlighted the specific enrichment of known hypoxia gene sets in ToF samples, pointing to a role for hypoxia in disease pathogenesis. The post-CPB myocardium exhibited significant alterations in the expression profile of genes related to transcription regulation, growth/apoptosis, inflammation, cell adhesion/matrix organization, and oxidative stress. Among DEGs, only 70 were common to the two disease groups, whereas 110 and 24 were unique in ToF and ASD, respectively. Interestingly, gene expression changes in ASD samples followed a consensus hypoxia profile. These data define ToF- and ASD-specific myocardial transcriptional signatures and demonstrate differential gene reprogramming in response to CPB in the two pathologies, with potential prognostic and therapeutic implications.
Project description:DG accounts for most of the identifiable cases of TOF. Exploring and comparing the transcriptome of hiPSC-CMs and hiPSC-CPs from TOF-DG and TOF-ND could provide a bettering understanding on TOF.