Tagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced... Show moreTagging of endogenous stress response genes can provide valuable in vitro models for chemical safety assessment. Here, we present the generation and application of a fluorescent human induced pluripotent stem cell (hiPSC) reporter line for Heme oxygenase-1 (HMOX1), which is considered a sensitive and reliable biomarker for the oxidative stress response. CRISPR/Cas9 technology was used to insert an enhanced green fluorescent protein (eGFP) at the C-terminal end of the endogenous HMOX1 gene. Individual clones were selected and extensively characterized to confirm precise editing and retained stem cell properties. Bardoxolone-methyl (CDDO-Me) induced oxidative stress caused similarly increased expression of both the wild-type and eGFP-tagged HMOX1 at the mRNA and protein level. Fluorescently tagged hiPSC-derived proximal tubule-like, hepatocyte-like, cardiomyocyte-like and neuron-like progenies were treated with CDDO-Me (5.62-1000 nM) or diethyl maleate (5.62-1000 µM) for 24 h and 72 h. Multi-lineage oxidative stress responses were assessed through transcriptomics analysis, and HMOX1-eGFP reporter expression was carefully monitored using live-cell confocal imaging. We found that eGFP intensity increased in a dose-dependent manner with dynamics varying amongst lineages and stressors. Point of departure modelling further captured the specific lineage sensitivities towards oxidative stress. We anticipate that the newly developed HMOX1 hiPSC reporter will become a valuable tool in understanding and quantifying critical target organ cell-specific oxidative stress responses induced by (newly developed) chemical entities. Show less
Krebs, A.; Vugt-Lussenburg, B.M.A. van; Waldmann, T.; Albrecht, W.; Boei, J.; Braak, B. ter; ... ; Leist, M. 2020
Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such... Show moreHazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity. Show less
Kumar, M.; Dijk, E.H.C. van; Raman, R.; Mehta, P.; Boon, C.J.F.; Goud, A.; ... ; Chhablani, J. 2020
Background To compare vision-related quality of life (VRQOL) between acute and chronic Central serous chorioretinopathy (CSC) and correlate this with Cohen's Perceived Stress Scale (PSS)... Show moreBackground To compare vision-related quality of life (VRQOL) between acute and chronic Central serous chorioretinopathy (CSC) and correlate this with Cohen's Perceived Stress Scale (PSS) questionnaire. Methods Patients who were diagnosed with both acute and chronic CSC were recruited in this study. Vision-related quality of life (VRQOL) was assessed with Rasch revised National Eye Institute Visual Functioning Questionnaire 25 (NEI-VFQ25) and perceived stress with Cohen's PSS questionnaire in 118 subjects with either acute or chronic CSC. The quality of life score was compared between patients with acute and chronic CSC. Correlations between the functional score and visual acuity (VA), stage of CSC, and stress were studied. Results There was no significant difference in VRQOL between Acute and Chronic CSC. In Acute CSC, affected eye VA correlated significantly with near vision question of the visual function subscale. Better eye VA correlated significantly with distance vision, social function, role limitation and dependency of the socioeconomic subscale. In chronic CSC, affected eye VA correlated with social function question of the socioemotional subscale and the better eye VA correlated with driving and distance vision of the visual function subscale. No other significant correlations with VA were noted. No correlations were observed between outcome of Cohen's PSS questionnaire and NEI-VFQ25 scores of acute and chronic CSC. Conclusion The VRQOL is similar between acute and chronic CSC. Perceived stress was not found to influence the VRQOL in CSC. Show less
Tewari, S.; Sabater Piqueres, C.; Kumar, M.; Stahl, S.; Crama, L.; Ruitenbeek, J.M. van 2017
We report on ALMA observations of continuum and molecular line emission with 0.''4 resolution towards the high-mass star-forming region G35.20-0.74 N. Two dense cores are detected in typical hot... Show moreWe report on ALMA observations of continuum and molecular line emission with 0.''4 resolution towards the high-mass star-forming region G35.20-0.74 N. Two dense cores are detected in typical hot-core tracers (e.g., CH$_{3}$CN) that reveal velocity gradients. In one of these cores, the velocity field can be fitted with an almost edge-on Keplerian disk rotating about a central mass of ~{}18 M$_{⊙}$. This finding is consistent with the results of a recent study of the CO first overtone bandhead emission at 2.3 {$μ$}m towards G35.20-0.74 N. The disk radius and mass are {gsim}2500 au and ~{}3 M$_{⊙}$. To reconcile the observed bolometric luminosity (~{}3 { imes} 10$^{4}$ L$_{⊙}$) with the estimated stellar mass of 18 M$_{⊙}$, we propose that the latter is the total mass of a binary system. Show less
Charge counting statistics (C.S.) of traversing electron in quantum devices like atomic-molecular junctions is sensitive to the local perturbation in the charge field at the contact and in the... Show moreCharge counting statistics (C.S.) of traversing electron in quantum devices like atomic-molecular junctions is sensitive to the local perturbation in the charge field at the contact and in the quantum channels. The first cumulant of C.S. i.e. current-voltage characteristic of such devices has been tool for such investigation since long time. Here we have used the second cumulant i.e. shot noise to study the electron-electron and electron-phonon interaction in the atomic contacts. The shot noise measurement on the Au atomic chain reveals the inelastic scattering in the noise. These signatures can provide vital information on the feedback of the local phonon population on electron transport. The current-voltage characteristic of the ferromagnetic atomic contacts unexpectedly shows zero bias anomalies. This observation is attributed to the interaction of traversing electron with localized magnetic moments within same host species. The observed connection between the Fano factor and the weight of the zero bias anomalies supports the view that the zero bias anomaly originates from spin scattering by localized magnetic moments. However, whether this is true Kondo scattering as suggested by Calvo et al. cannot be stated conclusively from our data. At the end of thesis we have presented a low noise high frequency broadband noise measurement setup suitable for break junction setup. Show less
Kumar, M.; Avriller, R.; Levy Yeyati, A.; Ruitenbeek, J.M. van 2012
