Summary To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T‐DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T‐DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T‐DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high‐quality template DNA accelerates the identification of T‐DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T‐DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T‐DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T‐DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M 2 family segregating a characterized gene mutation can be identified within 4 weeks.
Abstract Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)–catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5α-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5α-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.
Ziel/Aim Circulating tumor cells (CTCs) have emerged as predictive biomarker in PC therapies. In a cohort of patients with advanced metastastic castration-resistant PC (mCRPC), we aimed to elucidate the relationship between prostate-specific membrane antigen (PSMA) expression on CTCs, in metastases, and histological specimens, and its significance for the outcome of PSMA-targeted radioligand therapy (RLT).
Anhand von Komplementationsstudien der cpd-Mutante konnte die Funktionalitaet eines CPD-GFP-Fusionsproteins in planta demonstriert werden. Die Komplementation erfolgte unter Kontrolle des endogenen CPD-Promotors und unter Kontrolle des konstitutiven CaMV-35S-Promotors. Die transgenen Pflanzen wurden zur zellulaeren und gewebespezifischen Lokalisation des CPD-Proteins verwendet. Die Lokalisation des CPD-Proteins an der ER-Membran konnte sowohl ueber die Detektion der GFP-Fluoreszenz des generierten CPD-Fusionsproteins als auch mit einer Westen-Blot-Analyse eines differentiell zentrifugierten Zellextrakts bestaetigt werden. Konditionale Komplementationsstudien der cpd-Mutante verdeutlichen die wichtige Rolle von Brassinosteroiden bei der Pflanzenentwicklung. Durch konditionale, biochemische Komplementationen wurde demonstriert, das Brassinosteroide kontinuierlich waehrend der Pflanzenentwicklung benoetigt werden. Durch die konditionale genetische Komplementation der cpd-Mutante mit einem Oestradiol-induzierbaren Komplementationskonstrukt und der Verwendung eines unabhaengigen, induzierbaren GUS-Reporterkonstruktes war es moeglich einen Steroidtransport in planta nachzuweisen. Anhand des induziebaren GUS-Konstruktes wurde die inhomogene Oestradiol-Verteilung nach der lokal begrenzten Applikation mit Oestradiol-haltiger Lanolinpaste gezeigt. Verschiedene Studien belegen den Transport des Oestradiols. Die entsprechende lokale Komplementation der cpd-Mutante hat eine ganzheitliche Reaktion der Pflanze gezeigt, was einen Transport des Syntheseendprodukts Brassinolid oder eines nachgeschalteten Signals belegt. Die Analyse der CPD-Interaktionspartner ueber reverse Genetik hat zur Identifikation der iap2-1-Mutante gefuehrt, die eine T-DNA-Insertion in einem, fuer ein Ringfingerprotein-codierendes Gen traegt. Die Mutante weist einen vergleichbaren Phaenotyp wie CPD-Ueberexpressionslinien auf, die auf Kontrollmedium ein verstaerktes Wurzelwachstum entwickeln. Die iap2-1-Mutante zeigt auf Hochsteroidmedium eine hypersensitive Reaktion gegenueber Brassinolid, was auf einen veraenderten, endogenen Steroidgehalt oder eine modulierte Signaltransduktion hindeutet. Diese Ergebnisse stehen im Einklang mit der postulierten Funktion des IAP2-Ringfingerproteins, das als E3-Ubiquitin-Ligase an der Regulation der CPD-Proteinstabilitaet beteiligt sein kann. Es bleibt aber offen, ob IAP2 an der direkten Regulation des CPD-Proteins oder einer anderen Komponente der Brassinosteroidsignaltransduktion beteiligt ist. Die Analyse von T-DNA-Insertionsmutanten in fuenf OBP-Genen hat nicht zur Identifikation eines Steroidphaenotyps gefuehrt. Damit zeichnet sich eine redundante Funktionalitaet innerhalb der OBP-Proteinfamilie aus A. thaliana ab, wie sie z. B. bei den homologen OSH-Proteinen in Hefe beobachtet wurde ab. Analysen von Doppel- und Mehrfachmutanten der CPD-Interaktionspartner und insbesondere der obp-Mutanten werden ein genaueres Bild der IAP und OBP-Funktion geben.
The objective of this study is to evaluate the laser-tissue effects of laser radiation emitted by a newly developed high frequency pulsed Tm:YAG laser in comparison to the continuous wave Tm:YAG laser and the pulsed Ho:YAG laser.Ex-vivo experiments were performed on freshly slaughtered porcine kidneys in a physiological saline solution. Experiments were performed using two different laser devices in different settings: A Tm:YAG laser was operated in a pulsed mode up to 300 Hz and in a continuous wave (CW) mode. Results were compared with a 100 W standard pulsed Ho:YAG laser system. Comparative tissue experiments were performed at 5 W, 40 W and 80 W. The incision depth and the laser damage zone were measured under a microscope using a calibrated ocular scale.Increased laser power resulted in increased incision depth and increased laser damage zone for all investigated lasers in this set-up. The Ho:YAG created the largest combined tissue effect at the 5 W power setting and seems to be the least controllable laser at low power for soft tissue incisions. The CW Tm:YAG did not incise at all at 5 W, but created the largest laser damage zone. For the new pulsed Tm:YAG laser the tissue effect grew evenly with increasing power.Among the investigated laser systems in this setting the pulsed Tm:YAG laser shows the most controllable behavior, insofar as both the incision depth and the laser damage zone increase evenly with increasing laser power.
Collagenous spherulosis (CS) is a rare breast lesion of unknown histogenesis. Adenoid cystic carcinoma (ACC) is a rare basal-like breast carcinoma with low histological grade. CS is a benign lesion but resembles ACC. Both lesions show a similar histomorphology and feature bilineage differentiation. This study compared immunohistochemical markers in CS and ACC. We compiled n = 13 CS cases and n = 18 mammary ACCs. Fourteen marker proteins (ER, PR, HER2, GATA3, CK7, E-cadherin, CD117, CK5/14, p40, p63, SMA, CD10, calponin, P-cadherin) were evaluated by immunohistochemistry (IHC). MYB rearrangement, a common alteration in ACC, was assessed by fluorescence in situ hybridization. Patient age ranged between 40-60 years for CS lesions and 30-90 years for ACCs. 7/13 (54%) CS cases harbored a lobular carcinoma in situ (LCIS) in the luminal component. One CS/LCIS lesion occurred in a carrier of a pathogenic germline variant in CDH1/E-cadherin. MYB rearrangement was detected in 0/11 (0%) CS and 6/16 (37%) ACC cases (P = 0.054). CS was associated with expression of ER in the luminal component (P < 0.001), E-cadherin loss in the luminal component (P = 0.045), and expression of CD10 and calponin in the basal component (P < 0.001). Furthermore, CS was associated with GATA3 expression in the luminal component (12/13 [92%] versus 5/18 [27%], P < 0.001). In summary, IHC for GATA3 and E-cadherin may contribute to the differential diagnosis between CS and ACC, although these markers are not exclusively expressed in either lesion. Histologic evaluation has to take into account that CS is frequently colonized by LCIS, requiring thorough correlation of histomorphology and immunohistochemical features.
While a considerable number of tumor-specific hypermethylated loci have been identified in renal cell cancer (RCC), DNA methylation of loci showing successive increases in normal, tumoral, and metastatic tissues could point to genes with high relevance both for the process of tumor development and progression. Here, we report that DNA methylation of a locus in a genomic region corresponding to the 3'UTR of the transcription factor T-box brain 1 (TBR1) mRNA accumulates in normal renal tissues with age and possibly increased body mass index. Moreover, a further tissue-specific increase of methylation was observed for tumor and metastatic tissue samples.Biometric analyses of the TCGA KIRC methylation data revealed candidate loci for age-dependent and tumor-specific DNA methylation within the last exon and in a genomic region corresponding to the 3'UTR TBR1 mRNA. To evaluate whether methylation of TBR1 shows association with RCC carcinogenesis, we measured 15 tumor cell lines and 907 renal tissue samples including 355 normal tissues, 175 tissue pairs of normal tumor adjacent and corresponding tumor tissue as well 202 metastatic tissues samples of lung, bone, and brain metastases by the use of pyrosequencing. Statistical evaluation demonstrated age-dependent methylation in normal tissue (R = 0.72, p < 2 × 10-16), association with adiposity (P = 0.019) and tumor-specific hypermethylation (P = 6.1 × 10-19) for RCC tissues. Comparison of tumor and metastatic tissues revealed higher methylation in renal cancer metastases (P = 2.65 × 10-6).Our analyses provide statistical evidence of association between methylation of TBR1 and RCC development and disease progression.
Abstract The prognostication of individual disease trajectory and selection of optimal therapy in patients with localized, low-grade prostate cancer often presents significant difficulty. The phosphatase and tensin homolog on chromosome 10 (PTEN) has emerged as a potential novel biomarker in this clinical context, based on its demonstrated prognostic significance in multiple retrospective studies. Incorporation into standard clinical practice necessitates exceptional diagnostic accuracy, and PTEN’s binary readout—retention or loss—suggests its suitability as a biomarker. This multi-institutional ring trial aimed to validate the diagnostic precision of PTEN immunohistochemistry in localized, low- to intermediate-risk prostate cancer, across ten university pathology institutes in Germany. The trial incorporated 90 cases of patients diagnosed with acinar adenocarcinoma of the prostate of grade groups 1 ( n = 8, 8.9%) and 2 ( n = 82, 91.1%) post-radical prostatectomy. Remarkably, the interpretation of PTEN immunohistochemistry displayed substantial variation (12.5–51.2% PTEN loss rates) within an identical cohort of prostate cancer. Fluorescence in situ hybridization analysis demonstrated PTEN hemizygous deletions in 5.5% (5/90) of cases. All cases with hemizygous deletions presented a distinct loss of PTEN expression by immunohistochemistry and were unanimously identified as PTEN loss by all participants (sensitivity 100%). However, negative (loss) immunohistochemistry was relatively non-specific for an underlying genomic deletion. Improved inter-observer agreement was observed in a subsequent ring trial. Finally, we identify S473-pAKT immunohistochemistry as a useful marker in equivocal cases. In summary, this multi-institutional ring trial illustrates surprisingly heterogeneous outcomes in defining PTEN status by immunohistochemistry.
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