On best practices: The Institute of Medicine scheme for developing, validating, and demonstrating clinical utility of omics‐based diagnostic and predictive tests

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102242/1/prca1506.pd

Identification of a protein signature in renal allograft rejection

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102221/1/prca1489-sup-0001-tables2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102221/2/prca1489.pd

Single-nucleotide polymorphisms: analysis by mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry has evolved as a powerful method for analyzing nucleic acids. Here we provide protocols for genotyping single-nucleotide polymorphisms (SNPs) by MALDI based on PCR and primer extension to generate allele-specific products. Furthermore, we present three different approaches for sample preparation of primer-extension products before MALDI analysis and discuss their potential areas of application. The first approach, the 'GOOD' assay, is a purification-free procedure that uses DNA-modification chemistry, including alkylation of phosphorothioate linkages in the extension primers. The other two approaches use either solid-phase extraction or microarray purification for the purification of primer-extension products. Depending on the reaction steps of the various approaches, the protocols take about 6–8 hours

DNA polymorphisms and haplotype patterns of transcription factors involved in barley endosperm development are associated with key agronomic traits

<p>Abstract</p> <p>Background</p> <p>Association mapping is receiving considerable attention in plant genetics for its potential to fine map quantitative trait loci (QTL), validate candidate genes, and identify alleles of interest. In the present study association mapping in barley (<it>Hordeum vulgare </it>L.) is investigated by associating DNA polymorphisms with variation in grain quality traits, plant height, and flowering time to gain further understanding of gene functions involved in the control of these traits. We focused on the four loci <it>BLZ1</it>, <it>BLZ2</it>, <it>BPBF </it>and <it>HvGAMYB </it>that play a role in the regulation of B-hordein expression, the major fraction of the barley storage protein. The association was tested in a collection of 224 spring barley accessions using a two-stage mixed model approach.</p> <p>Results</p> <p>Within the sequenced fragments of four candidate genes we observed different levels of nucleotide diversity. The effect of selection on the candidate genes was tested by Tajima's D which revealed significant values for <it>BLZ1</it>, <it>BLZ2</it>, and <it>BPBF </it>in the subset of two-rowed barleys. Pair-wise LD estimates between the detected SNPs within each candidate gene revealed different intra-genic linkage patterns. On the basis of a more extensive examination of genomic regions surrounding the four candidate genes we found a sharp decrease of LD (<it>r</it>²<0.2 within 1 cM) in all but one flanking regions.</p> <p>Significant marker-trait associations between SNP sites within <it>BLZ1 </it>and flowering time, <it>BPBF </it>and crude protein content and <it>BPBF </it>and starch content were detected. Most haplotypes occurred at frequencies <0.05 and therefore were rejected from the association analysis. Based on haplotype information, <it>BPBF </it>was associated to crude protein content and starch content, <it>BLZ2 </it>showed association to thousand-grain weight and <it>BLZ1 </it>was found to be associated with flowering time and plant height.</p> <p>Conclusions</p> <p>Differences in nucleotide diversity and LD pattern within the candidate genes <it>BLZ1</it>, <it>BLZ2</it>, <it>BPBF</it>, and <it>HvGAMYB </it>reflect the impact of selection on the nucleotide sequence of the four candidate loci.</p> <p>Despite significant associations, the analysed candidate genes only explained a minor part of the total genetic variation although they are known to be important factors influencing the expression of seed quality traits. Therefore, we assume that grain quality as well as plant height and flowering time are influenced by many factors each contributing a small part to the expression of the phenotype. A genome-wide association analysis could provide a more comprehensive picture of loci involved in the regulation of grain quality, thousand grain weight and the other agronomic traits that were analyzed in this study. However, despite available high-throughput genotyping arrays the marker density along the barely genome is still insufficient to cover all associations in a whole genome scan. Therefore, the candidate gene-based approach will further play an important role in barley association studies.</p

Dark-adapted versus bleached state in fluorescence lifetime imaging ophthalmoscopy

Purpose: The (early) detection of diseases based on metabolic changes in the retina is the goal of the novel autofluorescence lifetime ophthalmoscopy (FLIO) technique. These metabolic changes can be detected as alterations in the fundus autofluorescence (FAF) lifetimes. The influences of the photopigment bleaching and photobleaching on the FAF lifetimes are unknown. Thus, we performed a volunteer study to investigate these influences. Methods: In 21 healthy volunteers (23.6±3.8 years) time-resolved FAF was measured with a FLIO device (30° of fundus, excitation at 473nm, detection in two spectral channels: 500-560nm (ch1) and 560-720nm (ch2), time-correlated single photon counting method). All subjects had a crystalline lens. The pupil was dilated with 0.5% Tropicamid. After volunteers adapted to the dark using a custom-made lightproof eyewear over a period of 30 min, the first FLIO measurement was recorded (dark-adapted state). Subsequently, one eye was bleached for 1 min using a luminance of 3200cd/m2, followed by a FLIO measurement (bleached state). The fluorescence lifetimes were estimated from the FAF decays, based on three exponential functions, using the software FLIMX (www.flimx.de). Average values from the central region, and the inner and outer rings of the ETDRS grid were utilized to compare both bleaching states using analysis of variance, Friedman, and post hoc tests. Results : Only ch2 yielded significant changes (p<0.05) for the fluorescence lifetime τ2 from all ETDRS regions (+19-28ps), for the fluorescence lifetime τ1 (+6ps) and the mean fluorescence lifetime (+6ps) in the central area that were less than 10% in magnitude. Additionally, the acquisition time in the bleached state was significantly reduced by approximately 20% on average, compared to the dark-adapted state. The fluorescence lifetime differences caused by bleaching were much smaller than pathological states known from literature. Conclusions: We conclude that bleaching is not relevant for current clinical FLIO applications because of the small magnitude of the elicited fluorescence lifetime changes. Thus, it is advisable to instruct patients to wait in a bright room before FLIO measurements. If the expected changes in the fluorescence lifetime in a specific experimental paradigm are small, FLIO users should follow a strict acquisition protocol in terms of the photopigment bleaching state of the patients to obtain the most reliable results

Dissecting the effect of genetic variation on the hepatic expression of drug disposition genes across the collaborative cross mouse strains

A central challenge in pharmaceutical research is to investigate genetic variation in response to drugs. The Collaborative Cross (CC) mouse reference population is a promising model for pharmacogenomic studies because of its large amount of genetic variation, genetic reproducibility, and dense recombination sites. While the CC lines are phenotypically diverse, their genetic diversity in drug disposition processes, such as detoxification reactions, is still largely uncharacterized. Here we systematically measured RNA-sequencing expression profiles from livers of 29 CC lines under baseline conditions. We then leveraged a reference collection of metabolic biotransformation pathways to map potential relations between drugs and their underlying expression quantitative trait loci (eQTLs). By applying this approach on proximal eQTLs, including eQTLs acting on the overall expression of genes and on the expression of particular transcript isoforms, we were able to construct the organization of hepatic eQTL-drug connectivity across the CC population. The analysis revealed a substantial impact of genetic variation acting on drug biotransformation, allowed mapping of potential joint genetic effects in the context of individual drugs, and demonstrated crosstalk between drug metabolism and lipid metabolism. Our findings provide a resource for investigating drug disposition in the CC strains, and offer a new paradigm for integrating biotransformation reactions to corresponding variations in DNA sequences

Serial interactome capture of the human cell nucleus

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present ‘serial RNA interactome capture’ (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)–RNA–protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA–RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs

Antidiabetic Effects of Chamomile Flowers Extract in Obese Mice through Transcriptional Stimulation of Nutrient Sensors of the Peroxisome Proliferator-Activated Receptor (PPAR) Family

Given the significant increases in the incidence of metabolic diseases, efficient strategies for preventing and treating of these common disorders are urgently needed. This includes the development of phytopharmaceutical products or functional foods to prevent or cure metabolic diseases. Plant extracts from edible biomaterial provide a potential resource of structurally diverse molecules that can synergistically interfere with complex disorders. In this study we describe the safe application of ethanolic chamomile (Matricaria recutita) flowers extract (CFE) for the treatment and prevention of type 2 diabetes and associated disorders. We show in vitro that this extract activates in particular nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) and its isotypes. In a cellular context, in human primary adipocytes CFE administration (300 µg/ml) led to specific expression of target genes of PPARγ, whereas in human hepatocytes CFE-induced we detected expression changes of genes that were regulated by PPARα. In vivo treatment of insulin-resistant high-fat diet (HFD)-fed C57BL/6 mice with CFE (200 mg/kg/d) for 6 weeks considerably reduced insulin resistance, glucose intolerance, plasma triacylglycerol, non-esterified fatty acids (NEFA) and LDL/VLDL cholesterol. Co-feeding of lean C57BL/6 mice a HFD with 200 mg/kg/d CFE for 20 weeks showed effective prevention of fatty liver formation and hepatic inflammation, indicating additionally hepatoprotective effects of the extract. Moreover, CFE treatment did not reveal side effects, which have otherwise been associated with strong synthetic PPAR-targeting molecules, such as weight gain, liver disorders, hemodilution or bone cell turnover. Taken together, modulation of PPARs and other factors by chamomile flowers extract has the potential to prevent or treat type 2 diabetes and related disorders

Foam Cell Specific LXRα Ligand

Objective The liver X receptor α (LXRα) is a ligand-dependent nuclear receptor and the major regulator of reverse cholesterol transport in macrophages. This makes it an interesting target for mechanistic study and treatment of atherosclerosis. Methods and Results We optimized a promising stilbenoid structure (STX4) in order to reach nanomolar effective concentrations in LXRα reporter-gene assays. STX4 displayed the unique property to activate LXRα effectively but not its subtype LXRβ. The potential of STX4 to increase transcriptional activity as an LXRα ligand was tested with gene expression analyses in THP1-derived human macrophages and oxLDL-loaded human foam cells. Only in foam cells but not in macrophage cells STX4 treatment showed athero- protective effects with similar potency as the synthetic LXR ligand T0901317 (T09). Surprisingly, combinatorial treatment with STX4 and T09 resulted in an additive effect on reporter-gene activation and target gene expression. In physiological tests the cellular content of total and esterified cholesterol was significantly reduced by STX4 without the undesirable increase in triglyceride levels as observed for T09. Conclusions STX4 is a new LXRα-ligand to study transcriptional regulation of anti-atherogenic processes in cell or ex vivo models, and provides a promising lead structure for pharmaceutical development

Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells

Background Monocytes and macrophages play essential role in innate immunity. Understanding the underlying mechanism of macrophage differentiation and the identification of regulatory mechanisms will help to find new strategies to prevent their harmful effects in chronic inflammatory diseases and sepsis. Results Maturation of blood monocytes into tissue macrophages and subsequent inflammatory response was mimicked in U937 cells of human histocytic lymphoma origin. Whole genome array analysis was employed to evaluate gene expression profile to identify underlying transcriptional networks implicated during the processes of differentiation and inflammation. In addition to already known transcription factors (i.e. MAFB, EGR, IRF, BCL6, NFkB, AP1, Nur77), gene expression analysis further revealed novel genes (i.e. MEF2, BRI, HLX, HDAC5, H2AV, TCF7L2, NFIL3) previously uncharacterized to be involved in the differentiation process. A total of 58 selected genes representing cytokines, chemokines, surface antigens, signaling molecules and transcription factors were validated by real time PCR and compared to primary monocyte-derived macrophages. Beside the verification of several new genes, the comparison reveals individual heterogeneity of blood donors. Conclusion Up regulation of MEF2 family, HDACs, and H2AV during cell differentiation and inflammation sheds new lights onto regulation events on transcriptional and epigenetic level controlling these processes. Data generated will serve as a source for further investigation of macrophages differentiation pathways and related biological responses