Turnover of PPP1R15A mRNA encoding GADD34 controls responsiveness and adaptation to cellular stress.

The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.

Transcriptomics-inferred dynamics of SARS-CoV-2 interactions with host epithelial cells.

Virus-host interactions can reveal potentially effective and selective therapeutic targets for treating infection. Here, we performed an integrated analysis of the dynamics of virus replication and the host cell transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using human Caco-2 colon cancer cells as a model. Time-resolved RNA sequencing revealed that, upon infection, cells immediately transcriptionally activated genes associated with inflammatory pathways that mediate the antiviral response, which was followed by an increase in the expression of genes involved in ribosome and mitochondria function, thus suggesting rapid alterations in protein production and cellular energy supply. At later stages, between 24 and 48 hours after infection, the expression of genes involved in metabolic processes-in particular, those related to xenobiotic metabolism-was decreased. Mathematical modeling incorporating SARS-CoV-2 replication suggested that SARS-CoV-2 proteins inhibited the host antiviral response and that virus transcripts exceeded the translation capacity of the host cells. Targeting kinase-dependent pathways that exhibited increases in transcription in host cells was as effective as a virus-targeted inhibitor at repressing viral replication. Our findings in this model system delineate a sequence of SARS-CoV-2 virus-host interactions that may facilitate the identification of druggable host pathways to suppress infection.

Cytokine release syndrome-like serum responses after COVID-19 vaccination are frequent and clinically inapparent under cancer immunotherapy.

Patients with cancer frequently receive immune-checkpoint inhibitors (ICIs), which may modulate immune responses to COVID-19 vaccines. Recently, cytokine release syndrome (CRS) was observed in a patient with cancer who received BTN162b2 vaccination under ICI treatment. Here, we analyzed adverse events and serum cytokines in patients with 23 different tumors undergoing (n = 64) or not undergoing (n = 26) COVID-19 vaccination under ICI therapy in a prospectively planned German single-center cohort study (n = 220). We did not observe clinically relevant CRS (≥grade 2) after vaccination (95% CI 0-5.6%; Common Terminology of Adverse Events v.5.0) in this small cohort. Within 4 weeks after vaccination, serious adverse events occurred in eight patients (12.5% 95% CI 5.6-23%): six patients were hospitalized due to events common under cancer therapy including immune related adverse events and two patients died due to conditions present before vaccination. Despite absence of CRS symptoms, a set of pairwise-correlated CRS-associated cytokines, including CXCL8 and interleukin-6 was >1.5-fold upregulated in 40% (95% CI 23.9-57.9%) of patients after vaccination. Hence, elevated cytokine levels are common and not sufficient to establish CRS diagnosis.

Temporal control of the integrated stress response by a stochastic molecular switch.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

Prospective multicentric validation of a novel prediction model for paroxysmal atrial fibrillation.

BACKGROUND: The early recognition of paroxysmal atrial fibrillation (pAF) is a major clinical challenge for preventing thromboembolic events. In this prospective and multicentric study we evaluated prediction scores for the presence of pAF, calculated from non-invasive medical history and echocardiographic parameters, in patients with unknown AF status. METHODS: The 12-parameter score with parameters age, LA diameter, aortic root diameter, LV,ESD, TDI A', heart frequency, sleep apnea, hyperlipidemia, type II diabetes, smoker, ß-blocker, catheter ablation, and the 4-parameter score with parameters age, LA diameter, aortic root diameter and TDI A' were tested. Presence of pAF was verified by continuous electrocardiogram (ECG) monitoring for up to 21 days in 305 patients. RESULTS: The 12-parameter score correctly predicted pAF in all 34 patients, in which pAF was newly detected by ECG monitoring. The 12- and 4-parameter scores showed sensitivities of 100% and 82% (95%-CI 65%, 93%), specificities of 75% (95%-CI 70%, 80%) and 67% (95%-CI 61%, 73%), and areas under the receiver operating characteristic (ROC) curves of 0.84 (95%-CI 0.80, 0.88) and 0.81 (95%-CI 0.74, 0.87). Furthermore, properties of AF episodes and durations of ECG monitoring necessary to detect pAF were analysed. CONCLUSIONS: The prediction scores adequately detected pAF using variables readily available during routine cardiac assessment and echocardiography. The model scores, denoted as ECHO-AF scores, represent simple, highly sensitive and non-invasive tools for detecting pAF that can be easily implemented in the clinical practice and might serve as screening test to initiate further diagnostic investigations for validating the presence of pAF. Prospective validation of a novel prediction model for paroxysmal atrial fibrillation based on echocardiography and medical history parameters by long-term Holter ECG.

Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

Principles of Cell Circuits for Tissue Repair and Fibrosis.

Tissue repair is a protective response after injury, but repetitive or prolonged injury can lead to fibrosis, a pathological state of excessive scarring. To pinpoint the dynamic mechanisms underlying fibrosis, it is important to understand the principles of the cell circuits that carry out tissue repair. In this study, we establish a cell-circuit framework for the myofibroblast-macrophage circuit in wound healing, including the accumulation of scar-forming extracellular matrix. We find that fibrosis results from multistability between three outcomes, which we term "hot fibrosis" characterized by many macrophages, "cold fibrosis" lacking macrophages, and normal wound healing. This framework clarifies several unexplained phenomena including the paradoxical effect of macrophage depletion, the limited time-window in which removing inflammation leads to healing, and why scar maturation takes months. We define key parameters that control the transition from healing to fibrosis, which may serve as potential targets for therapeutic reduction of fibrosis.

High-Density Cell Arrays for Genome-Scale Phenotypic Screening.

Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.

Unraveling mitotic protein networks by 3D multiplexed epitope drug screening.

Three-dimensional protein localization intricately determines the functional coordination of cellular processes. The complex spatial context of protein landscape has been assessed by multiplexed immunofluorescent staining or mass spectrometry, applied to 2D cell culture with limited physiological relevance or tissue sections. Here, we present 3D SPECS, an automated technology for 3D Spatial characterization of Protein Expression Changes by microscopic Screening. This workflow comprises iterative antibody staining, high-content 3D imaging, and machine learning for detection of mitoses. This is followed by mapping of spatial protein localization into a spherical, cellular coordinate system, a basis for model-based prediction of spatially resolved affinities of proteins. As a proof-of-concept, we mapped twelve epitopes in 3D-cultured spheroids and investigated the network effects of twelve mitotic cancer drugs. Our approach reveals novel insights into spindle fragility and chromatin stress, and predicts unknown interactions between proteins in specific mitotic pathways. 3D SPECS's ability to map potential drug targets by multiplexed immunofluorescence in 3D cell culture combined with our automated high-content assay will inspire future functional protein expression and drug assays.

Correlated receptor transport processes buffer single-cell heterogeneity.

Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

Stretch-activated two-pore-domain (K2P) potassium channels in the heart: Focus on atrial fibrillation and heart failure.

Two-pore-domain potassium (K2P) channels modulate cellular excitability. The significance of stretch-activated cardiac K2P channels (K2P2.1, TREK-1, KCNK2; K2P4.1, TRAAK, KCNK4; K2P10.1, TREK-2, KCNK10) in heart disease has not been elucidated in detail. The aim of this work was to assess expression and remodeling of mechanosensitive K2P channels in atrial fibrillation (AF) and heart failure (HF) patients in comparison to murine models. Cardiac K2P channel levels were quantified in atrial (A) and ventricular (V) tissue obtained from patients undergoing open heart surgery. In addition, control mice and mouse models of AF (cAMP-response element modulator (CREM)-IbΔC-X transgenic animals) or HF (cardiac dysfunction induced by transverse aortic constriction, TAC) were employed. Human and murine KCNK2 displayed highest mRNA abundance among mechanosensitive members of the K2P channel family (V > A). Disease-associated K2P2.1 remodeling was studied in detail. In patients with impaired left ventricular function, atrial KCNK2 (K2P2.1) mRNA and protein expression was significantly reduced. In AF subjects, downregulation of atrial and ventricular KCNK2 (K2P2.1) mRNA and protein levels was observed. AF-associated suppression of atrial Kcnk2 (K2P2.1) mRNA and protein was recapitulated in CREM-transgenic mice. Ventricular Kcnk2 expression was not significantly altered in mouse models of disease. In conclusion, mechanosensitive K2P2.1 and K2P10.1 K+ channels are expressed throughout the heart. HF- and AF-associated downregulation of KCNK2 (K2P2.1) mRNA and protein levels suggest a mechanistic contribution to cardiac arrhythmogenesis.

Inverse remodelling of K2P3.1 K+ channel expression and action potential duration in left ventricular dysfunction and atrial fibrillation: implications for patient-specific antiarrhythmic drug therapy.

AIMS: Atrial fibrillation (AF) prevalence increases with advanced stages of left ventricular (LV) dysfunction. Remote proarrhythmic effects of ventricular dysfunction on atrial electrophysiology remain incompletely understood. We hypothesized that repolarizing K2P3.1 K+ channels, previously implicated in AF pathophysiology, may contribute to shaping the atrial action potential (AP), forming a specific electrical substrate with LV dysfunction that might represent a target for personalized antiarrhythmic therapy. METHODS AND RESULTS: A total of 175 patients exhibiting different stages of LV dysfunction were included. Ion channel expression was quantified by real-time polymerase chain reaction and Western blot. Membrane currents and APs were recorded from atrial cardiomyocytes using the patch-clamp technique. Severely reduced LV function was associated with decreased atrial K2P3.1 expression in sinus rhythm patients. In contrast, chronic (c)AF resulted in increased K2P3.1 levels, but paroxysmal (p)AF was not linked to significant K2P3.1 remodelling. LV dysfunction-related suppression of K2P3.1 currents prolonged atrial AP duration (APD) compared with patients with preserved LV function. In individuals with concomitant LV dysfunction and cAF, APD was determined by LV dysfunction-associated prolongation and by cAF-dependent shortening, respectively, consistent with changes in K2P3.1 abundance. K2P3.1 inhibition attenuated APD shortening in cAF patients irrespective of LV function, whereas in pAF subjects with severely reduced LV function, K2P3.1 blockade resulted in disproportionately high APD prolongation. CONCLUSION: LV dysfunction is associated with reduction of atrial K2P3.1 channel expression, while cAF leads to increased K2P3.1 abundance. Differential remodelling of K2P3.1 and APD provides a basis for patient-tailored antiarrhythmic strategies.

A Simple, Non-Invasive Score to Predict Paroxysmal Atrial Fibrillation.

Paroxysmal atrial fibrillation (pAF) is a major risk factor for stroke but remains often unobserved. To predict the presence of pAF, we developed model scores based on echocardiographic and other clinical parameters from routine cardiac assessment. The scores can be easily implemented to clinical practice and might improve the early detection of pAF. In total, 47 echocardiographic and other clinical parameters were collected from 1000 patients with sinus rhythm (SR; n = 728), pAF (n = 161) and cAF (n = 111). We developed logistic models for classifying between pAF and SR that were reduced to the most predictive parameters. To facilitate clinical implementation, linear scores were derived. To study the pathophysiological progression to cAF, we analogously developed models for cAF prediction. For classification between pAF and SR, amongst 12 selected model parameters, the most predictive variables were tissue Doppler imaging velocity during atrial contraction (TDI, A'), left atrial diameter, age and aortic root diameter. Models for classifying between pAF and SR or between cAF and SR showed areas under the ROC curves of 0.80 or 0.93, which resembles classifiers with high discriminative power. The novel risk scores were suitable to predict the presence of pAF based on variables readily available from routine cardiac assessment. Modelling helped to quantitatively characterize the pathophysiologic transition from SR via pAF to cAF. Applying the scores may improve the early detection of pAF and might be used as decision aid for initiating preventive interventions to reduce AF-associated complications.

Separating fusion from rivalry.

Visual fusion is the process in which differing but compatible binocular information is transformed into a unified percept. Even though this is at the basis of binocular vision, the underlying neural processes are, as yet, poorly understood. In our study we therefore aimed to investigate neural correlates of visual fusion. To this end, we presented binocularly compatible, fusible (BF), and incompatible, rivaling (BR) stimuli, as well as an intermediate stimulus type containing both binocularly fusible and monocular, incompatible elements (BFR). Comparing BFR stimuli with BF and BR stimuli, respectively, we were able to disentangle brain responses associated with either visual fusion or rivalry. By means of functional magnetic resonance imaging, we measured brain responses to these stimulus classes in the visual cortex, and investigated them in detail at various retinal eccentricities. Compared with BF stimuli, the response to BFR stimuli was elevated in visual cortical areas V1 and V2, but not in V3 and V4 - implying that the response to monocular stimulus features decreased from V1 to V4. Compared to BR stimuli, the response to BFR stimuli decreased with increasing eccentricity, specifically within V3 and V4. Taken together, it seems that although the processing of exclusively monocular information decreases from V1 to V4, the processing of binocularly fused information increases from earlier to later visual areas. Our findings suggest the presence of an inhibitory neural mechanism which, depending on the presence of fusion, acts differently on the processing of monocular information.

Intra- and interdimeric caspase-8 self-cleavage controls strength and timing of CD95-induced apoptosis.

Apoptosis in response to the ligand CD95L (also known as Fas ligand) is initiated by caspase-8, which is activated by dimerization and self-cleavage at death-inducing signaling complexes (DISCs). Previous work indicated that the degree of substrate cleavage by caspase-8 determines whether a cell dies or survives in response to a death stimulus. To determine how a death ligand stimulus is effectively translated into caspase-8 activity, we assessed this activity over time in single cells with compartmentalized probes that are cleaved by caspase-8 and used multiscale modeling to simultaneously describe single-cell and population data with an ensemble of single-cell models. We derived and experimentally validated a minimal model in which cleavage of caspase-8 in the enzymatic domain occurs in an interdimeric manner through interaction between DISCs, whereas prodomain cleavage sites are cleaved in an intradimeric manner within DISCs. Modeling indicated that sustained membrane-bound caspase-8 activity is followed by transient cytosolic activity, which can be interpreted as a molecular timer mechanism reflected by a limited lifetime of active caspase-8. The activation of caspase-8 by combined intra- and interdimeric cleavage ensures weak signaling at low concentrations of CD95L and strongly accelerated activation at higher ligand concentrations, thereby contributing to precise control of apoptosis.