The Androgen Regulated lncRNA NAALADL2-AS2 Promotes Tumor Cell Survival in Prostate Cancer.

Castration resistance is the leading cause of death in men with prostate cancer. Recent studies indicate long noncoding RNAs (lncRNAs) to be important drivers of therapy resistance. The aim of this study was to identify differentially expressed lncRNAs in castration-resistant prostate cancer (CRPC) and to functionally characterize them in vitro. Tumor-derived RNA-sequencing data were used to quantify and compare the expression of 11,469 lncRNAs in benign, primary prostate cancer, and CRPC samples. CRPC-associated lncRNAs were selected for semi-quantitative PCR validation on 68 surgical tumor specimens. In vitro functional studies were performed by antisense-oligonucleotide-mediated lncRNA knockdown in hormone-sensitive prostate cancer (HSPC) and CRPC cell line models. Subsequently, cell proliferation, apoptosis, cell cycle, transcriptome and pathway analyses were performed using the appropriate assays. Transcriptome analysis of a prostate cancer tumor specimens unveiled NAALADL2-AS2 as a novel CRPC-upregulated lncRNA. The expression of NAALADL2-AS2 was found to be particularly high in HSPC in vitro models and to increase under androgen deprived conditions. NAALADL2-AS2 knockdown decreased cell viability and increased caspase activity and apoptotic cells. Cellular fractionization and RNA fluorescent in situ hybridization identified NAALADL2-AS2 as a nuclear transcript. Transcriptome and pathway analyses revealed that NAALADL2-AS2 modulates the expression of genes involved with cell cycle control and glycogen metabolism. We hypothesize that the nuclear lncRNA, NAALADL2-AS2, functions as a pro-survival signal in prostate cancer cells under pressure of targeted hormone therapy.

Implementation of initial emission mitigation measures for 1,4-dioxane in Germany: Are they taking effect?

Since our comprehensive investigation of finished drinking water in Germany obtained from managed aquifer recharge systems in the period 2015-2016, which revealed widespread contamination with 1,4-dioxane, mitigation measures (integration of AOP units, shutdown or alteration of production processes) have been implemented at some sites. In this study, we conducted follow-up tests on surface water concentrations and associated finished drinking water concentrations in 2017/2018, to evaluate the effectiveness of these measures. Our findings demonstrate that the emission mitigation measures had considerably reducing effects on the average 1,4-dioxane drinking water concentrations for some of the previously severely affected areas (Lower Franconia: -54%, Passau: -88%). Conversely, at notoriously contaminated sites where neither monitoring nor mitigation measures were introduced, the drinking water concentrations stagnated or even increased. Drinking water concentrations determined via a modified US EPA method 522 ranged from below LOQ (0.034 µg/L) up to 1.68 µg/L in all drinking water samples investigated. In river water samples, the maximum concentration exceeded 10 µg/L. Effluents of wastewater treatments plants containing 1,4-dioxane (5 µg/L-1.75 mg/L) were also analyzed for other similar cyclic ethers by suspected target screening. Thus, 1,3-dioxolane and three other derivatives were tentatively identified in effluents from the polyester processing or manufacturing industry. 1,3-Dioxolane was present in concentrations >1.2 mg/L at one site, exceeding up to sevenfold the 1,4-dioxane concentration found there. At another site 2-methyl-1,3-dioxolane was still found 13 km downstream of the discharge point, indicating that ethers analogous to 1,4-dioxane should be further considered regarding their occurrence and fate in wastewater treatment and the aquatic environment.

Consistency of the S5 DNA methylation classifier in formalin-fixed biopsies versus corresponding exfoliated cells for the detection of pre-cancerous cervical lesions.

Methylation biomarkers are promising tools for diagnosis and disease prevention. The S5 classifier is aimed at the prevention of cervical cancer by the early detection of cervical intraepithelial neoplasia (CIN). S5 is based on pyrosequencing a promoter region of EPB41L3 and five late regions of HPV types 16, 18, 31, and 33 following bisulfite conversion of DNA. Good biomarkers should perform well in a variety of sample types such as exfoliated cells, fresh frozen or formalin-fixed paraffin-embedded (FFPE) materials. Here, we tested the performance of S5 on 315 FFPE biopsies with paired exfoliated cervical samples using four different conversion kits (Epitect Bisulfite, Epitect Fast Bisulfite, EZ DNA Methylation, and EZ DNA Methylation-Lightning). The S5 values from FFPE biopsies for all kits were significantly correlated with those obtained from their paired exfoliated cells. For the EZ DNA Methylation kit, we observed an average increased methylation of 4.4% in FFPE. This was due to incomplete conversion of DNA (73% for FFPE vs. 95% for cells). The other kits had a DNA conversion rate in FFPE similar to the cells (95%-97%). S5 performed well at discriminating

1,4-Dioxane contamination of German drinking water obtained by managed aquifer recharge systems: Distribution and main influencing factors.

1,4-Dioxane, a cyclic ether that has been classified as a class 2B carcinogen by the US-EPA, is a substance of growing environmental concern because of its abundant occurrence in surface waters worldwide. Its high polarity and low biodegradability hamper its retardation in aquifer systems. Previous investigations in Germany have shown that 1,4-dioxane is already widely distributed in rivers and can be found in groundwater at contamination sites. Therefore, the present study shall provide an overview of the Germany-wide distribution of 1,4-dioxane in finished drinking water (FDW) obtained by managed aquifer recharge (MAR) systems. Thus, we investigated the 1,4-Dioxane levels in FDW obtained by MAR, such as river bank filtration (RBF) or artificial groundwater recharge (AGR), in regions that are supplied by surface water bodies (mainly rivers) with already known 1,4-dioxane contaminations. In total, 125 FDW samples and 33 samples of corresponding surface waters were analyzed for 1,4-dioxane content using solid phase extraction followed by gas chromatography-mass spectrometry (SIM-mode) using a slight modification to US-EPA method 522. About 80% of the investigated FDW samples contained 1,4-dioxane at levels exceeding the limit of quantification (0.034 µg/L); the maximum value was 2.05 µg/L. However, a maximum concentration of 3 µg/L was obtained in the surface water samples. Three main factors were associated with elevated levels of 1,4-dioxane in the FDW: A significant 1,4-dioxane contamination of the associated surface water, the application of RBF instead of AGR, and the proportion of available unpolluted groundwater and/or reservoir water blended in the individual waterworks. The results show that 1,4-dioxane should be critically monitored during FDW production by means of MAR not only in Germany. The findings are also of relevance to neighboring countries depending on the same river systems and for research in the field of small mobile substances in drinking water production in general.

Hydrocarbon Synthesis via Photoenzymatic Decarboxylation of Carboxylic Acids.

A recently discovered photodecarboxylase from Chlorella variabilis NC64A ( CvFAP) bears the promise for the efficient and selective synthesis of hydrocarbons from carboxylic acids. CvFAP, however, exhibits a clear preference for long-chain fatty acids thereby limiting its broad applicability. In this contribution, we demonstrate that the decoy molecule approach enables conversion of a broad range of carboxylic acids by filling up the vacant substrate access channel of the photodecarboxylase. These results not only demonstrate a practical application of a unique, photoactivated enzyme but also pave the way to selective production of short-chain alkanes from waste carboxylic acids under mild reaction conditions.

Molecular progression to cervical precancer, epigenetic switch or sequential model?

The evolution of precancerous cervical lesions is poorly understood. A widely held model of cervical intraepithelial neoplasia grade 3 (CIN3) development is sequential progression from normal through CIN1 and CIN2 to CIN3. Another hypothesis, the "molecular switch" model, postulates that CIN3 can evolve directly from human papillomavirus (HPV)-infected normal epithelium without progressing through CIN1 and CIN2. To shed light on this process, we compared DNA methylation of selected human biomarkers and HPV types in two groups of CIN1: CIN1 that were near or adjacent to CIN3 (adjacent-CIN1) and CIN1 that were the principal lesions with no CIN3 detected (principal-CIN1). 354 CIN (CIN1 and CIN3) and normal tissue areas were dissected and typed for HPV from 127 women who underwent loop electrosurgical excision procedures (LEEP). Methylation of genes EPB41L3 and the viral regions of HPV16-L1/L2, HPV18-L2, HPV31-L1, and HPV33-L2 were determined by a highly accurate quantitative pyrosequencing of bisulfite converted DNA. There was a significant trend of increased methylation with disease grade comparing normal to CIN1 and CIN3 (p < 0.0001). Adjacent-CIN1 predominantly shared the same HPV types as the CIN3, however, methylation differed substantially between adjacent-CIN1 and CIN3 (p = 0.008). In contrast diagnostically principal-CIN1 had an indistinguishable methylation distribution compared to adjacent-CIN1 (EPB41L3: p = 0.49; HPVme-All: p = 0.11). Our results suggest that progression from normal epithelium to CIN1 or CIN3 is usually promoted by the same HPV type but occurs via distinct DNA epigenotypes, thus favoring the "molecular switch" model.

Identification of long non-coding RNAs that stimulate cell survival in bladder cancer.

For many years, research on the biology underlying bladder cancer focused on protein-coding genes which cover only about 3% of the human genome. Recently, it was discovered that a large part of the human genome is actively transcribed as long non-coding RNAs (lncRNAs). LncRNAs are master regulators of gene expression and several lncRNAs were shown to play a role in bladder cancer development and progression. Here, we analyzed lncRNA expression in muscle-invasive bladder cancer (MIBC) using the MiTranscriptome database of cancer lncRNA expression profiles, and we studied their function in bladder cancer-derived tumor cells. Analysis of the MiTranscriptome lncRNA expression data revealed four MIBC subgroups, which partially overlapped with the four mRNA clusters identified by The Cancer Genome Atlas consortium. Up-regulation of three lncRNAs CAT266, CAT1297, and CAT1647 in bladder cancer, in comparison to normal urothelium, was confirmed in an independent series of normal, non-muscle invasive (NMIBC) and MIBC tissue samples. Furthermore, expression levels of CAT1297 were found to be correlated with disease-free and overall survival in MIBC. Knockdown of CAT266, CAT1297, and CAT1647 decreased cell viability and colony formation, due to the induction of apoptosis. In conclusion, our data show that lncRNAs expression is de-regulated in MIBC and three aberrantly expressed transcripts regulate proliferation and apoptosis. Our data indicate that lncRNAs play an important role in MIBC development and progression and are a treasure chest for the discovery of new biomarkers.