Bias in Intracellular Luminescence Thermometry: The Case of the Green Fluorescent Protein

Measurement of intracellular temperature in a fast, accurate, reliable, and remote manner is crucial for the understanding of cellular processes. Nanothermometers based on the green fluorescence protein (GFP) are of special interest because intracellular temperature readouts can be obtained from the analysis of the polarization state of its luminescence. Despite the good results provided by GFP thermometers, the reliability of their intracellular thermal readouts is still a question of debate. Here, light is shed on this issue by introducing cell activity as a relevant bias mechanism that prevents the use of GFP for reliable intranuclear thermal measurements. Experimental evidence that this lack of reliability can affect not only GFP but also other widely used thermometers such as semiconductor nanocrystals is provided. It is discussed how differences observed between calibration curves obtained in presence and absence of cell activity can inform about the presence of bias. The presented results and discussion are aimed to warn the community working in intracellular thermometry and encourage authors to approach the issue in a conscious manner. The performance and reliability of the chosen intracellular thermometers must be judiciously assessed. This is the only way intracellular thermometry can progress and deliver indisputable resultsThis work was financed by the Spanish Ministerio de Innovación y Ciencias under Project Nos. RTI2018-101050-J-I00, NANONERV PID2019- 106211RB-I00, and EIN2020-112419. Additional funding was provided by the European Union Horizon 2020 FETOpen project NanoTBTech (Grant No. 801305). P.R.-S. is grateful for a Juan de la Cierva – Incorporación scholarship (Grant No. IJC2019-041915-I). A.E. is grateful to Retos Projects Program of the Spanish Ministry of Science, Innovation, and Universities, the Spanish State Research Agency, co-funded by the European Regional Development Fund (A.E. is an EMBO Young Investigator). S.T. is grateful to AECC (Spanish Association Against Cancer) IDEAS21989THOM

Telomerase reverse transcriptase promoter mutations in bladder cancer: High frequency across stages, detection in urine, and lack of association with outcome

Background Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). D

Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across Stages, Detection in Urine, and Lack of Association with Outcome

Background: Hotspot mutations in the promoter of the gene coding for telomerase reverse transcriptase (TERT) have been described and proposed to activate gene expression. Objectives: To investigate TERT mutation frequency, spectrum, association with expression and clinical outcome, and potential for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). Design, setting, and participants: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription-quantitative polymerase chain reaction. The two most frequent mutations were investigated, using a SNaPshot assay, in an independent set of 184 non-muscle-invasive and 173 muscle-invasive UBC (median follow-up: 53 mo and 21 mo, respectively). Voided urine from patients with suspicion of incident UBC (n = 174), or under surveillance after diagnosis of non-muscle-invasive UBC (n = 194), was tested using a SNaPshot assay. Outcome measurements and statistical analysis: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease-specific survival, and overall survival. Results and limitations: In the two series, 78 of 111 (70%) and 283 of 357 (79%) tumors harbored TERT mutations, C228T being the most frequent substitution (83% for both series). TERT mutations were not associated with clinical or pathologic parameters, but were more frequent among FGFR3 mutant tumors (p = 0.0002). There was no association between TERT mutations and mRNA expression (p = 0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC patients. The sensitivity was 62% in incident and 42% in recurrent UBC. A limitation of the study is its retrospective nature. Conclusions: Somatic TERT promoter mutations are an early, highly prevalent genetic event in UBC and are not associated with TERT mRNA levels or disease outcomes. A SNaPshot assay in urine may help to detect UBC recurrences. (C) 2013 European Association of Urology. Published by Elsevier B. V. All rights reserved

Recurrent inactivation of STAG2 in bladder cancer is not associated with aneuploidy

Urothelial bladder cancer (UBC) is heterogeneous at the clinical, pathological and genetic levels. Tumor invasiveness (T) and grade (G) are the main factors associated with outcome and determine patient management(1). A discovery exome sequencing screen (n = 17), followed by a prevalence screen (n = 60), identified new genes mutated in this tumor coding for proteins involved in chromatin modification (MLL2, ASXL2 and BPTF), cell division (STAG2, SMC1A and SMC1B) and DNA repair (ATM, ERCC2 and FANCA). STAG2, a subunit of cohesin, was significantly and commonly mutated or lost in UBC, mainly in tumors of low stage or grade, and its loss was associated with improved outcome. Loss of expression was often observed in chromosomally stable tumors, and STAG2 knockdown in bladder cancer cells did not increase aneuploidy. STAG2 reintroduction in non-expressing cells led to reduced colony formation. Our findings indicate that STAG2 is a new UBC tumor suppressor acting through mechanisms that are different from its role in preventing aneuploidy

Engineering Erg10 Thiolase from <i>Saccharomyces cerevisiae</i> as a Synthetic Toolkit for the Production of Branched-Chain Alcohols

Thiolases catalyze the condensation of acyl-CoA thioesters through the Claisen condensation reaction. The best described enzymes usually yield linear condensation products. Using a combined computational/experimental approach, and guided by structural information, we have studied the potential of thiolases to synthesize branched compounds. We have identified a bulky residue located at the active site that blocks proper accommodation of substrates longer than acetyl-CoA. Amino acid replacements at such a position exert effects on the activity and product selectivity of the enzymes that are highly dependent on a protein scaffold. Among the set of five thiolases studied, Erg10 thiolase from <i>Saccharomyces cerevisiae</i> showed no acetyl-CoA/butyryl-CoA branched condensation activity, but variants at position F293 resulted the most active and selective biocatalysts for this reaction. This is the first time that a thiolase has been engineered to synthesize branched compounds. These novel enzymes enrich the toolbox of combinatorial (bio)­chemistry, paving the way for manufacturing a variety of α-substituted synthons. As a proof of concept, we have engineered <i>Clostridium</i>’s 1-butanol pathway to obtain 2-ethyl-1-butanol, an alcohol that is interesting as a branched model compound