The LIFE-Adult-Study is a population-based cohort study, which has recently completed the baseline examination of 10,000 randomly selected participants from Leipzig, a major city with 550,000 inhabitants in the east of Germany. It is the first study of this kind and size in an urban population in the eastern part of Germany. The study is conducted by the Leipzig Research Centre for Civilization Diseases (LIFE). Our objective is to investigate prevalences, early onset markers, genetic predispositions, and the role of lifestyle factors of major civilization diseases, with primary focus on metabolic and vascular diseases, heart function, cognitive impairment, brain function, depression, sleep disorders and vigilance dysregulation, retinal and optic nerve degeneration, and allergies. The study covers a main age range from 40-79 years with particular deep phenotyping in elderly participants above the age of 60. The baseline examination was conducted from August 2011 to November 2014. All participants underwent an extensive core assessment programme (5-6 h) including structured interviews, questionnaires, physical examinations, and biospecimen collection. Participants over 60 underwent two additional assessment programmes (3-4 h each) on two separate visits including deeper cognitive testing, brain magnetic resonance imaging, diagnostic interviews for depression, and electroencephalography. The participation rate was 33 %. The assessment programme was accepted well and completely passed by almost all participants. Biomarker analyses have already been performed in all participants. Genotype, transcriptome and metabolome analyses have been conducted in subgroups. The first follow-up examination will commence in 2016.

Stratification of head and neck squamous cell carcinomas (HNSCC) based on HPV16 DNA and RNA status, gene expression patterns, and mutated candidate genes may facilitate patient treatment decision. We characterize head and neck squamous cell carcinomas (HNSCC) with different HPV16 DNA and RNA (E6*I) status from 290 consecutively recruited patients by gene expression profiling and targeted sequencing of 50 genes. We show that tumors with transcriptionally inactive HPV16 (DNA+ RNA‐) are similar to HPV‐negative (DNA‐) tumors regarding gene expression and frequency ofTP53mutations (47%, 8/17 and 43%, 72/167, respectively). We also find that an immune response‐related gene expression cluster is associated with lymph node metastasis, independent of HPV16 status and that disruptiveTP53mutations are associated with lymph node metastasis in HPV16 DNA‐ tumors. We validate each of these associations in another large data set. Four gene expression clusters which we identify differ moderately but significantly in overall survival. Our findings underscore the importance of measuring the HPV16 RNA (E6*I) andTP53‐mutation status for patient stratification and identify associations of an immune response‐related gene expression cluster andTP53mutations with lymph node metastasis in HNSCC.

Genetics of gene expression (eQTLs or expression QTLs) has proved an indispensable tool for understanding biological pathways and pathomechanisms of trait-associated SNPs. However, power of most genome-wide eQTL studies is still limited. We performed a large eQTL study in peripheral blood mononuclear cells of 2112 individuals increasing the power to detect trans-effects genome-wide. Going beyond univariate SNP-transcript associations, we analyse relations of eQTLs to biological pathways, polygenetic effects of expression regulation, trans-clusters and enrichment of co-localized functional elements. We found eQTLs for about 85% of analysed genes, and 18% of genes were trans-regulated. Local eSNPs were enriched up to a distance of 5 Mb to the transcript challenging typically implemented ranges of cis-regulations. Pathway enrichment within regulated genes of GWAS-related eSNPs supported functional relevance of identified eQTLs. We demonstrate that nearest genes of GWAS-SNPs might frequently be misleading functional candidates. We identified novel trans-clusters of potential functional relevance for GWAS-SNPs of several phenotypes including obesity-related traits, HDL-cholesterol levels and haematological phenotypes. We used chromatin immunoprecipitation data for demonstrating biological effects. Yet, we show for strongly heritable transcripts that still little trans-chromosomal heritability is explained by all identified trans-eSNPs; however, our data suggest that most cis-heritability of these transcripts seems explained. Dissection of co-localized functional elements indicated a prominent role of SNPs in loci of pseudogenes and non-coding RNAs for the regulation of coding genes. In summary, our study substantially increases the catalogue of human eQTLs and improves our understanding of the complex genetic regulation of gene expression, pathways and disease-related processes.

Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding. The interactions of the helicase with the DNA were investigated using a series of forked DNAs. Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA. When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3′-tail and 35-nt 5′-tail was unwound with optimal rates close to 60 base pairs/s at 18 °C. When the helicase was not preassembled on the DNA, a >65-nt long 5′-tail was required for maximal unwinding rates of 12 base pairs/s. We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region. These studies have provided new insights into the mechanism of DNA unwinding. We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5′-strand and excluding the 3′-strand from the hole.

Purpose: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. Experimental Design: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. Results: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomibenhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP_L and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. Conclusion: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.