SmartFlares fail to reflect their target transcripts levels

Abstract SmartFlare probes have recently emerged as a promising tool for visualisation and quantification of specific RNAs in living cells. They are supposed to overcome the common drawbacks of current methods for RNA analysis: the need of cell fixation or lysis, or the requirements for genetic manipulations. In contrast to the traditional methods, SmartFlare probes are also presumed to provide information on RNA levels in single cells. Disappointingly, the results of our comprehensive study involving probes specific to five different transcripts, HMOX1, IL6, PTGS2, Nrg1, and ERBB4, deny the usefulness of SmartFlare probes for RNA analysis. We report a total lack of correlation between fluorescence intensities of SmartFlare probes and the levels of corresponding RNAs assessed by RT-qPCR. To ensure strong differences in the levels of analysed RNAs, their expression was modified via: (i) HMOX1-knockdown generated by CRISPR-Cas9 genome editing, (ii) hemin-mediated stimulation of HMOX1- and IL1β-mediated stimulation of IL6- and PTGS2 transcription, (iii) lentiviral vector-mediated Nrg1 overexpression. Additionally, ERBB4-specific SmartFlare probe failed to distinguish between ERBB4-expressing and non-expressing cell lines. Finally, we demonstrated that fluorescence intensity of HMOX1-specific SmartFlare probe corresponds to the efficacy of its uptake and/or accumulation

Construction of a set of novel transposon vectors for efficient silencing of protein and lncRNA genes via CRISPR interference

In recent years, CRISPR interference (CRISPRi) technology of gene silencing has emerged as a promising alternative to RNA interference (RNAi) surpassing the latter in terms of efficiency and accuracy. Here, we describe the construction of a set of transposon vectors suitable for constitutive or tetracycline (doxycycline)-inducible silencing of genes of interest via CRISPRi method and conferring three different antibiotic resistances, using vectors available via Addgene repository. We have analyzed the performance of the new vectors in the silencing of mouse Adam10 and human lncRNA, NORAD. The empty vector variants can be used to efficiently silence any genes of interest

Pharmacological versus genetic inhibition of heme oxygenase-1 : the comparison of metalloporphyrins, shRNA and CRISPR/Cas9 system

Inhibition of heme oxygenase-1 (HO-1, encoded by HMOX1), a cytoprotective, anti-apoptotic and anti-inflammatory enzyme, may serve as a valuable therapy in various pathophysiological processes, including tumorigenesis. We compared the effect of chemical inhibitors - metalloporphyrins, with genetic tools - shRNA and CRISPR/Cas9 systems, to knock-down (KD)/knock-out (KO) HO-1 expression/activity. 293T cells were incubated with metalloporphyrins, tin and zinc protoporphyrins (SnPPIX and ZnPPIX, respectively) or were either transduced with lentiviral vectors encoding different shRNA sequences against HO-1 or were modified by CRISPR/Cas9 system targeting HMOX1. Metalloporphyrins decreased HO activity but concomitantly strongly induced HO-1 mRNA and protein in 293T cells. On the other hand, only slight basal HO-1 inhibition in shRNA KD 293T cell lines was confirmed on mRNA and protein level with no significant effect on enzyme activity. Nevertheless, silencing effect was much stronger when CRISPR/Cas9-mediated knock-out was performed. Most of the clones harboring mutations within HMOX1 locus did not express HO-1 protein and failed to increase bilirubin concentration after hemin stimulation. Furthermore, CRISPR/Cas9-mediated HO-1 depletion decreased 293T viability, growth, clonogenic potential and increased sensitivity to H2O2 treatment. In summary, we have shown that not all technologies can be used for inhibition of HO activity in vitro with the same efficiency. In our hands, the most potent and comprehensible results can be obtained using genetic tools, especially CRISPR/Cas9 approach

ADAM17 Silencing in Mouse Colon Carcinoma Cells: The Effect on Tumoricidal Cytokines and Angiogenesis

ADAM17 (a disintegrin and metalloprotease 17) is a major sheddase for numerous growth factors, cytokines, receptors, and cell adhesion molecules and is often overexpressed in malignant cells. It is generally accepted that ADAM17 promotes tumor development via activating growth factors from the EGF family, thus facilitating autocrine stimulation of tumor cell proliferation and migration. Here we show, using MC38CEA murine colon carcinoma model, that ADAM17 also regulates tumor angiogenesis and cytokine profile. When ADAM17 was silenced in MC38CEA cells, in vivo tumor growth and in vitro cell motility were significantly diminished, but no effect was seen on in vitro cell proliferation. ADAM17-silencing was accompanied by decreased in vitro expression of vascular endothelial growth factor-A and matrix metalloprotease-9, which was consistent with the limited angiogenesis and slower growth seen in ADAM17-silenced tumors. Among the growth factors susceptible to shedding by ADAM17, neuregulin-1 was the only candidate to mediate the effects of ADAM17 on MC38CEA motility and tumor angiogenesis. Concentrations of TNF and IFN gamma, cytokines that synergistically induced proapoptotic effects on MC38CEA cells, were significantly elevated in the lysates of ADAM17-silenced tumors compared to mock transfected controls, suggesting a possible role for ADAM17 in host immune suppression. These results introduce new, complex roles of ADAM17 in tumor progression, including its impact on the anti-tumor immune response

A global experiment on motivating social distancing during the COVID-19 pandemic

Finding communication strategies that effectively motivate social distancing continues to be a global public health priority during the COVID-19 pandemic. This cross-country, preregistered experiment (n = 25,718 from 89 countries) tested hypotheses concerning generalizable positive and negative outcomes of social distancing messages that promoted personal agency and reflective choices (i.e., an autonomy-supportive message) or were restrictive and shaming (i.e., a controlling message) compared with no message at all. Results partially supported experimental hypotheses in that the controlling message increased controlled motivation (a poorly internalized form of motivation relying on shame, guilt, and fear of social consequences) relative to no message. On the other hand, the autonomy-supportive message lowered feelings of defiance compared with the controlling message, but the controlling message did not differ from receiving no message at all. Unexpectedly, messages did not influence autonomous motivation (a highly internalized form of motivation relying on one’s core values) or behavioral intentions. Results supported hypothesized associations between people’s existing autonomous and controlled motivations and self-reported behavioral intentions to engage in social distancing. Controlled motivation was associated with more defiance and less long-term behavioral intention to engage in social distancing, whereas autonomous motivation was associated with less defiance and more short- and long-term intentions to social distance. Overall, this work highlights the potential harm of using shaming and pressuring language in public health communication, with implications for the current and future global health challenges

The psychological science accelerator’s COVID-19 rapid-response dataset

In response to the COVID-19 pandemic, the Psychological Science Accelerator coordinated three large-scale psychological studies to examine the effects of loss-gain framing, cognitive reappraisals, and autonomy framing manipulations on behavioral intentions and affective measures. The data collected (April to October 2020) included specific measures for each experimental study, a general questionnaire examining health prevention behaviors and COVID-19 experience, geographical and cultural context characterization, and demographic information for each participant. Each participant started the study with the same general questions and then was randomized to complete either one longer experiment or two shorter experiments. Data were provided by 73,223 participants with varying completion rates. Participants completed the survey from 111 geopolitical regions in 44 unique languages/dialects. The anonymized dataset described here is provided in both raw and processed formats to facilitate re-use and further analyses. The dataset offers secondary analytic opportunities to explore coping, framing, and self-determination across a diverse, global sample obtained at the onset of the COVID-19 pandemic, which can be merged with other time-sampled or geographic data

A multi-country test of brief reappraisal interventions on emotions during the COVID-19 pandemic.

The COVID-19 pandemic has increased negative emotions and decreased positive emotions globally. Left unchecked, these emotional changes might have a wide array of adverse impacts. To reduce negative emotions and increase positive emotions, we tested the effectiveness of reappraisal, an emotion-regulation strategy that modifies how one thinks about a situation. Participants from 87 countries and regions (n = 21,644) were randomly assigned to one of two brief reappraisal interventions (reconstrual or repurposing) or one of two control conditions (active or passive). Results revealed that both reappraisal interventions (vesus both control conditions) consistently reduced negative emotions and increased positive emotions across different measures. Reconstrual and repurposing interventions had similar effects. Importantly, planned exploratory analyses indicated that reappraisal interventions did not reduce intentions to practice preventive health behaviours. The findings demonstrate the viability of creating scalable, low-cost interventions for use around the world

The Psychological Science Accelerator’s COVID-19 rapid-response dataset

In response to the COVID-19 pandemic, the Psychological Science Accelerator coordinated three large-scale psychological studies to examine the effects of loss-gain framing, cognitive reappraisals, and autonomy framing manipulations on behavioral intentions and affective measures. The data collected (April to October 2020) included specific measures for each experimental study, a general questionnaire examining health prevention behaviors and COVID-19 experience, geographical and cultural context characterization, and demographic information for each participant. Each participant started the study with the same general questions and then was randomized to complete either one longer experiment or two shorter experiments. Data were provided by 73,223 participants with varying completion rates. Participants completed the survey from 111 geopolitical regions in 44 unique languages/dialects. The anonymized dataset described here is provided in both raw and processed formats to facilitate re-use and further analyses. The dataset offers secondary analytic opportunities to explore coping, framing, and self-determination across a diverse, global sample obtained at the onset of the COVID-19 pandemic, which can be merged with other time-sampled or geographic data