Serial stereotactic biopsy of brainstem lesions in adults improves diagnostic accuracy compared with MRI only.

Objective: The aim of the current prospective study was to analyse the validity of MRI based diagnosis of brainstem gliomas which was verified by stereotactic biopsy and follow-up evaluation as well as to assess prognostic factors and risk profile. Methods: Between 1998 and 2007, all consecutive adult patients with radiologically suspected brainstem glioma were included. The MRI based diagnosis of the lesions was made independently by an experienced neuroradiologist. Histopathological evaluation was performed in all patients from paraffin embedded specimens obtained by multimodal image guided stereotactic serial biopsy technique. Histopathological results were compared with prior radiological assessment. Length of survival was estimated with the Kaplan–Meier method and prognostic factors were calculated using the Cox model. Results: 46 adult patients were included. Histological evaluation revealed pilocytic astrocytoma (n=2), WHO grade II glioma (n=14), malignant glioma (n=12), metastasis (n=7), lymphoma (n=5), cavernoma (n=1), inflammatory disease (n=2) or no tumour/ gliosis (n=3). Perioperative morbidity was 2.5% (n=1). There was no permanent morbidity and no mortality. All patients with ‘‘no tumour’’ or ‘‘inflammatory disease’’ survived. Patients with low grade glioma and malignant glioma showed a 1 year survival rate of 75% and 25%, respectively; the 1 year survival rate for patients with lymphoma or metastasis was 30%. In the subgroup with a verified brainstem glioma, negative predictors for length of survival were higher tumour grade (p=0.002) and Karnofsky performance score (70 (p=0.004). Conclusion: Intra-axial brainstem lesions with a radiological pattern of glioma represent a very heterogeneous tumour group with completely different outcomes. Radiological features alone are not reliable for diagnostic classification. Stereotactic biopsy is a safe method to obtain a valid tissue diagnosis, which is indispensible for treatment decision

MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors

Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin

The carbon catabolite repressor protein 4 (Ccr4)–Negative on TATA (Not) complex controls gene expression at two levels. In the nucleus, it regulates the basal transcription machinery, nuclear receptor-mediated transcription and histone modifications. In the cytoplasm, the complex is required for messenger RNA (mRNA) turnover through its two associated deadenylases, Ccr4 and Caf1. Not1 is the largest protein of the Ccr4–Not complex and serves as a scaffold for other subunits of the complex. Here, we provide evidence that human Not1 in the cytoplasm associates with the C-terminal domain of tristetraprolin (TTP), an RNA binding protein that mediates rapid degradation of mRNAs containing AU-rich elements (AREs). Not1 shows extensive interaction through its central region with TTP, whereas binding of Caf1 is restricted to a smaller central domain within Not1. Importantly, Not1 is required for the rapid decay of ARE-mRNAs, and TTP can recruit the Caf1 deadenylase only in presence of Not1. Thus, cytoplasmic Not1 provides a platform that allows a specific RNA binding protein to recruit the Caf1 deadenylase and thereby trigger decay of its target mRNAs

Prognostic Value of Three Different Methods of MGMT Promoter Methylation Analysis in a Prospective Trial on Newly Diagnosed Glioblastoma

Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O6-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost effectiveness and ease of use, we recommend pyrosequencing for analyses of MGMT promoter methylation in high-throughput settings and MSP for clinical routine diagnostics with low sample numbers

Genome-Wide Assessment of AU-Rich Elements by the AREScore Algorithm

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila

Aircraft noise determination of novel wing configurations

During different investigations the aircraft slats were identified as one of the major sources for airframe noise. This suggests that design modifications of the wing especially the slats could reduce aircraft noise. For this purpose different wing model configurations from a standard to a slatless wing were designed. Within the German Aerospace Center (DLR) project LEISA these wing configurations will be investigated. The wind tunnel measured lift and drag coefficients can be integrated in the fast time flight simulation tool (NAPSim) developed by DLR. With this tool it is possible to simulate noise abatement flight procedures flown by generic airplanes. The tool has an interface to the DLR noise calculation software SIMUL to compute and compare the different noise impact produced by these configurations. Furthermore the simulation can calculate economical aspects like time and fuel consumption, which are used to evaluate the different configurations, because one constraint is to achieve nearly the same performance that results from a standard wing design. In this paper the calculation of flight paths and the flight mechanics of an airplane with different wings and thus resulting noise is described

Analysis of Tis11-sensitive mRNAs in <i>D. melanogaster</i> SL2 cells.

<p>(A) <i>D. melanogaster</i> SL2 cells were treated over a period of 4 days with 12.5 µg/ml dsRNA in order to knock down Tis11, or with dsRNA targeting GFP as a control. Total RNA was extracted from three biological repeats for microarray analysis using the Affymetrix Drosophila Genome 2.0 array. After normalization of the signal intensities using Robust Multi-array Analysis (RMA), the fold change of expression by Tis11 kd (signal in Tis11 kd/signal in GFP kd) was calculated. The list shows all 53 mRNAs with a log2-transformed fold change of >0.5, i.e. a fold change of >1.41. Statistical significance was determined by rank products (RP) test and independently by Student's T-test (TT). A heat map of the signal intensities in the three biological repeats is provided on the left side, the ARE<i>Score</i> is shown on the right side. (B) The ARE<i>Score</i> distribution is depicted for 49 out of the 53 Tis11-sensitive mRNAs identified in panel A. Only mRNAs with an annotated 3′UTR length ≥10 nt were included. The ARE<i>Score</i> distribution of the entire <i>D. melanogaster</i> transcriptome serves as the control group.</p

ARE<i>Score</i> distribution in comparison to randomized controls.

<p>(A) The ARE<i>Score</i> distribution of the <i>H. sapiens</i> transcriptome (every annotated transcript with a 3′UTR length ≥10 nt) was compared to a fully adjusted, randomized control set of sequences with identical lengths and A/T/G/C-content. Percentage of transcripts is depicted on a logarithmic scale. (B) The same analysis was done with the <i>D. melanogaster</i> transcriptome, as in panel A. Frequencies of 0 were omitted from the graph. (C) The same analysis was done with the <i>C. elegans</i> transcriptome, as in panel B. (D) The frequency of mRNAs with an ARE<i>Score</i> ≥10 in the actual transcriptome of 14 species was compared to the frequency in fully adjusted, randomized control sequences. The analysis was carried out for <i>Amphimedon queenslandica</i> (demosponge), <i>Hydra magnipapillata</i> (freshwater polyp), <i>Aplysia californica</i> (California sea hare), <i>Caenorhabditis elegans</i> (roundworm), <i>Ixodes scapularis</i> (deer tick), <i>Drosophila melanogaster</i> (fruit fly), <i>Strongylocentrotus purpuratus</i> (purple sea urchin), <i>Ciona intestinalis</i> (vase tunicate), <i>Branchiostoma floridae</i> (Florida lancelet), <i>Danio rerio</i> (zebrafish), <i>Xenopus laevis</i> (African clawed frog), <i>Gallus gallus</i> (chicken), <i>Mus musculus</i> (common house mouse) and <i>Homo sapiens</i> (man). The Φ coefficient serves as a measure for how strongly ARE<i>Scores</i> ≥10 are associated with the actual transcriptome as compared to the randomized control. P-values were calculated by χ²-test, n represents the number of transcripts. Species labeled in red show a significant enrichment of mRNAs with ARE<i>Scores</i> ≥10.</p

<i>Drosophila</i> mRNA half-lives in SL2 cells treated with GFP and Tis11 dsRNA.

<p><i>Drosophila</i> mRNA half-lives in SL2 cells treated with GFP and Tis11 dsRNA.</p

Relationship among ARE<i>Score</i>, 3′UTR length, and mRNA half-lives.

<p>(A) The half-lives of 26 Tis11-sensitive and control mRNAs, as measured by qPCR in SL2 cells subjected to control GFP dsRNA, were set in relation to their ARE<i>Score</i>. Half-lives above 240 minutes could not be determined accurately, and were presented without scale in the white area at the top of the graph. R_S, Spearman rank correlation coefficient. (B) The half-lives of the same mRNAs were compared to their 3′UTR length. Half-lives above 240 minutes could not be determined accurately, and were presented without scale in the white area at the top of the graph. (C) ROC analysis was applied to the 26 mRNAs, testing the ability of both ARE<i>Score</i> and 3′UTR length to discriminate mRNAs with a half-life <140 minutes from mRNAs with a half-life >240 minutes. AUC, area under the curve.</p