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AAbs provide value in identifying individuals at risk, stratifying patients with different clinical courses, improving our understanding of autoimmune destructions, identifying antigens for cellular immune response and providing candidates for prevention trials in T1D. A two-stage serological AAb screening against 6,000 human proteins was performed. A dual specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) was validated with 36% sensitivity at 98% specificity by an orthogonal immunoassay. This is the first systematic screening for novel AAbs against large number of human proteins by protein arrays in T1D. A more comprehensive search for novel AAbs was performed using a knowledge-based approach by ELISA and a screening-based approach against 10,000 human proteins by NAPPA. Six AAbs were identified and validated with sensitivities ranged from 16% to 27% at 95% specificity. These two studies enriched the T1D “autoantigenome” and provided insights into T1D pathophysiology in an unprecedented breadth and width.
The rapid rise of T1D incidence suggests the potential involvement of environmental factors including viral infections. Sero-reactivity to 646 viral antigens was assessed in new-onset T1D patients. Antibody positive rate of EBV was significantly higher in cases than controls that suggested a potential role of EBV in T1D development. A high density-NAPPA platform was demonstrated with high reproducibility and sensitivity in profiling anti-viral antibodies.
This dissertation shows the power of a protein-array based immunoproteomics approach to characterize humoral immunoprofile against human and viral proteomes. The identification of novel T1D-specific AAbs and T1D-associated viruses will help to connect the nodes in T1D etiology and provide better understanding of T1D pathophysiology.
Mild Cognitive Impairment (MCI) is a transitional stage between normal aging and dementia and people with MCI are at high risk of progression to dementia. MCI is attracting increasing attention, as it offers an opportunity to target the disease process during an early symptomatic stage. Structural magnetic resonance imaging (MRI) measures have been the mainstay of Alzheimer's disease (AD) imaging research, however, ventricular morphometry analysis remains challenging because of its complicated topological structure. Here we describe a novel ventricular morphometry system based on the hyperbolic Ricci flow method and tensor-based morphometry (TBM) statistics. Unlike prior ventricular surface parameterization methods, hyperbolic conformal parameterization is angle-preserving and does not have any singularities. Our system generates a one-to-one diffeomorphic mapping between ventricular surfaces with consistent boundary matching conditions. The TBM statistics encode a great deal of surface deformation information that could be inaccessible or overlooked by other methods. We applied our system to the baseline MRI scans of a set of MCI subjects from the Alzheimer's Disease Neuroimaging Initiative (ADNI: 71 MCI converters vs. 62 MCI stable). Although the combined ventricular area and volume features did not differ between the two groups, our fine-grained surface analysis revealed significant differences in the ventricular regions close to the temporal lobe and posterior cingulate, structures that are affected early in AD. Significant correlations were also detected between ventricular morphometry, neuropsychological measures, and a previously described imaging index based on fluorodeoxyglucose positron emission tomography (FDG-PET) scans. This novel ventricular morphometry method may offer a new and more sensitive approach to study preclinical and early symptomatic stage AD.
We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase β subunit (β-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, β-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple β-F1-ATPase peptides. There were three β-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to β-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R[superscript 2] = 0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the β-F1-ATPase[subscript 134-143] peptide. Measured stable-isotope enrichment was highly reproducible for the β-F1-ATPase[subscript 134-143] peptide (CV = 2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R[superscript 2] = 0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the β-F1-ATPase[subscript 134-143] peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle β-F1-ATPase based on the determination of the enrichment of the β-F1-ATPase[subscript 134-143] peptide.
Background: Healthy individuals on the lower end of the insulin sensitivity spectrum also have a reduced gene expression response to exercise for specific genes. The goal of this study was to determine the relationship between insulin sensitivity and exercise-induced gene expression in an unbiased, global manner.
Methods and Findings: Euglycemic clamps were used to measure insulin sensitivity and muscle biopsies were done at rest and 30 minutes after a single acute exercise bout in 14 healthy participants. Changes in mRNA expression were assessed using microarrays, and miRNA analysis was performed in a subset of 6 of the participants using sequencing techniques. Following exercise, 215 mRNAs were changed at the probe level (Bonferroni-corrected P<0.00000115). Pathway and Gene Ontology analysis showed enrichment in MAP kinase signaling, transcriptional regulation and DNA binding. Changes in several transcription factor mRNAs were correlated with insulin sensitivity, including MYC, r=0.71; SNF1LK, r=0.69; and ATF3, r= 0.61 (5 corrected for false discovery rate). Enrichment in the 5’-UTRs of exercise-responsive genes suggested regulation by common transcription factors, especially EGR1. miRNA species of interest that changed after exercise included miR-378, which is located in an intron of the PPARGC1B gene.
Conclusions: These results indicate that transcription factor gene expression responses to exercise depend highly on insulin sensitivity in healthy people. The overall pattern suggests a coordinated cycle by which exercise and insulin sensitivity regulate gene expression in muscle.
Although insulin resistance in skeletal muscle is well-characterized, the role of circulating whole blood in the metabolic syndrome phenotype is not well understood. We set out to test the hypothesis that genes involved in inflammation, insulin signaling and mitochondrial function would be altered in expression in the whole blood of individuals with metabolic syndrome. We further wanted to examine whether similar relationships that we have found previously in skeletal muscle exist in peripheral whole blood cells. All subjects (n=184) were Latino descent from the Arizona Insulin Resistance registry. Subjects were classified based on the metabolic syndrome phenotype according to the National Cholesterol Education Program’s Adult Treatment Panel III. Of the 184 Latino subjects in the study, 74 were classified with the metabolic syndrome and 110 were without. Whole blood gene expression profiling was performed using the Agilent 4x44K Whole Human Genome Microarray. Whole blood microarray analysis identified 1,432 probes that were altered in expression ≥1.2 fold and P<0.05 after Benjamini-Hochberg in the metabolic syndrome subjects. KEGG pathway analysis revealed significant enrichment for pathways including ribosome, oxidative phosphorylation and MAPK signaling (all Benjamini-Hochberg P<0.05). Whole blood mRNA expression changes observed in the microarray data were confirmed by quantitative RT-PCR. Transcription factor binding motif enrichment analysis revealed E2F1, ELK1, NF-kappaB, STAT1 and STAT3 significantly enriched after Bonferroni correction (all P<0.05). The results of the present study demonstrate that whole blood is a useful tissue for studying the metabolic syndrome and its underlying insulin resistance although the relationship between blood and skeletal muscle differs.
Background: Although the effect of the fat mass and obesity-associated (FTO) gene on adiposity is well established, there is a lack of evidence whether physical activity (PA) modifies the effect of FTO variants on obesity in Latino populations. Therefore, the purpose of this study was to examine PA influences and interactive effects between FTO variants and PA on measures of adiposity in Latinos.
Results: After controlling for age and sex, participants who did not engage in regular PA exhibited higher BMI, fat mass, HC, and WC with statistical significance (P < 0.001). Although significant associations between the three FTO genotypes and adiposity measures were found, none of the FTO genotype by PA interaction assessments revealed nominally significant associations. However, several of such interactive influences exhibited considerable trend towards association.
Conclusions: These data suggest that adiposity measures are associated with PA and FTO variants in Latinos, but the impact of their interactive influences on these obesity measures appear to be minimal. Future studies with large sample sizes may help to determine whether individuals with specific FTO variants exhibit differential responses to PA interventions.
Background: Carriers of the APOE ε4 allele are at increased risk of developing Alzheimer’s disease (AD), and have been shown to have reduced cerebral metabolic rate of glucose (CMRgl) in the same brain areas frequently affected in AD. These individuals also exhibit reduced plasma levels of apolipoprotein E (apoE) attributed to a specific decrease in the apoE4 isoform as determined by quantification of individual apoE isoforms in APOE ε4 heterozygotes. Whether low plasma apoE levels are associated with structural and functional brain measurements and cognitive performance remains to be investigated.
Methods: Using quantitative mass spectrometry we quantified the plasma levels of total apoE and the individual apoE3 and apoE4 isoforms in 128 cognitively normal APOE ε3/ε4 individuals included in the Arizona APOE cohort. All included individuals had undergone extensive neuropsychological testing and 25 had in addition undergone FDG-PET and MRI to determine CMRgl and regional gray matter volume (GMV).
Results: Our results demonstrated higher apoE4 levels in females versus males and an age-dependent increase in the apoE3 isoform levels in females only. Importantly, a higher relative ratio of apoE4 over apoE3 was associated with GMV loss in the right posterior cingulate and with reduced CMRgl bilaterally in the anterior cingulate and in the right hippocampal area. Additional exploratory analysis revealed several negative associations between total plasma apoE, individual apoE isoform levels, GMV and CMRgl predominantly in the frontal, occipital and temporal areas. Finally, our results indicated only weak associations between apoE plasma levels and cognitive performance which further appear to be affected by sex.
Conclusions: Our study proposes a sex-dependent and age-dependent variation in plasma apoE isoform levels and concludes that peripheral apoE levels are associated with GMV, CMRgl and possibly cognitive performance in cognitively healthy individuals with a genetic predisposition to AD.