Serum Metabolites Responding in a Dose-Dependent Manner to the Intake of a High-Fat Meal in Normal Weight Healthy Men Are Associated with Obesity

Although the composition of the human blood metabolome is influenced both by the health status of the organism and its dietary behavior, the interaction between these two factors has been poorly characterized. This study makes use of a previously published randomized controlled crossover acute intervention to investigate whether the blood metabolome of 15 healthy normal weight (NW) and 17 obese (OB) men having ingested three doses (500, 1000, 1500 kcal) of a high-fat (HF) meal can be used to identify metabolites differentiating these two groups. Among the 1024 features showing a postprandial response, measured between 0 h and 6 h, in the NW group, 135 were dose-dependent. Among these 135 features, 52 had fasting values that were significantly different between NW and OB men, and, strikingly, they were all significantly higher in OB men. A subset of the 52 features was identified as amino acids (e.g., branched-chain amino acids) and amino acid derivatives. As the fasting concentration of most of these metabolites has already been associated with metabolic dysfunction, we propose that challenging normal weight healthy subjects with increasing caloric doses of test meals might allow for the identification of new fasting markers associated with obesity

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency

Inflammatory and metabolic responses to high-fat meals with and without dairy products in men.

Postprandial inflammation is an important factor for human health since chronic low-grade inflammation is associated with chronic diseases. Dairy products have a weak but significant anti-inflammatory effect on postprandial inflammation. The objective of the present study was to compare the effect of a high-fat dairy meal (HFD meal), a high-fat non-dairy meal supplemented with milk (HFM meal) and a high-fat non-dairy control meal (HFC meal) on postprandial inflammatory and metabolic responses in healthy men. A cross-over study was conducted in nineteen male subjects. Blood samples were collected before and 1, 2, 4 and 6 h after consumption of the test meals. Plasma concentrations of insulin, glucose, total cholesterol, LDL-cholesterol, HDL-cholesterol, TAG and C-reactive protein (CRP) were measured at each time point. IL-6, TNF-α and endotoxin concentrations were assessed at baseline and endpoint (6 h). Time-dependent curves of these metabolic parameters were plotted, and the net incremental AUC were found to be significantly higher for TAG and lower for CRP after consumption of the HFM meal compared with the HFD meal; however, the HFM and HFD meals were not different from the HFC meal. Alterations in IL-6, TNF-α and endotoxin concentrations were not significantly different between the test meals. The results suggest that full-fat milk and dairy products (cheese and butter) have no significant impact on the inflammatory response to a high-fat meal

Structural effects of linkage disequilibrium on the transcriptome

A majority of SNPs (single nucleotide polymorphisms) map to noncoding and intergenic regions of the genome. Noncoding SNPs are often identified in genome-wide association studies (GWAS) as strongly associated with human disease. Two such disease-associated SNPs in the 5′ UTR of the human FTL (Ferritin Light Chain) gene are predicted to alter the ensemble of structures adopted by the mRNA. High-accuracy single nucleotide resolution chemical mapping reveals that these SNPs result in substantial changes in the structural ensemble in agreement with the computational prediction. Furthermore six rescue mutations are correctly predicted to restore the mRNA to its wild-type ensemble. Our data confirm that the FTL 5′ UTR is a “RiboSNitch,” an RNA that changes structure if a particular disease-associated SNP is present. The structural change observed is analogous to that of a bacterial Riboswitch in that it likely regulates translation. These data further suggest that specific pairs of SNPs in high linkage disequilibrium (LD) will form RNA structure-stabilizing haplotypes (SSHs). We identified 484 SNP pairs that form SSHs in UTRs of the human genome, and in eight of the 10 SSH-containing transcripts, SNP pairs stabilize RNA protein binding sites. The ubiquitous nature of SSHs in the transcriptome suggests that certain haplotypes are conserved to avoid RiboSNitch formation

Structural divergence creates new functional features in alphavirus genomes

Alphaviruses are mosquito-borne pathogens that cause human diseases ranging from debilitating arthritis to lethal encephalitis. Studies with Sindbis virus (SINV), which causes fever, rash, and arthralgia in humans, and Venezuelan equine encephalitis virus (VEEV), which causes encephalitis, have identified RNA structural elements that play key roles in replication and pathogenesis. However, a complete genomic structural profile has not been established for these viruses. We used the structural probing technique SHAPE-MaP to identify structured elements within the SINV and VEEV genomes. Our SHAPE-directed structural models recapitulate known RNA structures, while also identifying novel structural elements, including a new functional element in the nsP1 region of SINV whose disruption causes a defect in infectivity. Although RNA structural elements are important for multiple aspects of alphavirus biology, we found the majority of RNA structures were not conserved between SINV and VEEV. Our data suggest that alphavirus RNA genomes are highly divergent structurally despite similar genomic architecture and sequence conservation; still, RNA structural elements are critical to the viral life cycle. These findings reframe traditional assumptions about RNA structure and evolution: rather than structures being conserved, alphaviruses frequently evolve new structures that may shape interactions with host immune systems or co-evolve with viral proteins

A novel application of pattern recognition for accurate SNP and indel discovery from high-throughput data: Targeted resequencing of the glucocorticoid receptor co-chaperone FKBP5 in a Caucasian population

The detection of single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) with precision from high-throughput data remains a significant bioinformatics challenge. Accurate detection is necessary before next-generation sequencing can routinely be used in the clinic. In research, scientific advances are inhibited by gaps in data, exemplified by the underrepresented discovery of rare variants, variants in non-coding regions and indels. The continued presence of false positives and false negatives prevents full automation and requires additional manual verification steps. Our methodology presents applications of both pattern recognition and sensitivity analysis to eliminate false positives and aid in the detection of SNP/indel loci and genotypes from high-throughput data. We chose FK506-binding protein 51(FKBP5) (6p21.31) for our clinical target because of its role in modulating pharmacological responses to physiological and synthetic glucocorticoids and because of the complexity of the genomic region. We detected genetic variation across a160 kb region encompassing FKBP5. 613 SNPs and 57 indels, including a 3.3 kb deletion were discovered. We validated our method using three independent data sets and, with Sanger sequencing and Affymetrix and Illumina microarrays, achieved 99% concordance. Furthermore we were able to detect 267 novel rare variants and assess linkage disequilibrium. Our results showed both a sensitivity and specificity of 98%, indicating near perfect classification between true and false variants. The process is scalable and amenable to automation, with the downstream filters taking only 1.5 hours to analyze 96 individuals simultaneously. We provide examples of how our level of precision uncovered the interactions of multiple loci, their predicted influences on mRNA stability, perturbations of the hsp90 binding site, and individual variation in FKBP5 expression. Finally we show how our discovery of rare variants may change current conceptions of evolution at this locus

A functional riboSNitch in the 3' untranslated region of FKBP5 alters MicroRNA-320a binding efficiency and mediates vulnerability to chronic post-traumatic pain

Previous studies have shown that common variants of the gene coding for FK506-binding protein 51 (FKBP5), a critical regulator of glucocorticoid sensitivity, affect vulnerability to stress-related disorders. In a previous report, FKBP5 rs1360780 was identified as a functional variant because of its effect on gene methylation. Here we report evidence for a novel functional FKBP5 allele, rs3800373. This study assessed the association between rs3800373 and post-traumatic chronic pain in 1607 women and men from two ethnically diverse human cohorts. The molecular mechanism through which rs3800373 affects adverse outcomes was established via in silico, in vivo, and in vitro analyses. The rs3800373 minor allele predicted worse adverse outcomes after trauma exposure, such that individuals with the minor (risk) allele developed more severe post-traumatic chronic musculoskeletal pain. Among these individuals, peritraumatic circulating FKBP5 expression levels increased as cortisol and glucocorticoid receptor (NR3C1) mRNA levels increased, consistent with increased glucocorticoid resistance. Bioinformatic, in vitro, and mutational analyses indicate that the rs3800373 minor allele reduces the binding of a stress-and pain-associated microRNA, miR-320a, to FKBP5 via altering the FKBP5 mRNA 3'UTR secondary structure (i.e., is a riboSNitch). This results in relatively greater FKBP5 translation, unchecked by miR-320a. Overall, these results identify an important gene–miRNA interaction influencing chronic pain risk in vulnerable individuals and suggest that exogenous methods to achieve targeted reduction in poststress FKBP5 mRNA expression may constitute useful therapeutic strategies

An RNA structure-mediated, posttranscriptional model of human α-1-antitrypsin expression

Protein and mRNA expression are in most cases poorly correlated, which suggests that the posttranscriptional regulatory program of a cell is an important component of gene expression. This regulatory network is still poorly understood, including how RNA structure quantitatively contributes to translational control. We present here a series of structural and functional experiments that together allow us to derive a quantitative, structure-dependent model of translation that accurately predicts translation efficiency in reporter assays and primary human tissue for a complex and medically important protein, α-1-antitrypsin. Our model demonstrates the importance of accurate, experimentally derived RNA structural models partnered with Kozak sequence information to explain protein expression and suggests a strategy by which α-1-antitrypsin expression may be increased in diseased individuals

Disease-Associated Mutations That Alter the RNA Structural Ensemble

Genome-wide association studies (GWAS) often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs) from the Human Gene Mutation Database (HGMD) that map to the untranslated regions (UTRs) of a gene. Rather than using minimum free energy approaches (e.g. mFold), we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, β-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD), and Hypertension), we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5′ UTRs of FTL and RB1) SNP–induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a “RiboSNitch,” that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble

Sharing and archiving nucleic acid structure mapping data

Nucleic acids are particularly amenable to structural characterization using chemical and enzymatic probes. Each individual structure mapping experiment reveals specific information about the structure and/or dynamics of the nucleic acid. Currently, there is no simple approach for making these data publically available in a standardized format. We therefore developed a standard for reporting the results of single nucleotide resolution nucleic acid structure mapping experiments, or SNRNASMs. We propose a schema for sharing nucleic acid chemical probing data that uses generic public servers for storing, retrieving, and searching the data. We have also developed a consistent nomenclature (ontology) within the Ontology of Biomedical Investigations (OBI), which provides unique identifiers (termed persistent URLs, or PURLs) for classifying the data. Links to standardized data sets shared using our proposed format along with a tutorial and links to templates can be found at http://snrnasm.bio.unc.edu