Biochemische und physiologische Untersuchung der Phytochelatinsynthasen von Arabidopsis thaliana

Nicht-essenzielle Schwermetalle wie Cd werden vorwiegend durch die Bildung von kleinen Cystein-reichen Peptiden, den Phytochelatinen (PC), in der Pflanze entgiftet. Die PC werden durch die Phytochelatinsynthasen (PCS) nicht-ribosomal in einer metallabhängigen Reaktion aus Glutathion (GSH) gebildet. Ihre generelle Struktur ist (γ-Glutamyl-Cystein)n-Glycin, wobei eine Anzahl n der γ-Glutamyl-Cystein-Einheit von 2 bis 11 Wiederholungen möglich ist. In verschiedenen Organismen konnte gezeigt werden, dass die PCS essenziell sind für die Detoxifizierung von nicht-essenziellen Schwermetallen wie Cd2+, Hg2+ und dem Metalloid As. Bis heute gibt es aber keinen direkten Nachweis, dass die PCS eine Funktion in der Metallhomöostase essenzieller Metalle haben. Aufgrund sehr viel sensitiveren Methoden konnte der Einfluss der PC-Bildung und der PC-Defizienz auf die Zn2+-Homöostase in Arabidopsis thaliana erneut untersucht werden. So wurde für die bekannte Cd2+-hypersensitive und PC-defiziente cad1-3-Mutante, welche eine Punktmutation im AtPCS1-Gen besitzt, eine erhöhte Zn2+-Sensitivität nachgewiesen. Nach Isolierung einer neuen Mutante, cad1-6, in der das AtPCS1-Gen durch eine T-DNA-Insertion in Exon 8 inaktiviert wurde, konnte die Cd2+- und Zn2+-Hypersensitivität der cad1-3-Mutante bestätigt werden. In Massenspektrometrischen Analysen (capLC-ESI-QTOF-MS) wurde nachgewiesen, dass Zn2+, wie auch Cd2+ als der stärkste Aktivator, die PC-Bildung in Wildtyp-Pflanzen (WT) Ecotyp Columbia induziert. Auch in Abwesenheit von erhöhten Schwermetallen im Pflanzenmedium wurde eine geringe PC-Bildung in WT Pflanzen gemessen. Die Mutanten cad1-3 und cad1-6 zeigten weiterhin eine signifikante reduzierte Cd2+- und Zn2+-Aufnahme in die Wurzeln als WT-Pflanzen. Diese Ergebnisse deuten darauf hin, dass die PC maßgeblich an der Akkumulation und Detoxifikation von Zn2+ beteiligt sind. Diese Funktion erklärt möglicherweise die evolutionäre Konservierung der PCS im Pflanzenreich und anderen Organismen. AtPCS2 ist das zweite funktionale PCS-Gen in Arabidopsis. AtPCS2 ist wie auch AtPCS1 konstitutiv exprimiert, jedoch deutlich schwächer. Eine Funktion von AtPCS2 in Arabidopsis ist bisher nicht bekannt. Durch Überexpression in der bekannten cad1-3-Mutante sollten Funktionen von AtPCS2 identifiziert werden. Ergebnisse zeigten, dass AtPCS2 auch in einer stark erhöhten Expression die Cd2+- und Zn2+-Hypersensitivität der cad1-3-Mutante nicht komplementieren kann. In capLC-ESI-QTOF-MS-Analysen konnte für AtPCS2 eine Aktivität nach Cd2+- und Zn2+-Behandlung in planta nachgewiesen werden. Die nachgewiesene PC-Bildung in transgenen cad1-3-Pflanzen, die AtPCS2 überexprimieren, ist aber nicht ausreichend für eine erhöhte Toleranz gegenüber diesen Schwermetallen. Eine spezifische Funktion für AtPCS2 ist weiterhin unbekannt. Weiterhin ist bekannt dass AtPCS2 in einer heterologen Expression in Hefen nach Cd2+-Behandlung Aktivität zeigt. Nach Cu2+-Behandlung konnte jedoch keine Aktivität nachgewiesen werden. In Sequenzvergleichen konnte gezeigt werden, dass bestimmt Aminosäuren in AtPCS2 verändert sind, die in anderen PCS zu 100 % konserviert sind. Durch Mutation von AtPCS1 und der Expression dieser Mutantenversionen in der Cd2+- und Cu2+-sensitiven Saccharomyces cerevisiae Δcup1-Mutante, wurde eine Aminosäure (Cys113) identifiziert, die für die Cu2+-Aktivierung essenziell ist. Diese Mutation in AtPCS1 kann die erhöhte Cu2+-Sensitivät der Δcup1-Mutante nicht mehr vollständig komplementieren, während die Cd2+-Aktivierung dieser Mutation unbeeinflusst war. Die Ergebnisse unterstützen das Modell einer direkten Metallbindung an bestimmte Sequenzbereiche des PCS-Proteins wodurch die Aktivierung der PC-Synthese erfolgt.Non essential heavy metals like Cd2+ are mainly detoxified in plants by the synthesis of small Cystein-rich peptides the phytochelatins (PC). PC are synthesized non-ribosomal from Glutathione (GSH) by the enzyme phytochelatin synthase in a metal dependent manner. Their general structure is (gγ-Glutamyl-Cysteine)n-Glycine, where n grows from 2 to 11. PCS are essential for detoxification of non-essential heavy metals like Cd2+, Pb2+ or the metalloid As in different organism. But to date, no direct evidence was reported for a role of PCS in the metal homeostasis of essential heavy metals. We were able to re-investigate the influence of PC synthesis and PC deficiency in Arabidopsis in zinc homeostasis because of more sensitive methods. We found that the kwon Cd2+ hypersensitive and PC deficient cad1-3 mutant, which had a point mutation in the AtPCS1 gene, show a pronounced Zn2+ hypersensitivity, The mutant phenotype of cad1-3 was confirmed in a new isolated mutant, cad1-6, with a T-DNA insertion in AtPCS1. In cap-LC-ESI-QTOF-MS analysis was shown that Zn2+ also like Cd2+ as a strong activator induce the PC synthesis in wild type (WT) plants. PC were also detectable in the absence of heavy metals in the growth medium in WT plants. It was also shown that cad1-3 and cad1-6 have a significant reduction in root Zn2+ accumulation. These results indicate an essential role of PC in the accumulation and detoxification of Zn2+. This function could explain the evolutionary conservation of PCS in the plant kingdom and also other organisms. AtPCS2 is the second functional PCS in Arabidopsis. AtPCS2 is like AtPCS1 constitutively expressed albeit at lower level. To date no function of AtPCS2 in Arabidopsis is known. To identify function of AtPCS2 the gene was over expressed in the known cad1-3-mutant. Results show that AtPCS2 is not able to complement the Cd2+ and Zn2+ hypersensitivity of cad1-3 by over expression. In CapLC-ESI-QTOF-MS analysis activity of AtPCS2 was measured in planta after treatment with Cd2+ and Zn2+. The measured PC accumulation in transgenic cad1-3 plants which over expressed AtPCS2 was not sufficient for an increased tolerance to these heavy metals. A specific function for AtPCS2 is further unknown. Furthermore is known that AtPCS2 shows activity in a heterologous expression in yeast after Cd2+ treatment. After Cu2+ treatment no activity was measurable. In Sequence alignments was shown that determined amino acid are changed in AtPCS2 which are 100 % conserved in other PCS. After mutation of these amino acids in AtPCS1 and expression of the mutant version in the Cd2+ and Cu2+ sensitive Saccharomyces cerevisiae Δcup1 mutant one amino acid, Cystein 113, was identified essential for Cu2+ activity. This mutation in AtPCS1 could not complement the Cu2+ hypersensitivity of Δcup1 while the Cd2+ activation was not influenced. The results support the model of direct metal binding to specific binding sites of the PCS protein to activate PC synthesis.von Pierre Tennsted

New strategy for the identification of prostate cancer: The combination of Proclarix and the prostate health index

Prostate health index (PHI) and, more recently, Proclarix have been proposed as serum biomarkers for prostate cancer (PCa). In this study, we aimed to evaluate Proclarix and PHI for predicting clinically significant prostate cancer (csPCa)

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

The Prognostic PDE4D7 Score in a Diagnostic Biopsy Prostate Cancer Patient Cohort with Longitudinal Biological Outcomes

Purpose. To further validate the prognostic power of the biomarker PDE4D7, we investigated the correlation of PDE4D7 scores adjusted for presurgical clinical variables with longitudinal postsurgical biological outcomes. Methods. RNA was extracted from biopsy punches of resected tumors (550 patients; RP cohort) and diagnostic needle biopsies (168 patients; DB cohort). Cox regression and survival were applied to correlate PDE4D7 scores with patient outcomes. Logistic regression was used to combine the clinical CAPRA score with PDE4D7. Results. In univariate analysis, the PDE4D7 score was significantly associated with PSA recurrence after prostatectomy in both studied patient cohorts' analysis (HR 0.53; 95% CI 0.41-0.67; p<1.0E-04 and HR 0.47; 95% CI 0.33-0.65; p<1.0E-04, respectively). After adjustment for the presurgical clinical variables preoperative PSA, PSA density, biopsy Gleason, clinical stage, percentage tumor in the biopsy (data only available for RP cohort), and percentage of positive biopsies, the HR was 0.49 (95% CI 0.38-0.64; p<1.0E-04) and 0.43 (95% CI 0.29-0.63; p<1.0E-04), respectively. The addition of the PDE4D7 to the clinical CAPRA score increased the AUC by 5% over the CAPRA score alone (0.82 versus 0.77; p=0.004). This combination model stratified 14.6% patients of the DB cohort to no risk of biochemical relapse (NPV 100%) over a follow-up period of up to 15 years. Conclusions. The PDE4D7 score provides independent risk information for pretreatment risk stratification. Combining CAPRA with PDE4D7 scores significantly improved the clinical risk stratification before surgery

Abdominal aortic aneurysm is associated with a variant in low-density lipoprotein receptor-related protein 1

Abdominal aortic aneurysm (AAA) is a common cause of morbidity and mortality and has a significant heritability. We carried out a genome-wide association discovery study of 1866 patients with AAA and 5435 controls and replication of promising signals (lead SNP with a p value &lt; 1 × 10-5) in 2871 additional cases and 32,687 controls and performed further follow-up in 1491 AAA and 11,060 controls. In the discovery study, nine loci demonstrated association with AAA (p &lt; 1 × 10-5). In the replication sample, the lead SNP at one of these loci, rs1466535, located within intron 1 of low-density-lipoprotein receptor-related protein 1 (LRP1) demonstrated significant association (p = 0.0042). We confirmed the association of rs1466535 and AAA in our follow-up study (p = 0.035). In a combined analysis (6228 AAA and 49182 controls), rs1466535 had a consistent effect size and direction in all sample sets (combined p = 4.52 × 10-10, odds ratio 1.15 [1.10-1.21]). No associations were seen for either rs1466535 or the 12q13.3 locus in independent association studies of coronary artery disease, blood pressure, diabetes, or hyperlipidaemia, suggesting that this locus is specific to AAA. Gene-expression studies demonstrated a trend toward increased LRP1 expression for the rs1466535 CC genotype in arterial tissues; there was a significant (p = 0.029) 1.19-fold (1.04-1.36) increase in LRP1 expression in CC homozygotes compared to TT homozygotes in aortic adventitia. Functional studies demonstrated that rs1466535 might alter a SREBP-1 binding site and influence enhancer activity at the locus. In conclusion, this study has identified a biologically plausible genetic variant associated specifically with AAA, and we suggest that this variant has a possible functional role in LRP1 expression