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We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads.

Summary: AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier.

Introduction Chronic postsurgical pain (CPSP) is defined as pain that persists after a surgical procedure and has a significant impact on quality of life. Previous studies show the importance of psychological factors in CPSP, yet the majority of studies focused solely on negative emotions. This longitudinal observational study aims to broaden this knowledge base by examining the role of emotional state, emotion variability, emotion regulation and emotion differentiation on the child and the parent level for the development CPSP, and to describe pain and emotion-related trajectories following surgery.Methods and analysis We intend to include 280 children and adolescents aged 8–18 years with a planned orthopaedic surgery and their parents. A total of five assessment time points is planned: 3 weeks before surgery (baseline), 2 weeks after surgery (post) and 3 months (follow-up (FU) 1), 6 months and 12 months after surgery. At baseline and post only, children and parents are asked to complete a daily diary thrice a day for a week where they rate their current emotional state and their pain severity (children only). Emotional state ratings will be used to calculate indices of emotion variability, emotion regulation and emotion differentiation. Children and parents will complete questionnaires at each time point, including measures on quality of life, social support, sleep, and symptoms of anxiety and depression.To predict development of CPSP, generalised linear regression models will be used, resulting in ORs and 95% CIs. Pearson product-moment correlations between predictors and outcomes will be evaluated at each time point. The primary outcome of the prediction model is CPSP at FU1. For the trajectory analysis, the classification method K-means for longitudinal data will be used to determine clusters in the data.Ethics and dissemination The Ethics Committee of the Canton of Zurich, Switzerland, has approved the study (ID: 2023-01475). Participants will be compensated, and a dissemination workshop will be held.Trial registration number NCT05816174.

Abstract The transcription factor complex, consisting of LMO2, TAL1 or LYL1, and GATA2, plays an important role in capillary sprouting by regulating VEGFR2, DLL4, and angiopoietin 2 in tip cells. Overexpression of the basic helix-loop-helix transcription factor LYL1 in transgenic mice results in shortened tails. This phenotype is associated with vessel hyperbranching and a relative paucity of straight vessels due to DLL4 downregulation in tip cells by forming aberrant complex consisting of LMO2 and LYL1. Knockdown of LMO2 or TAL1 inhibits capillary sprouting in spheroid-based angiogenesis assays, which is associated with decreased angiopoietin 2 secretion. In the same assay using mixed TAL1- and LYL1-expressing endothelial cells, TAL1 was found to be primarily located in tip cells, while LYL1-expressing cells tended to occupy the stalk position in sprouts by upregulating VEGFR1 than TAL1. Thus, the interaction between LMO2 and TAL1 in tip cells plays a key role in angiogenic switch of sprouting angiogenesis.

Abstract Background Spinal fusion surgery is associated with severe acute postsurgical pain and high rates of chronic postsurgical pain in adolescents. Psychological distress, sleep disturbance, and low pain self-efficacy predict higher acute pain and likelihood of developing chronic postsurgical pain. Interventions targeting baseline psychosocial risk factors have potential to interrupt a negative trajectory of continued pain and poor health-related quality of life (HRQL) over time but have not yet been developed and evaluated. This randomized controlled trial will test effectiveness of a digital peri-operative cognitive-behavioral intervention (SurgeryPalTM) vs. education-control delivered to adolescents and their parents to improve acute and chronic pain and health outcomes in adolescents undergoing spine surgery. Methods Adolescents 12–18 years of age undergoing spinal fusion for idiopathic conditions, and their parent, will be recruited from pediatric centers across the USA, for a target complete sample of 400 dyads. Adolescents will be randomized into 4 study arms using a factorial design to SurgeryPalTM or education control during 2 phases of treatment: (1) pre-operative phase (one-month before surgery) and (2) post-operative phase (1 month after surgery). Acute pain severity and interference (primary acute outcomes) and opioid use will be assessed daily for 14 days following hospital discharge. Chronic pain severity and interference (primary acute outcomes), as well as HRQL, parent and adolescent distress, sleep quality, and opioid use/misuse (secondary outcomes), will be assessed at 3 months and 6 months post-surgery. Discussion Demonstration of effectiveness and understanding optimal timing of perioperative intervention will enable implementation of this scalable psychosocial intervention into perioperative care. Ultimately, the goal is to improve pain outcomes and reduce reliance on opioids in adolescents after spine surgery. Trial registration NCT04637802 ClinicalTrials.gov. Registered on November 20, 2020

Abstract Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.

Abstract Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.

The application of antibodies in cells was first shown in the early 1990s, and subsequently, the field of intracellular antibodies has expanded to encompass antibody fragments and their use in target validation and as engineered molecules that can be fused to moieties (referred to as warheads) to replace the Fc effector region of a whole immunoglobulin to elicit intracellular responses, such as cell death pathways or protein degradation. These various forms of intracellular antibodies have largely been used as research tools to investigate function within cells by perturbing protein activity. New applications of such molecules are on the horizon, namely their use as drugs per se and as templates for small-molecule drug discovery. The former is a potential new pharmacology that could harness the power and flexibility of molecular biology to generate new classes of drugs (herein referred to as macrodrugs when used in the context of disease control). Delivery of engineered intracellular antibodies, and other antigen-binding macromolecules formats, into cells to produce a therapeutic effect could be applied to any therapeutic area where regulation, degradation or other kinds of manipulation of target proteins can produce a therapeutic effect. Further, employing single-domain antibody fragments as competitors in small-molecule screening has been shown to enable identification of drug hits from diverse chemical libraries. Compounds selected in this way can mimic the effects of the intracellular antibodies that have been used for target validation. The capability of intracellular antibodies to discriminate between closely related proteins lends a new dimension to drug screening and drug development.

Abstract Intracellular antibodies are valuable tools for target validation studies for clinical situations such as cancer. Recently we have shown that antibodies can be used for drug discovery in screening for chemical compounds surrogates by showing that compounds could be developed to the so-called undruggable RAS protein family. This method, called Antibody-derived compound (Abd) technology, employed intracellular antibodies binding to RAS in a competitive surface plasmon resonance chemical library screen. Success with this method requires a high affinity interaction between the antibody and the target. We now show that reduction in the affinity (dematuration) of the anti-active RAS antibody facilitates the screening of a chemical library using an in vitro AlphaScreen method. This identified active RAS specific-binding Abd compounds that inhibit the RAS-antibody interaction. One compound is shown to be a pan-RAS binder to KRAS, HRAS and NRAS-GTP proteins with a Kd of average 37 mM, offering the possibility of a new chemical series that interacts with RAS in the switch region where the intracellular antibody binds. This simple approach shows the druggability of RAS and is generally applicable to antibody-derived chemical library screening by affording flexibility through simple antibody affinity variation. This approach can be applied to find Abd compounds as surrogates of antibody-combining sites for novel drug development in a range of human diseases.

Background: Adolescents who undergo major surgery experience high rates of disabling acute and chronic postsurgical pain (CPSP). However, little is known about the subacute period when acute to chronic pain transition occurs. Aims: The aims of this study were to examine feasibility of electronic daily monitoring of pain and opioid use in adolescents during the first 30 days at home following major surgery and identify target features of subacute pain curves associated with CPSP at 4 months. Methods: Twenty-five youth (10–18 years) undergoing major musculoskeletal surgery reported daily pain severity, interference, and opioid use on the Brief Pain Inventory each evening for 30 days after hospital discharge to form short time series trajectories. At 4 months, youth reported on pain intensity and health-related quality of life. Characteristics of subacute pain severity and interference curves were compared by 4-month CPSP status. Results: At 4 months, 20.8% of youth met criteria for CPSP. During the 30-day monitoring period, youth who went on to develop CPSP reported high pain severity on 45.9% of days compared to 2.9% of days in youth who recovered (P = 0.005) and high pain interference on 49.4% of days vs. 9.7% in youth who recovered (P = 0.01). Pain variability and rate of change were not significantly associated with CPSP in our pilot sample. Conclusions: We found it feasible to collect daily pain data in youth recovering at home after major surgery. Pilot findings suggest that daily electronic monitoring may identify early recovery problems at home after surgery. Larger studies are needed to validate subacute pain trajectory features to identify risk for CPSP.

Targeted protein degradation is an attractive therapeutic strategy to deplete oncogenic proteins in tumours. Here the authors demonstrate the specific targeting of endogenous KRAS protein for degradation from cancer cells, and regression of tumours expressing mutant KRAS in a mouse model.

Targeting hard-to-drug proteins, such as proteins functioning by protein-protein interactions (PPIs) with small molecules, is difficult because of the lack of well-defined pockets. Fragment or computational-based methods are usually employed for the discovery of such compounds, but no generic method is available to quickly identify small molecules interfering with PPIs. Here, we provide a protocol describing a generic method to discover small molecules inhibiting the interaction between an intracellular antibody and its target, in particular for proteins that are hard to make in recombinant form. This protocol reports a versatile and generic method that can be applied to any target/intracellular antibody. Because it is a cell-based assay, it identifies chemical matters that are already displaying advantageous cell permeability properties.Graphic abstract:Cell-based intracellular antibody-guided small molecule screening.

Background Spinal fusion surgery is a common and painful musculoskeletal surgery performed in the adolescent population. Despite the known risk for developing chronic post-surgical pain, few perioperative psychosocial interventions have been evaluated in this population, and none have been delivered remotely (via the Internet) to improve accessibility. Aims The aim of this single-arm pilot study was to evaluate the feasibility and acceptability of the first Internet-based psychological intervention delivered during the perioperative period to adolescents undergoing major spinal fusion surgery and their parents. Methods : 13 adolescents (M age = 14.3; 69.2% female) scheduled for spine fusion surgery and their parents were provided access to the online psychosocial intervention program. The program included 6 lessons delivering cognitive-behavioral therapy skills targeting anxiety, sleep, and acute pain management using a model of pre- and post-operative skills training. Feasibility indicators included recruitment rate, intervention engagement, and measure completion. Acceptability was assessed via quantitative ratings and qualitative interviews. Results Our recruitment rate was 81.2% of families approached for screening. Among participating adolescent-parent dyads, high levels of engagement were demonstrated (100% completed all 6 lessons). All participants completed outcome measures. High treatment acceptability was demonstrated via survey ratings and qualitative feedback, with families highlighting numerous strengths of the program as well as areas for improvement. Conclusions These findings suggest that this online psychosocial intervention delivered during the perioperative period is feasible and acceptable to adolescents and their parents. Given favorable feasibility outcomes, an important next step is to evaluate the intervention in a full-scale randomized controlled trial.

Mutant RAS family members occur in a wide range of tumour types, and there is a great interest in identifying isoform-specific inhibitors. Here, the authors characterise two designed ankyrin repeat proteins (DARPins) that specifically inhibit the KRAS isoform by binding to the region around the KRAS-specific residue histidine 95 and show that they affect KRAS/effector interactions in different ways.

Background Assessing features of centralized pain may prove to be clinically meaningful in pediatric populations. However, we are currently limited by the lack of validated pediatric measures. Aim We examined the psychometric properties of the Widespread Pain Index (WPI) and Symptom Severity (SS) scale to assess features of centralized pain in youth with painful conditions from three clinical samples: (1) musculoskeletal surgery, (2) headache, and (3) chronic pain. Methods Participants were 240 youth aged 10 to 18 years (Mage = 14.8, SD = 1.9) who completed the WPI and SS scale. Subsets of participants also completed additional measures of pain region, pain intensity, quality of life, pain interference, and physical function. Results Increased features of centralized pain by age were seen for the WPI (r = 0.27, P

Metagenomics, the study of microbial genomes within diverse environments, is a rapidly developing field. The identification of microbial sequences within a host organism enables the study of human intestinal, respiratory, and skin microbiota, and has allow