Search

SummaryDevelopment of functional neurons is a complex orchestration of several signaling pathways controlling cell proliferation, differentiation, and homeostasis1. However, details about the involved factors are not fully understood. The balance of antioxidants and vitamins is important for neuronal survival, synaptic plasticity, and early neuronal development; thus, we hypothesized that ferroptosis—a lipid peroxidation dependent cell death modality that is inhibited by antioxidanats2,3—needs to be suppressed to gain neurons. Our study shows that removal of antioxidants diminishes neuronal development and laminar organization of cortical organoids. Intriguingly, impaired neuronal development in conditions lacking antioxidants can be fully restored when ferroptosis is specifically inhibited by ferrostatin-1, or neuronal differentiation occurs in the presence of sufficient amounts of vitamin A. Mechanistically, vitamin A activates the heterodimeric nuclear receptor complex Retinoic Acid Receptor (RAR)/Retinoid X Receptor (RXR)4, which upregulates expression of the ferroptosis regulators GPX4, FSP1, GCH1, and ACSL3, amongst others. Therefore, our study reveals that above a certain threshold, vitamin A increases expression of essential cellular gatekeepers of lipid peroxidation. This study uncovers a critical process during early neuronal development, where suppression of ferroptosis by radical-trapping antioxidants or vitamin A is required to obtain maturing neurons and proper laminar organization in cortical organoids.

Ferroptosis is a regulated form of non-apoptotic cell death implicated in pathological settings. To be exploited clinically, ferroptosis requires reagents that unequivocally detect ferroptosis in human and animal tissues. Such tools may enable development of ferroptosis-based medicines for diverse diseases.

ObjectiveFerroptosis is a regulated cell death modality that occurs upon iron-dependent lipid peroxidation. The recent decade of research has uncovered many regulators driving ferroptosis as well as cellular gatekeepers preventing ferroptosis. Yet, many processes and networks remain to be elucidated.Methods and resultsIn this study, we performed a chemical screen using small molecules with known mode of action and identified two agonists (Turofexorate and Fexaramine) of the nuclear receptor Farnesoid X Receptor (FXR), also known as NR1H4, to suppress ferroptosis, but not apoptosis or necroptosis. Further, we demonstrate that in liver cells with high FXR protein levels, inhibition of FXR sensitizes cells to undergo ferroptotic cell death, while activation of FXR inhibits ferroptosis. Importantly, FXR also inhibits ferroptosis in ex vivo primary mouse hepatocytes. Activation of FXR by Turofexorate and Fexaramine significantly reduces lipid peroxidation. Mechanistically, overexpression of FXR or activation of FXR by bile acids upregulates the ferroptosis-inhibitory regulators FSP1, PPARα, GPX4, SCD1, and ACSL3 to reduce peroxidized lipids and to counteract ferroptosis.ConclusionIn this study, we demonstrate that activation of FXR inhibits ferroptotic cell death via upregulation of a number of ferroptosis-inhibitory proteins (FSP1, PPARα, GPX4, SCD1, and ACSL3) to reduce lipid peroxidation. Hence, modulating FXR activity may be beneficial to overcome ferroptosis-mediated degenerative diseases.

ABCA3 (ATP-binding cassette subfamily A member 3) is a lipid transporter expressed in alveolar type II cells and localized in the limiting membrane of lamellar bodies. It is crucial for pulmonary surfactant storage and homeostasis. Mutations in the ABCA3 gene are the most common genetic cause of respiratory distress syndrome in mature newborns and of interstitial lung disease in children. Apart from lung transplant, there is no cure available. To address the lack of causal therapeutic options for ABCA3 deficiency, a rapid and reliable approach is needed to investigate variant-specific molecular mechanisms and to identify pharmacologic modulators for monotherapies or combination therapies. To this end, we developed a phenotypic cell-based assay to autonomously identify ABCA3 wild-type-like or mutant-like cells by using machine learning algorithms aimed at identifying morphologic differences in wild-type and mutant cells. The assay was subsequently used to identify new drug candidates for ABCA3-specific molecular correction by using high-content screening of 1,280 Food and Drug Administration-approved small molecules. Cyclosporin A was identified as a potent corrector, specific for some but not all ABCA3 variants. Results were validated by using our previously established functional small-format assays. Hence, cyclosporin A may be selected for orphan drug evaluation in controlled repurposing trials in patients.

Small-molecule screening is a powerful approach to identify modulators of either specific biological targets or cellular pathways with phenotypic endpoints. A myriad of assay technologies are available to assess the activity of enzymes, monitor protein-protein interactions, measure transcription factor activity in reporter assays, or detect cellular features and activities using high-content imaging. A common challenge during small-molecule screening is, however, the presence of hit compounds generating assay interference, thereby producing false-positive hits. Thus, efforts are needed to uncover such interferences to prioritize high-quality hits for further analysis. This process encompasses (1) computational approaches to flag undesirable compounds, and (2) the use of experimental approaches like counter, orthogonal, and cellular fitness screens to identify and eliminate artifacts. In this brief guide, we provide an overview for first-time users, highlighting experimental screening strategies to prioritize high-quality bioactive hits from high-throughput screening/high-content screening (HTS/HCS) campaigns.

Biallelic variants in ABCA3, the gene encoding the lipid transporter ATP-binding cassette subfamily A member 3 (ABCA3) that is predominantly expressed in alveolar type II cells, may cause interstitial lung diseases in children (chILD) and adults. Currently, there is no proven therapy, but, frequently, hydroxychloroquine (HCQ) is used empirically. We hypothesized that the in vitro responsiveness to HCQ might correlate to patients’ clinical outcomes from receiving HCQ therapy. The clinical data of the subjects with chILD due to ABCA3 deficiency and treated with HCQ were retrieved from the literature and the Kids Lung Register data base. The in vitro experiments were conducted on wild type (WT) and 16 mutant ABCA3-HA-transfected A549 cells. The responses of the functional read out were assessed as the extent of deviation from the untreated WT. With HCQ treatment, 19 patients had improved or unchanged respiratory conditions, and 20 had respiratory deteriorations, 5 of whom transiently improved then deteriorated. The in vitro ABCA3 functional assays identified two variants with complete response, five with partial response, and nine with no response to HCQ. The variant-specific HCQ effects in vivo closely correlated to the in vitro data. An ABCA3+ vesicle volume above 60% of the WT volume was linked to responsiveness to HCQ; the HCQ treatment response was concentration dependent and differed for variants in vitro. We generated evidence for an ABCA3 variant-dependent impact of the HCQ in vitro. This may also apply for HCQ treatment in vivo, as supported by the retrospective and uncontrolled data from the treatment of chILD due to ABCA3 deficiency.

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity (“nuisance compounds”) are routinely encountered in cellular assays, including phenotypic and high-content screening assays. Much is known regarding compound-dependent assay interferences in cell-free assays. However, despite the essential role of cellular assays in chemical biology and drug discovery, there is considerably less known about nuisance compounds in more complex cell-based assays. In our view, a major obstacle to realizing the full potential of chemical biology will not just be difficult-to-drug targets or even the sheer number of targets, but rather nuisance compounds, due to their ability to waste significant resources and erode scientific trust. In this review, we summarize our collective academic, government, and industry experiences regarding cellular nuisance compounds. We describe assay design strategies to mitigate the impact of nuisance compounds and suggest best practices to efficiently address these compounds in complex biological settings. Nuisance compounds can waste significant resources by producing promising bioactivities that are attributable to undesirable mechanisms of action. Addressing nuisance compounds is particularly challenging in cellular assays. Dahlin et al. summarize academic, government, and industry experiences with assay design and hit triage to specifically address cellular nuisance compounds.

Trypanosomiases are neglected tropical diseases caused by Trypanosoma (sub)species. Available treatments are limited and have considerable adverse effects and questionable efficacy in the chronic stage of the disease, urgently calling for the identification of new targets and drug candidates. Recently, we have shown that impairment of glycosomal protein import by the inhibition of the PEX5-PEX14 protein-protein interaction (PPI) is lethal to Trypanosoma. Here, we report the development of a novel dibenzo[b,f][1,4]oxazepin-11(10H)-one scaffold for small molecule inhibitors of PEX5-PEX14 PPI. The initial hit was identified by a high throughput screening (HTS) of a library of compounds. A bioisosteric replacement approach allowed to replace the metabolically unstable sulphur atom from the initial dibenzo[b,f][1,4]thiazepin-11(10H)-one HTS hit with oxygen. A crystal structure of the hit compound bound to PEX14 surface facilitated the rational design of the compound series accessible by a straightforward chemistry for the initial structure-activity relationship (SAR) analysis. This guided the design of compounds with trypanocidal activity in cell-based assays providing a promising starting point for the development of new drug candidates to tackle trypanosomiases.

While target-based drug discovery strategies rely on the precise knowledge of the identity and function of the drug targets, phenotypic drug discovery (PDD) approaches allow the identification of novel drugs based on knowledge of a distinct phenotype. Image-based high-content screening (HCS) is a potent PDD strategy that characterizes small-molecule effects through the quantification of features that depict cellular changes among or within cell populations, thereby generating valuable data sets for subsequent data analysis. However, these data can be complex, making image analysis from large HCS campaigns challenging. Technological advances in image acquisition, processing, and analysis as well as machine-learning (ML) approaches for the analysis of multidimensional data sets have rendered HCS as a viable technology for small-molecule drug discovery. Here, we discuss HCS concepts, current workflows as well as opportunities and challenges of image-based phenotypic screening and data analysis.

Determining cell death mechanisms occurring in patient and animal tissues is a longstanding goal that requires suitable biomarkers and accurate quantification. However, effective methods remain elusive. To develop more powerful and unbiased analytic frameworks, we developed a machine learning approach for automated cell death classification. Image sets were collected of HT-1080 fibrosarcoma cells undergoing ferroptosis or apoptosis and stained with an anti-transferrin receptor 1 (TfR1) antibody, together with nuclear and F-actin staining. Features were extracted using high-content-analysis software, and a classifier was constructed by fitting a multinomial logistic lasso regression model to the data. The prediction accuracy of the classifier within three classes (control, ferroptosis, apoptosis) was 93%. Thus, TfR1 staining, combined with nuclear and F-actin staining, can reliably detect both apoptotic and ferroptotis cells when cell features are analyzed in an unbiased manner using machine learning, providing a method for unbiased analysis of modes of cell death.

The COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort and available vaccines, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identify acriflavine (ACF) as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLpro catalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models, in vivo in mice and ex vivo in human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks., Graphical abstract, Napolitano et al. discovered acriflavine (ACF), a clinically approved drug, as an effective inhibitor of SARS-CoV-2 papain-like protease (PLpro). ACF inhibits viral replication at nanomolar concentrations in vitro and ex vivo, as well as in vivo. These findings open a promising therapeutic approach against COVID-19 and other betacoronaviruses.

Trypanosomiases are life-threatening infections of humans and livestock, and novel effective therapeutic approaches are needed. Trypanosoma compartmentalize glycolysis into specialized organelles termed glycosomes. Most of the trypanosomal glycolytic enzymes harbor a peroxisomal targeting signal-1 (PTS1) which is recognized by the soluble receptor PEX5 to facilitate docking and translocation of the cargo into the glycosomal lumen. Given its pivotal role in the glycosomal protein import, the PEX5–PTS1 interaction represents a potential target to inhibit import of glycolytic enzymes and thus kill the parasite. We developed a fluorescence polarization (FP)-based assay for monitoring the PEX5–PTS1 interaction and performed a High Throughput Screening (HTS) campaign to identify small molecule inhibitors of the interaction. Six of the identified hits passed orthogonal selection criteria and were found to inhibit parasite growth in cell culture. Our results validate PEX5 as a target for small molecule inhibitors and provide scaffolds suitable for further pre-clinical development of novel trypanocidal compounds.

Description, Innovative high-content screening platform identified N-(2-butoxyphenyl)-3-(phenyl)acrylamides as novel antifibrotics., Fibrogenic processes instigate fatal chronic diseases leading to organ failure and death. Underlying biological processes involve induced massive deposition of extracellular matrix (ECM) by aberrant fibroblasts. We subjected diseased primary human lung fibroblasts to an advanced three-dimensional phenotypic high-content assay and screened a repurposing drug library of small molecules for inhibiting ECM deposition. Fibrotic Pattern Detection by Artificial Intelligence identified tranilast as an effective inhibitor. Structure-activity relationship studies confirmed N-(2-butoxyphenyl)-3-(phenyl)acrylamides (N23Ps) as a novel and highly potent compound class. N23Ps suppressed myofibroblast transdifferentiation, ECM deposition, cellular contractility, and altered cell shapes, thus advocating a unique mode of action. Mechanistically, transcriptomics identified SMURF2 as a potential therapeutic target network. Antifibrotic activity of N23Ps was verified by proteomics in a human ex vivo tissue fibrosis disease model, suppressing profibrotic markers SERPINE1 and CXCL8. Conclusively, N23Ps are a novel class of highly potent compounds inhibiting organ fibrosis in patients.

Background: Studies suggest that general anesthetics like isoflurane and sevoflurane may aggravate Alzheimer’s disease (AD) neuropathogenesis, e.g., increased amyloid-β (Aβ) protein aggregation resulting in synaptotoxicity and cognitive dysfunction. Other studies showed neuroprotective effects, e.g., with xenon. Objective: In the present study, we want to detail the interactions of inhalational anesthetics with Aβ-derived pathology. We hypothesize xenon-mediated beneficial mechanisms regarding Aβ oligomerization and Aβ-mediated neurotoxicity on processes related to cognition. Methods: Oligomerization of Aβ1–42 in the presence of anesthetics has been analyzed by means of TR-FRET and silver staining. For monitoring changes in neuronal plasticity due to anesthetics and Aβ1–42, Aβ1–40, pyroglutamate-modified amyloid-(AβpE3), and nitrated Aβ (3NTyrAβ), we quantified long-term potentiation (LTP) and spine density. We analyzed network activity in the hippocampus via voltage-sensitive dye imaging (VSDI) and cognitive performance and Aβ plaque burden in transgenic AD mice (ArcAβ) after anesthesia. Results: Whereas isoflurane and sevoflurane did not affect Aβ1–42 aggregation, xenon alleviated the propensity for aggregation and partially reversed AβpE3 induced synaptotoxic effects on LTP. Xenon and sevoflurane reversed Aβ1–42-induced spine density attenuation. In the presence of Aβ1–40 and AβpE3, anesthetic-induced depression of VSDI-monitored signaling recovered after xenon, but not isoflurane and sevoflurane removal. In slices pretreated with Aβ1–42 or 3NTyrAβ, activity did not recover after washout. Cognitive performance and plaque burden were unaffected after anesthetizing WT and ArcAβ mice. Conclusion: None of the anesthetics aggravated Aβ-derived AD pathology in vivo. However, Aβ and anesthetics affected neuronal activity in vitro, whereby xenon showed beneficial effects on Aβ1–42 aggregation, LTP, and spine density.

Background and purpose: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/β-catenin signaling is reduced in emphysema and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/β-catenin signaling and asses its potential to activate lung epithelial cells and repair. Experimental approach: We screened 1216 human-approved compounds for Wnt/β-catenin signaling activation using luciferase reporter cells, and selected candidates based on their computational predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell derived lung organoids and in vivo using a murine elastase-induced emphysema model. Key results: The primary screen identified 16 compounds that significantly induced Wnt/β-catenin-dependent luciferase activity. Selected compounds activated Wnt/β-catenin signaling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/β-catenin inhibition, confirming the Wnt β-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signaling in emphysema. Conclusion and implications: Using a drug screen based on Wnt/β-catenin activity, organoid assays, and a murine emphysema model, amlexanox was identified as a novel potential therapeutic for emphysema.

AlphaScreen is one of the most widely used assay technologies in drug discovery due to its versatility, dynamic range and sensitivity. However, a presence of false positives and frequent hitters contributes to difficulties with an interpretation of measured HTS data. Although filters do exist to identify frequent hitters for AlphaScreen, they are frequently based on privileged scaffolds. The development of such filters is time consuming and requires deep domain knowledge. Recently, machine learning and artificial intelligence methods are emerging as important tools to advance drug discovery and chemoinformatics, including their application to identification of frequent hitters in screening assays. However, the relative performance and complementarity of the Machine Learning and scaffold-based techniques has not yet been comprehensively compared. In this study, we analysed filters based on the privileged scaffolds with filters built using machine learning. Our results demonstrate that machine-learning methods provide more accurate filters for identification of frequent hitters in AlphaScreen assays than scaffold-based methods and can be easily redeveloped once new data are measured. We present highly accurate models to identify frequent hitters in AlphaScreen assays.

By accentuating drug efficacy and impeding resistance mechanisms, combinatorial, multi‐agent therapies have emerged as key approaches in the treatment of complex diseases, most notably cancer. Using high‐throughput drug screens, we uncovered distinct metabolic vulnerabilities and thereby identified drug combinations synergistically causing a starvation‐like lethal catabolic response in tumor cells from different cancer entities. Domperidone, a dopamine receptor antagonist, as well as several tricyclic antidepressants (TCAs), including imipramine, induced cancer cell death in combination with the mitochondrial uncoupler niclosamide ethanolamine (NEN) through activation of the integrated stress response pathway and the catabolic CLEAR network. Using transcriptome and metabolome analyses, we characterized a combinatorial response, mainly driven by the transcription factors CHOP and TFE3, which resulted in cell death through enhanced pyrimidine catabolism as well as reduced pyrimidine synthesis. Remarkably, the drug combinations sensitized human organoid cultures to the standard‐of‐care chemotherapy paclitaxel. Thus, our combinatorial approach could be clinically implemented into established treatment regimen, which would be further facilitated by the advantages of drug repurposing., This study identifies novel combinatorial drug treatments to induce death of different tumor cells, and defines the mechanisms of synergism between a mitochondrial uncoupler and antidepressants or dopamine receptor antagonists.

SummaryThe COVID-19 pandemic caused by SARS-CoV-2 has been socially and economically devastating. Despite an unprecedented research effort, effective therapeutics are still missing to limit severe disease and mortality. Using high-throughput screening, we identified acriflavine as a potent papain-like protease (PLpro) inhibitor. NMR titrations and a co-crystal structure confirm that acriflavine blocks the PLprocatalytic pocket in an unexpected binding mode. We show that the drug inhibits viral replication at nanomolar concentration in cellular models,in vivoin mice andex vivoin human airway epithelia, with broad range activity against SARS-CoV-2 and other betacoronaviruses. Considering that acriflavine is an inexpensive drug approved in some countries, it may be immediately tested in clinical trials and play an important role during the current pandemic and future outbreaks.

Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.

Protein-protein interactions (PPI) are involved in a myriad of cellular processes, and their deregulation can lead to many diseases. One such process is protein ubiquitination that requires an orchestrated action of three key enzymes to add ubiquitin moieties to substrate proteins. Importantly, this process is reversible through deubiquitinating enzymes. Both ubiquitination and deubiquitination require many PPIs that once classified can be utilized to identify small molecule inhibitors counteracting these reactions. Here, we study the protein-protein interaction between the two deubiquitinating enzymes OTUB1 and OTUD6B and report for the first time that both proteins directly interact with each other. We describe the GFP-Trap immunoprecipitation as a cell-based method to analyze the OTUD6B-OTUB1 interaction in the cellular context and the AlphaScreen (amplified luminescent proximity homogeneous assay) assay as a tool to detect direct interactions and to search for PPI inhibitors.

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program., High-throughput chemical screening identifies retinoic acid signaling as a regulatory pathway of 2-cell-like cell reprogramming and early mouse development.

Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. GBM has a poor prognosis, and currently, there are no curative options exist. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of the fundamental biology of GSCs is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit an elevated level of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components at the ciliary base is sufficient to induce ciliogenesis in a subset of GSCs. Importantly, restoring ciliogenesis caused GSCs to behave like healthy NPCs switching from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a crucial role for cilium as a molecular switch in determining GSCs’ fate and suggest that cilium induction is an attractive strategy to intervene in GSCs proliferation.

The biological function and disease association of human endogenous retroviral (HERV) elements is largely elusive. HERV-K(HML-2) has been associated with neurotoxicity but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation by using CRISPR engineering to activate or repress its expression levels in a human pluripotent stem cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell type-specific role for HERV-K(HML-2) in cortical neuron development.

Ferroptosis is a regulated form of cell death that occurs when phospholipids with polyunsaturated fatty acyl tails are oxidized in an iron-dependent manner. Research in recent years has uncovered complex cellular networks that induce and suppress lethal lipid peroxidation. This SnapShot provides an overview of ferroptosis-related pathways, including relevant biomolecules and small-molecule modulators regulating them.

The extracellular matrix is known to modulate cell adhesion and migration during tissue regeneration. However, the molecular mechanisms that fine-tune cells to extra-cellular matrix dynamics during regeneration of the peripheral nervous system remain poorly understood. Using the RSC96 Schwann cell line, we show that Sox2 directly controls fibronectin fibrillogenesis in Schwann cells in culture, to provide a highly oriented fibronectin matrix, which supports their organization and directional migration. We demonstrate that Sox2 regulates Schwann cell behaviour through the upregulation of multiple extracellular matrix and migration genes as well as the formation of focal adhesions during cell movement. We find that mouse primary sensory neurons and human induced pluripotent stem cell-derived motoneurons require the Sox2-dependent fibronectin matrix in order to migrate along the oriented Schwann cells. Direct loss of fibronectin in Schwann cells impairs their directional migration affecting the alignment of the axons in vitro. Furthermore, we show that Sox2 and fibronectin are co-expressed in proregenerative Schwann cells in vivo in a time-dependent manner during sciatic nerve regeneration. Taken together, our results provide new insights into the mechanisms by which Schwann cells regulate their own extracellular microenvironment in a Sox2-dependent manner to ensure the proper migration of neurons.

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are ...

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions., Surgical adhesions are organ-joining bands of scar tissue that remain clinically untreatable. Here, the authors show that adhesions are formed through expansive mesothelial membrane bridges, and that blocking these with small molecules prevents formation of adhesions in mice.