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Imaging-based methods are widely used for studying the subcellular localization of proteins in living cells. While routine for individual proteins, global monitoring of protein dynamics following perturbation typically relies on arrayed panels of fluorescently tagged cell lines, limiting throughput and scalability. Here, we describe a strategy that combines high-throughput microscopy, computer vision and machine learning to detect perturbation-induced changes in multicolour tagged visual proteomics cell (vpCell) pools. We use genome-wide and cancer-focused intron-targeting sgRNA libraries to generate vpCell pools and a large, arrayed collection of clones each expressing two different endogenously tagged fluorescent proteins. Individual clones can be identified in vpCell pools by image analysis using the localization patterns and expression level of the tagged proteins as visual barcodes, enabling simultaneous live-cell monitoring of large sets of proteins. To demonstrate broad applicability and scale, we test the effects of antiproliferative compounds on a pool with cancer-related proteins, on which we identify widespread protein localization changes and new inhibitors of the nuclear import/export machinery. The time-resolved characterization of changes in subcellular localization and abundance of proteins upon perturbation in a pooled format highlights the power of the vpCell approach for drug discovery and mechanism-of-action studies. Reicher, Reiniš et al. report a method for multicolour tagging using genome-scale intron-targeting sgRNA libraries that, in combination with computer vision, enables the systematic detection of protein localization changes.

SMNDC1 is a Tudor domain protein that recognizes di-methylated arginines and controls gene expression as an essential splicing factor. Here, we study the specific contributions of the SMNDC1 Tudor domain to protein-protein interactions, subcellular localization, and molecular function. To perturb the protein function in cells, we develop small molecule inhibitors targeting the dimethylarginine binding pocket of the SMNDC1 Tudor domain. We find that SMNDC1 localizes to phase-separated membraneless organelles that partially overlap with nuclear speckles. This condensation behavior is driven by the unstructured C-terminal region of SMNDC1, depends on RNA interaction and can be recapitulated in vitro. Inhibitors of the protein’s Tudor domain drastically alter protein-protein interactions and subcellular localization, causing splicing changes for SMNDC1-dependent genes. These compounds will enable further pharmacological studies on the role of SMNDC1 in the regulation of nuclear condensates, gene regulation and cell identity. SMNDC1 is a splicing factor that binds arginine methylation with its Tudor domain. Here, the authors study the protein’s phase-separating behavior and develop small-molecule Tudor domain inhibitors that perturb SMNDC1 function.

In this Tools of the Trade article, Reiniš and Reicher (Kubicek Lab) describe the development of a multicolour protein-tagging strategy that allows live-cell monitoring of multiple proteins.

Whether 2 nd -line-chemotherapy (2LCTX) + best-supportive-care (BSC) benefits patients with advanced biliary tract cancer (aBTC) more than BSC alone is unclear. We therefore conducted a propensity-score-based comparative effectiveness analysis of overall survival (OS) outcomes in 80 patients with metastatic, recurrent, or inoperable aBTC, of whom 38 (48%) were treated with BSC + 2LCTX and 42 (52%) with BSC alone. After a median follow-up of 14.8 months and 49 deaths, the crude 6-, 12-, and 18-month Kaplan-Meier OS estimates were 77%, 53% and 23% in the BSC + 2LCTX group, and 29%, 21%, and 14% in patients in the BSC group (p = 0.0003; Hazard ratio (HR) = 0.36, 95%CI:0.20–0.64, p = 0.001). An inverse-probability-of-treatment-weighted (IPTW) analysis was conducted to rigorously account for the higher prevalence of favorable prognostic variables in the 2LCTX + BSC group. After IPTW-weighting, the favorable association between 2LCTX and OS prevailed (adjusted HR = 0.40, 95%CI: 0.17–0.95, p = 0.037). IPTW-weighted 6-, 12-, and 18-month OS estimates were 77%, 58% and 33% in the BSC + 2LCTX group, and 39%, 28% and 22% in the BSC group (p = 0.037). Moreover, the benefit of 2LCTX was consistent across several clinically-relevant subgroups. Within the limitations of an observational study, these findings support the concept that 2LCTX + BSC is associated with an OS benefit over BSC alone in aBTC.

Fibroblast growth factor receptor 2 (FGFR2) gene fusions are bona fide oncogenic drivers in 10–15% of intrahepatic cholangiocarcinoma (CCA), yet currently there are no cell lines publically available to study endogenous FGFR2 gene fusions. The ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to generate large yet precise chromosomal rearrangements has presented the possibility of engineering endogenous gene fusions for downstream studies. In this technical report, we describe the generation of an endogenous FGFR2–Bicaudal family RNA binding protein 1 (BICC1) fusion in multiple independent cholangiocarcinoma and immortalized liver cell lines using CRISPR. BICC1 is the most common FGFR2 fusion partner in CCA, and the fusion arises as a consequence of a 58-megabase-sized inversion on chromosome 10. We replicated this inversion to generate a fusion product that is identical to that seen in many human CCA. Our results demonstrate the feasibility of generating large megabase-scale inversions that faithfully reproduce human cancer aberrations.

Peripheral leukocytes can exacerbate brain damage by release of cytotoxic mediators that disrupt blood-brain barrier (BBB) function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) formed via the myeloperoxidase (MPO)-H2O2-Cl(-) system. In the present study we examined the role of leukocyte activation, leukocyte-derived MPO and MPO-generated oxidants on BBB function in vitro and in vivo. In a mouse model of lipopolysaccharide (LPS)-induced systemic inflammation, neutrophils that had become adherent released MPO into the cerebrovasculature. In vivo, LPS-induced BBB dysfunction was significantly lower in MPO-deficient mice as compared to wild-type littermates. Both, fMLP-activated leukocytes and the MPO-H2O2-Cl(-) system inflicted barrier dysfunction of primary brain microvascular endothelial cells (BMVEC) that was partially rescued with the MPO inhibitor 4-aminobenzoic acid hydrazide. BMVEC treatment with the MPO-H2O2-Cl(-) system or activated neutrophils resulted in the formation of plasmalogen-derived chlorinated fatty aldehydes. 2-chlorohexadecanal (2-ClHDA) severely compromised BMVEC barrier function and induced morphological alterations in tight and adherens junctions. In situ perfusion of rat brain with 2-ClHDA increased BBB permeability in vivo. 2-ClHDA potently activated the MAPK cascade at physiological concentrations. An ERK1/2 and JNK antagonist (PD098059 and SP600125, respectively) protected against 2-ClHDA-induced barrier dysfunction in vitro. The current data provide evidence that interference with the MPO pathway could protect against BBB dysfunction under (neuro)inflammatory conditions.

ObjectivesTo investigate whether citizens’ adherence to health-protective non-pharmaceutical interventions (NPIs) during the COVID-19 pandemic is predicted by identity leadership, wherein leaders are perceived to create a sense of shared national identity.DesignObservational two-wave study. Hypotheses testing was conducted with structural equation modelling.SettingData collection during the COVID-19 pandemic in China, Germany, Israel and the USA in April/May 2020 and four weeks later.ParticipantsAdults in China (n=548, 66.6% women), Germany (n=182, 78% women), Israel (n=198, 51.0% women) and the USA (n=108, 58.3% women).MeasuresIdentity leadership (assessed by the four-item Identity Leadership Inventory Short-Form) at Time 1, perceived shared national identification (PSNI; assessed with four items) and adherence to health-protective NPIs (assessed with 10 items that describe different health-protective interventions; for example, wearing face masks) at Time 2.ResultsIdentity leadership was positively associated with PSNI (95% CI 0.11 to 0.30, p<0.001) in all countries. This, in turn, was related to more adherence to health-protective NPIs in all countries (95% CI 0.03 to 0.36, 0.001≤p≤0.017) except Israel (95% CI −0.03 to 0.27, p=0.119). In Germany, the more people saw Chancellor Merkel as engaging in identity leadership, the more they adhered to health-protective NPIs (95% CI 0.04 to 0.18, p=0.002). In the USA, in contrast, the more people perceived President Trump as engaging in identity leadership, the less they adhered to health-protective NPIs (95% CI −0.17 to −0.04, p=0.002).ConclusionsNational leaders can make a difference by promoting a sense of shared identity among their citizens because people are more inclined to follow health-protective NPIs to the extent that they feel part of a united ‘us’. However, the content of identity leadership (perceptions of what it means to be a nation’s citizen) is essential, because this can also encourage people to disregard such recommendations.

The second phase of the Fifth International Ice Nucleation Workshop (FIN-02) involved the gathering of a large number of researchers at the Karlsruhe Institute of Technology's Aerosol Interactions and Dynamics of the Atmosphere (AIDA) facility to promote characterization and understanding of ice nucleation measurements made by a variety of methods used worldwide. Compared to the previous workshop in 2007, participation was doubled, reflecting a vibrant research area. Experimental methods involved sampling of aerosol particles by direct processing ice nucleation measuring systems from the same volume of air in separate experiments using different ice nucleating particle (INP) types, and collections of aerosol particle samples onto filters or into liquid for sharing amongst measurement techniques that post-process these samples. In this manner, any errors introduced by differences in generation methods when samples are shared across laboratories were mitigated. Furthermore, as much as possible, aerosol particle size distribution was controlled so that the size limitations of different methods were minimized. The results presented here use data from the workshop to assess the comparability of immersion freezing measurement methods activating INPs in bulk suspensions, methods that activate INPs in condensation and/or immersion freezing modes as single particles on a substrate, continuous flow diffusion chambers (CFDCs) directly sampling and processing particles well above water saturation to maximize immersion and subsequent freezing of aerosol particles, and expansion cloud chamber simulations in which liquid cloud droplets were first activated on aerosol particles prior to freezing. The AIDA expansion chamber measurements are expected to be the closest representation to INP activation in atmospheric cloud parcels in these comparisons, due to exposing particles freely to adiabatic cooling. The different particle types used as INPs included the minerals illite NX and potassium feldspar (K-feldspar), two natural soil dusts representative of arable sandy loam (Argentina) and highly erodible sandy dryland (Tunisia) soils, respectively, and a bacterial INP (Snomax®). Considered together, the agreement among post-processed immersion freezing measurements of the numbers and fractions of particles active at different temperatures following bulk collection of particles into liquid was excellent, with possible temperature uncertainties inferred to be a key factor in determining INP uncertainties. Collection onto filters for rinsing versus directly into liquid in impingers made little difference. For methods that activated collected single particles on a substrate at a controlled humidity at or above water saturation, agreement with immersion freezing methods was good in most cases, but was biased low in a few others for reasons that have not been resolved, but could relate to water vapor competition effects. Amongst CFDC-style instruments, various factors requiring (variable) higher supersaturations to achieve equivalent immersion freezing activation dominate the uncertainty between these measurements, and for comparison with bulk immersion freezing methods. When operated above water saturation to include assessment of immersion freezing, CFDC measurements often measured at or above the upper bound of immersion freezing device measurements, but often underestimated INP concentration in comparison to an immersion freezing method that first activates all particles into liquid droplets prior to cooling (the PIMCA-PINC device, or Portable Immersion Mode Cooling chAmber–Portable Ice Nucleation Chamber), and typically slightly underestimated INP number concentrations in comparison to cloud parcel expansions in the AIDA chamber; this can be largely mitigated when it is possible to raise the relative humidity to sufficiently high values in the CFDCs, although this is not always possible operationally. Correspondence of measurements of INPs among direct sampling and post-processing systems varied depending on the INP type. Agreement was best for Snomax® particles in the temperature regime colder than −10 ∘C, where their ice nucleation activity is nearly maximized and changes very little with temperature. At temperatures warmer than −10 ∘C, Snomax® INP measurements (all via freezing of suspensions) demonstrated discrepancies consistent with previous reports of the instability of its protein aggregates that appear to make it less suitable as a calibration INP at these temperatures. For Argentinian soil dust particles, there was excellent agreement across all measurement methods; measures ranged within 1 order of magnitude for INP number concentrations, active fractions and calculated active site densities over a 25 to 30 ∘C range and 5 to 8 orders of corresponding magnitude change in number concentrations. This was also the case for all temperatures warmer than −25 ∘C in Tunisian dust experiments. In contrast, discrepancies in measurements of INP concentrations or active site densities that exceeded 2 orders of magnitude across a broad range of temperature measurements found at temperatures warmer than −25 ∘C in a previous study were replicated for illite NX. Discrepancies also exceeded 2 orders of magnitude at temperatures of −20 to −25 ∘C for potassium feldspar (K-feldspar), but these coincided with the range of temperatures at which INP concentrations increase rapidly at approximately an order of magnitude per 2 ∘C cooling for K-feldspar. These few discrepancies did not outweigh the overall positive outcomes of the workshop activity, nor the future utility of this data set or future similar efforts for resolving remaining measurement issues. Measurements of the same materials were repeatable over the time of the workshop and demonstrated strong consistency with prior studies, as reflected by agreement of data broadly with parameterizations of different specific or general (e.g., soil dust) aerosol types. The divergent measurements of the INP activity of illite NX by direct versus post-processing methods were not repeated for other particle types, and the Snomax® data demonstrated that, at least for a biological INP type, there is no expected measurement bias between bulk collection and direct immediately processed freezing methods to as warm as −10 ∘C. Since particle size ranges were limited for this workshop, it can be expected that for atmospheric populations of INPs, measurement discrepancies will appear due to the different capabilities of methods for sampling the full aerosol size distribution, or due to limitations on achieving sufficient water supersaturations to fully capture immersion freezing in direct processing instruments. Overall, this workshop presents an improved picture of present capabilities for measuring INPs than in past workshops, and provides direction toward addressing remaining measurement issues.

The purpose of this study was to investigate which social groups are perceived as a threat target and which are perceived as a threat source during the COVID-19 outbreak. In a German sample (N = 1454) we examined perceptions of social groups ranging from those that are psychologically close and smaller (family, friends, neighbors) to those that are more distal and larger (people living in Germany, humankind). We hypothesized that psychologically closer groups would be perceived as less affected by COVID-19 as well as less threatening than more psychologically distal groups. Based on social identity theorizing, we also hypothesized that stronger identification with humankind would change these patterns. Furthermore, we explored how these threat perceptions relate to adherence to COVID-19 health guidelines. In line with our hypotheses, latent random-slope modelling revealed that psychologically distal and larger groups were perceived as more affected by COVID-19 and as more threatening than psychologically closer and smaller groups. Including identification with humankind as a predictor into the threat target model resulted in a steeper increase in threat target perception patterns, whereas identification with humankind did not predict differences in threat source perceptions. Additiolly, an increase in threat source perceptions across social groups was associated with more adherence to health guidelines, whereas an increase in threat target perceptions was not. We fully replicated these findings in a subgroup from the origil sample (N = 989) four weeks later. We argue that societal recovery from this and other crises will be supported by an inclusive approach informed by a sense of our common identity as human beings.

Telemonitoring of patients with chronic heart failure (CHF) is an emerging concept to detect early warning signs of impending acute decompensation in order to prevent hospitalization. The goal of the MOBIle TELemonitoring in Heart Failure Patients Study (MOBITEL) was to evaluate the impact of home-based telemonitoring using Internet and mobile phone technology on the outcome of heart failure patients after an episode of acute decompensation. Patients were randomly allocated to pharmacological treatment (control group) or to pharmacological treatment with telemedical surveillance for 6 months (tele group). Patients randomized into the tele group were equipped with mobile phone-based patient terminals for data acquisition and data transmission to the monitoring center. Study physicians had continuous access to the data via a secure Web portal. If transmitted values went outside individually adjustable borders, study physicians were sent an email alert. Primary endpoint was hospitalization for worsening CHF or death from cardiovascular cause. The study was stopped after randomization of 120 patients (85 male, 35 female); median age was 66 years (IQR 62-72). The control group comprised 54 patients (39 male, 15 female) with a median age of 67 years (IQR 61-72), and the tele group included 54 patients (40 male, 14 female) with a median age of 65 years (IQR 62-72). There was no significant difference between groups with regard to baseline characteristics. Twelve tele group patients were unable to begin data transmission due to the inability of these patients to properly operate the mobile phone (\"never beginners\"). Four patients did not finish the study due to personal reasons. Intention-to-treat analysis at study end indicated that 18 control group patients (33%) reached the primary endpoint (1 death, 17 hospitalizations), compared with 11 tele group patients (17%, 0 deaths, 11 hospitalizations; relative risk reduction 50%, 95% CI 3-74%, P = .06). Per-protocol analysis revealed that 15% of tele group patients (0 deaths, 8 hospitalizations) reached the primary endpoint (relative risk reduction 54%, 95% CI 7-79%, P= .04). NYHA class improved by one class in tele group patients only (P< .001). Tele group patients who were hospitalized for worsening heart failure during the study had a significantly shorter length of stay (median 6.5 days, IQR 5.5-8.3) compared with control group patients (median 10.0 days, IQR 7.0-13.0; P= .04). The event rate of never beginners was not higher than the event rate of control group patients. Telemonitoring using mobile phones as patient terminals has the potential to reduce frequency and duration of heart failure hospitalizations. Providing elderly patients with an adequate user interface for daily data acquisition remains a challenging component of such a concept.