Peter Ahnert

Bortezomib Sensitizes Primary Human Astrocytoma Cells of WHO Grades I to IV for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

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 cFLIPL 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.

Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5'- and 3'-tails of the forked DNA substrate.

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.

A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein.

We have investigated the mechanism of binding single‐stranded DNA (ssDNA) into the central channel of the ring‐shaped T7 gp4A′ helicase–primase hexamer. Presteady‐state kinetic studies show a facilitated five‐step mechanism and provide understanding of how a ring‐shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA‐binding site is facilitated by transient binding to the primase DNA‐binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring‐opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein–DNA complex is kinetically stable, only the last species in the five‐step mechanism is poised to function as a helicase at the unwinding junction.

The role of gene DCDC2 in German dyslexics

Dyslexia is a complex reading and writing disorder with a strong genetic component. In a German case-control cohort, we studied the influence of the suspected dyslexia-associated gene DCDC2. For the first time in a German cohort, we describe association of a 2445 basepair deletion, first identified in an American study. Evidence of association for three DCDC2 single nucleotide polymorphisms (rs807724, rs793862, rs807701), previously identified in German or American cohorts, was replicated. A haplotype of these polymorphisms showed evidence for association as well. Thus, our data further corroborate association of DCDC2 with dyslexia. Analysis of functional subgroups suggests association of investigated DCDC2 variants mainly with nondysphonetic, nonsevere, but probably dyseidetic (surface) dyslexia. Based on the presumed function of DCDC2, our findings point to a role of impaired neuronal migration in the etiology of the disease.

The role of HPV RNA transcription, immune response-related gene expression and disruptive TP53 mutations in diagnostic and prognostic profiling of head and neck cancer

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 of TP53 mutations (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 disruptive TP53 mutations 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) and TP53‐mutation status for patient stratification and identify associations of an immune response‐related gene expression cluster and TP53 mutations with lymph node metastasis in HNSCC.

No evidence of major effects in several Toll-like receptor gene polymorphisms in rheumatoid arthritis

IntroductionThe objective was to study the potential genetic contribution of Toll-like receptor (TLR) genes in rheumatoid arthritis (RA). TLRs bind to pathogen-associated molecular patterns, and TLR genes influence both proinflammatory cytokine production and autoimmune responses. Host–pathogen interactions are involved in RA physiopathology.MethodsWe tested SNPs of five TLR genes (TLR9, TLR2, TLR6, TLR1, and TLR4) in a cohort of 100 French families with RA. Genotypes were analyzed using the transmission disequilibrium test. As TLR2, TLR6, and TLR1 are located on chromosome 4, we determined the haplotype relative risk. Analyses were performed in subgroups defined by status for rheumatoid factor, anti-cyclic citrullinated peptide autoantibodies, and erosions.ResultsWe found no disequilibrium in allele transmission for any of the SNPs of the five TLR genes. In subgroup analyses, no associations were detected linking TLR9, TLR2, or TLR9/TLR2 to rheumatoid factor, anti-cyclic citrullinated peptide autoantibodies, or erosions. Haplotype analysis of the polymorphisms showed no haplotype associations in any of the subgroups.ConclusionsWe found no evidence of major effects of TLR gene polymorphisms in RA, although we tested different TLR phenotypes. Moreover, no associations were noted with autoantibody production or erosions.

Dissecting the genetics of the human transcriptome identifies novel trait-related trans-eQTLs and corroborates the regulatory relevance of non-protein coding loci

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.

Imaging genetics of FOXP2 in dyslexia.

Dyslexia is a developmental disorder characterised by extensive difficulties in the acquisition of reading or spelling. Genetic influence is estimated at 50–70%. However, the link between genetic variants and phenotypic deficits is largely unknown. Our aim was to investigate a role of genetic variants of FOXP2, a prominent speech and language gene, in dyslexia using imaging genetics. This technique combines functional magnetic resonance imaging (fMRI) and genetics to investigate relevance of genetic variants on brain activation. To our knowledge, this represents the first usage of fMRI-based imaging genetics in dyslexia. In an initial case/control study (n=245) for prioritisation of FOXP2 polymorphisms for later use in imaging genetics, nine SNPs were selected. A non-synonymously coding mutation involved in verbal dyspraxia was also investigated. SNP rs12533005 showed nominally significant association with dyslexia (genotype GG odds ratio recessive model=2.1 (95% confidence interval 1.1–3.9), P=0.016). A correlated SNP was associated with altered expression of FOXP2 in vivo in human hippocampal tissue. Therefore, influence of the rs12533005-G risk variant on brain activity was studied. fMRI revealed a significant main effect for the factor ‘genetic risk’ in a temporo-parietal area involved in phonological processing as well as a significant interaction effect between the factors ‘disorder’ and ‘genetic risk’ in activation of inferior frontal brain areas. Hence, our data may hint at a role of FOXP2 genetic variants in dyslexia-specific brain activation and demonstrate use of imaging genetics in dyslexia research.

The Pore Size of PLGA Bone Implants Determines the De Novo Formation of Bone Tissue in Tibial Head Defects in Rats

The influence of the pore size of biodegradable poly(lactic‐co‐glycolic acid) scaffolds on bone regeneration was investigated.

Association of the X-Chromosomal Genes TIMP1 and IL9R with Rheumatoid Arthritis

Objective. Rheumatoid arthritis (RA) is an inflammatory joint disease with features of an autoimmune disease with female predominance. Candidate genes located on the X-chromosome were selected for a family trio-based association study. Methods. A total of 1452 individuals belonging to 3 different sample sets were genotyped for 16 single-nucleotide polymorphisms (SNP) in 7 genes. The first 2 sets consisted of 100 family trios, each of French Caucasian origin, and the third of 284 additional family trios of European Caucasian origin. Subgroups were analyzed according to sex of patient and presence of anti-cyclic citrullinated peptide (anti-CCP) autoantibodies. Results. Four SNP were associated with RA in the first sample set and were genotyped in the second set. In combined analysis of sets 1 and 2, evidence remained for association of 3 SNP in the genes UBA1, TIMP1, and IL9R. These were again genotyped in the third sample set. Two SNP were associated with RA in the joint analysis of all samples: rs6520278 (TIMP1) was associated with RA in general (p = 0.035) and rs3093457 (IL9R) with anti-CCP-positive RA patients (p = 0.037) and male RA patients (p = 0.010). A comparison of the results with data from whole-genome association studies further supports an association of RA with TIMPL The sex-specific association of rs3093457 (IL9R) was supported by the observation that men homozygous for rs3093457-CC are at a significantly higher risk to develop RA than women (risk ratio male/female = 2.98; p = 0.048). Conclusion. We provide evidence for an association of at least 2 X-chromosomal genes with RA: TIMP1 (rs6520278) and IL9R (rs3093457).