Mechanism Of Activation Research Articles

A novel dual-detection electrochemiluminescence sensor for the selective detection of Hg2⁺ and Zn2⁺: Signal suppression and activation mechanisms

In this study, we developed a novel covalent organic framework (COF) material, termed RuCOFs, specifically designed and synthesized for electrochemiluminescence (ECL) sensor applications. RuCOFs are based on the classic ECL emitter Ru(dcbpy)32+, ingeniously integrating 4,4′,4''-(1,3,5-triazine-2,4,6-triyl) triphenylamine (TAPT) with [2,2′-bipyridine]-5,5′-diamine (BPYDA), forming a structure with a high specific surface area. This configuration not only significantly enhances the stability of the ECL signal but also provides ideal N,N′-bipyridine chelating sites for efficient metal ion recognition. Utilizing Ru(dcbpy)32+-functionalized COF (RuCOFs), a novel dual-function ECL sensor was developed, achieving high sensitivity and selectivity in detecting mercury (Hg2⁺) and zinc (Zn2⁺) ions. Experimental results indicate that Hg2⁺ significantly quenches the ECL signal, while Zn2⁺ markedly enhances it, with detection limits of 4.71 nM for Hg2⁺ and 6.57 nM for Zn2⁺ across a wide linear response range from 1 μM to 1 nM. This research not only demonstrates the significant advantages of COF-based ECL sensing platforms in tracking environmental metal ions but also opens new possibilities for environmental monitoring.

Novel insight into mechanisms of ROS1 catalytic activation via loss of the extracellular domain.

The ROS1 receptor tyrosine kinase (RTK) possesses the largest extracellular amino-terminal domain (ECD) among the human RTK family, yet the mechanisms regulating its activation are not fully understood. While chimeric ROS1 fusion proteins, resulting from chromosomal rearrangements, are well-known oncogenic drivers, their activation mechanisms also remain underexplored. To elucidate the role of the ROS1 ECD in catalytic regulation, we engineered a series of amino-terminal deletion mutants. Our functional studies compared the full-length ROS1 receptor, the CD74-ROS1 oncogenic fusion, and ECD-deleted ROS1 constructs, identifying the ECD regions that inhibit ROS1 tyrosine kinase activity. Notably, we found that deletion of the ROS1 ECD alone significantly increases constitutive catalytic activation and neoplastic transformation in the absence of an amino-terminal fusion partner, challenging the presumed necessity for a dimerization domain in the activation mechanism of kinase fusions in cancer. Our data suggest that inter-genic deletions resulting in the loss of the ECD may be underappreciated oncogenic drivers in cancer. Furthermore, our studies demonstrate that RNASE7 is not a ligand for the ROS1 receptor as previously reported, confirming that ROS1 remains an orphan receptor. Thus, the discovery of a ROS1 ligand remains an important future priority. These findings highlight the potential for disease-associated somatic aberrations or splice variants that modify the ROS1 ECD to promote constitutive receptor activation, warranting further investigation.

Cryo-EM structure of small-molecule agonist bound delta opioid receptor-Gi complex enables discovery of biased compound

Delta opioid receptor (δOR) plays a pivotal role in modulating human sensation and emotion. It is an attractive target for drug discovery since, unlike Mu opioid receptor, it is associated with low risk of drug dependence. Despite its potential applications, the pharmacological properties of δOR, including the mechanisms of activation by small-molecule agonists and the complex signaling pathways it engages, as well as their relation to the potential side effects, remain poorly understood. In this study, we use cryo-electron microscopy (cryo-EM) to determine the structure of the δOR-Gi complex when bound to a small-molecule agonist (ADL5859). Moreover, we design a series of probes to examine the key receptor-ligand interaction site and identify a region involved in signaling bias. Using ADL06 as a chemical tool, we elucidate the relationship between the β-arrestin pathway of the δOR and its biological functions, such as analgesic tolerance and convulsion activities. Notably, we discover that the β-arrestin recruitment of δOR might be linked to reduced gastrointestinal motility. These insights enhance our understanding of δOR’s structure, signaling pathways, and biological functions, paving the way for the structure-based drug discovery.

Task decomposition strategy based on machine learning for boosting performance and identifying mechanisms in heterogeneous activation of peracetic acid process

Heterogeneous activation of peracetic acid (PAA) process is a promising method for removing organic pollutants from water. Nevertheless, this process is constrained by several complex factors, such as the selection of catalysts, optimization of reaction conditions, and identification of mechanism. In this study, a task decomposition strategy was adopted by combining a catalyst and reaction condition optimization machine learning (CRCO-ML) model and a mechanism identification machine learning (MI-ML) model to address these issues. The Categorical Boosting (CatBoost) model was identified as the best-performing model for the dataset (1024 sets and 7122 data points) in this study, achieving an R2 of 0.92 and an RMSE of 1.28. Catalyst composition, PAA dosage, and catalyst dosage were identified as the three most important features through SHAP analysis in the CRCO-ML model. The HCO3– is considered the most influential water matrix affecting the k value. The errors between all reverse experiment results and the predictions of the CRCO-ML and MI-ML models were <10 % and 15 %, respectively. This interdisciplinary work provides novel insights into the design and application of the heterogeneous activation of PAA process, significantly contributing to the rapid development of this technology.

Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia

AbstractOncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T-cell acute lymphoblastic leukemia. Loss of this CTCF-bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (−644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that “promoter tethering” of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

Tuning RXR Modulators for PGC1α Recruitment.

The molecular activation mechanism of the nuclear retinoid X receptors (RXRs) crucially involves ligand-induced corepressor release and coactivator recruitment which mediate transcriptional repression or activation. The ability of RXR to bind diverse coactivators suggests that a coregulator-selective modulation by ligands may open an avenue to tissue- or gene-selective RXR activation. Here, we identified strong induction of peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) binding to RXR by a synthetic agonist but not by the endogenous ligand 9-cis retinoic acid. Structure-guided diversification of this lead resulted in a set of three structurally related RXR agonists with different ability to promote PGC1α recruitment in cell-free and cellular context. These results demonstrate that selective modulation of coregulator recruitment to RXR can be achieved with molecular glues and potentially open new therapeutic opportunities by targeting the ligand-induced RXR-PGC1α interaction.

Enhancing T-cell recruitment in renal cell carcinoma with cytokine-armed adenoviruses

ABSTRACT Immunotherapy has emerged as a promising approach for cancer treatment, with oncolytic adenoviruses showing power as immunotherapeutic agents. In this study, we investigated the immunotherapeutic potential of an adenovirus construct expressing CXCL9, CXCL10, or IL-15 in clear cell renal cell carcinoma (ccRCC) tumor models. Our results demonstrated robust cytokine secretion upon viral treatment, suggesting effective transgene expression. Subsequent analysis using resistance-based transwell migration and microfluidic chip assays demonstrated increased T-cell migration in response to chemokine secretion by infected cells in both 2D and 3D cell models. Flow cytometry analysis revealed CXCR3 receptor expression across T-cell subsets, with the highest percentage found on CD8+ T-cells, underscoring their key role in immune cell migration. Alongside T-cells, we also detected NK-cells in the tumors of immunocompromised mice treated with cytokine-encoding adenoviruses. Furthermore, we identified potential immunogenic antigens that may enhance the efficacy and specificity of our armed oncolytic adenoviruses in ccRCC. Overall, our findings using ccRCC cell line, in vivo humanized mice, physiologically relevant PDCs in 2D and patient-derived organoids (PDOs) in 3D suggest that chemokine-armed adenoviruses hold promise for enhancing T-cell migration and improving immunotherapy outcomes in ccRCC. Our study contributes to the development of more effective ccRCC treatment strategies by elucidating immune cell infiltration and activation mechanisms within the tumor microenvironment (TME) and highlights the usefulness of PDOs for predicting clinical relevance and validating novel immunotherapeutic approaches. Overall, our research offers insights into the rational design and optimization of viral-based immunotherapies for ccRCC.

Copper-Catalyzed Enantioselective [4π + 2σ] Cycloaddition of Bicyclobutanes with Nitrones.

The selective construction of bridged bicyclic scaffolds has garnered increasing attention because of their extensive use as saturated bioisosteres of arene in pharmaceutical industry. However, in sharp contrast to their racemic counterparts, assembling chiral bridged bicyclic structures in an enantioselective and regioselective manner remains challenging. Herein, we describe our protocol for constructing chiral 2-oxa-3-azabicyclo[3.1.1]heptanes (BCHeps) by enantioselective [4π + 2σ] cycloadditions of bicyclo[1.1.0]butanes (BCBs) and nitrones taking advantage of a chiral copper(II) complex as a Lewis acid catalyst. This method features mild conditions, good functional group tolerance, high yield (up to 99%), and excellent enantioselectivity (up to 99% ee). Density functional theory (DFT) calculation elucidates the origin of the reaction's enantioselectivity and the mechanism of BCB activation by Cu(II) complex.

Structural insights into the molecular recognition of integrin αVβ3 by RGD-containing ligands: The role of the specificity-determining loop (SDL).

Integrin αVβ3 is a prominent member of the "RGD-recognizing" integrin family of cell surface receptors. αVβ3 binds to various extracellular matrix (ECM) proteins and oxysterols such as 25-hydroxycholesterol, is implicated in several diseases, including cancer metastasis, lung fibrosis, inflammation, and autoimmune diseases, and is pursued as a valuable therapeutic target. Despite enormous efforts to seek a pure antagonist, to date, no single drug candidate has successfully reached clinics due to associated partial agonism and toxicity issues. Developing effective and safe inhibitors require a thorough understanding of the molecular interactions and structural changes related to the receptor's activation and inhibition mechanisms. This study offers a comprehensive residue-residue contact and network analyses of the ligand-binding β-propeller βI domains (headpiece) based on all available experimental structures of integrin αVβ3 in unliganded, agonist-, antagonist-, and antibody-bound states. The analyses reveal many critical interactions that were not reported before and show that specific orientation and interactions of residues from the specificity-determining loop (SDL) are critical in molecular recognition and regulation. Also, the network analysis reveals that residues from the nearby allosteric site (site II) connect to the primary RGD-binding site via SDL, which likely acts as an interface between the two sites. Our results provide valuable insights into molecular interactions, structural changes, distinct features of the active and inactive headpiece conformations, the role of SDL in ligand recognition, and SDL-mediated allostery. Thus, the insights from this study may facilitate the designing of pure antagonists or site II-mediated allosteric modulators to integrin αVβ3 to treat various diseases.

Hydroxylamine hydrochloride-driven activation of NiFe2O4 for the degradation of phenol via peroxymonosulfate

In this study, hydroxylamine hydrochloride (HA) was employed to enhance the activation of NiFe2O4 towards peroxymonosulfate (PMS) for the effective degradation of phenol. NiFe2O4 particles were synthesized via a simple hydrothermal method, followed by characterization of their surface morphology, and microstructure. Upon the addition of HA to the system of NiFe2O4/PMS, the degradation activity is significantly enhanced. The degradation efficiency of phenol reached 98.5% after 60 min using 0.6 g/L NiFe2O4, 3 mmol/L PMS, 2 mmol/L HA at pH 7, which increased by 4.76 times compared to the system without HA. The study further explored the activation mechanism of the NiFe2O4/HA/PMS system, revealing that HA significantly enhanced the conversion of Fe3+/Fe2+ and the leaching of metal ions, thereby accelerating the reaction rate. In addition, the NiFe2O4/HA/PMS system proved effective across a broad range of pH values. This study provides new insights and perspectives into enhancing peroxymonosulfate activation coupled with metal oxides catalysts through the use of reducing agents.

Plant-Derived Flavonoids as AMPK Activators: Unveiling Their Potential in Type 2 Diabetes Management through Mechanistic Insights, Docking Studies, and Pharmacokinetics

Type 2 diabetes mellitus (T2DM) remains a significant global health issue, marked by insulin resistance and disrupted glucose metabolism. AMP-activated protein kinase (AMPK) serves as a key regulator of cellular energy balance, playing a crucial role in enhancing insulin sensitivity, promoting glucose uptake, and reducing glucose production in the liver. Recently, there has been growing interest in plant-derived flavonoids as natural activators of AMPK, offering a promising complementary approach to conventional diabetes treatments. This review delves into ten flavonoids identified as AMPK activators, including baicalein, dihydromyricetin, bavachin, 7-O-MA, derrone, and alpinumisoflavone. Their activation mechanisms are explored, which include both direct binding to the AMPK complex and indirect pathways involving upstream signaling. Through molecular docking studies, the binding affinities and interaction profiles of these flavonoids with AMPK are assessed, revealing varying levels of activation potential. Notably, baicalein and dihydromyricetin showed strong binding to the α1 subunit of AMPK, indicating high potential for robust activation. Additionally, this review provides a thorough analysis of the pharmacokinetic properties and drug-likeness of these flavonoids using the SwissADME tool, focusing on aspects such as ADME (Absorption, Distribution, Metabolism, and Excretion). While the overall profiles of these compounds are promising, issues like solubility and possible drug–drug interactions are areas that need further refinement. In summary, plant-derived flavonoids emerge as a promising avenue for developing new natural therapies for T2DM. Moving forward, research should aim at optimizing these compounds for clinical application, elucidating their specific mechanisms of AMPK activation, and confirming their efficacy in T2DM treatment. This review highlights the potential of flavonoids as safer and more holistic alternatives or adjuncts to current diabetes therapies.

Study on the Mechanism of FOXA2 Activation on Glutathione Metabolic Reprogramming Mediated by ETV4 Transcription to Facilitate Colorectal Cancer Malignant Progression.

The metabolic imbalance of glutathione (GSH) has been widely recognized in most cancers, but the specific molecular mechanism of GSH metabolic regulation in the malignant progression of colorectal cancer (CRC) is unexplored. The objective of our project is to elucidate whether ETV4 affects the malignant progression of CRC through GSH metabolic reprogramming. Bioinformatics and molecular experiments measured the expression of ETV4 in CRC, and in vitro experiments explored the impact of ETV4 on CRC malignant progression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) identified the pathway of ETV4 enrichment. The bioinformatics approach identified FOXA2 as an upstream regulatory factor of ETV4. The dual-luciferase assay, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) experiment verified the binding relationship between ETV4 and FOXA2. Cell viability, migration, and invasion abilities were determined by conducting CCK-8, wound healing, and Transwell assays, respectively. The expression levels of N-cadherin, E-cadherin, and vimentin were determined by utilizing immunofluorescence (IF). Metabolism-related enzymes GCLM, GCLC, and GSTP1 levels were detected to evaluate the GSH metabolism level by analyzing the GSH/GSSG ratio. In vivo experiments were performed to explore the effect of FOXA2/ETV4 on CRC progression, and the expression of related proteins was detected by western blot. ETV4 was highly expressed in CRC. Knocking down ETV4 suppressed CRC cell viability, migration, invasion, and epithelial-mesenchymal transition (EMT) progression in vitro. ETV4 was abundant in the GSH metabolic pathway, and overexpression of ETV4 facilitated CRC malignant progression through activation of the GSH metabolism. In addition, in vitro cellular experiments and in vivo experiments in nude mice confirmed that FOXA2 transcriptionally activated ETV4. Knocking down FOXA2 repressed the malignant phenotype of CRC cells by suppressing GSH metabolism. These effects were reversed by overexpressing ETV4. Our results indicated that FOXA2 transcriptionally activates ETV4 to facilitate CRC malignant progression by modulating the GSH metabolic pathway. Targeting the FOXA2/ETV4 axis or GSH metabolism may be an effective approach for CRC treatment.

Selective oxidation of organic pollutants by unactivated and carbonate-activated peroxymonosulfate (PMS): Mechanism, kinetics, and transformation pathway

Although the activation mechanisms of peroxymonosulfate (PMS) by various homogeneous and heterogeneous catalysts have been reported, the chemistry of PMS in catalyst-free systems and its interaction with background oxygenated anions remains poorly understood. In this study, the unactivated PMS and carbonate-activated PMS (CO32–/PMS) systems for removal of various organic pollutant (including oxytetracycline (OTC), metronidazole (MNZ), methylene blue (MB), and acid orange G (OG)) were investigated. The results showed that 74.5 %, 90.21 %, 1.22 %, and 2.25 % of OTC, MB, OG, and MNZ were removed, respectively within 180 min in the unactivated PMS system due to the formation of ·OH and 1O2. 95.88 %, 100 %, 100 %, and 6.09 % of OTC, MB, OG, and MNZ were removed, respectively in the CO32–/PMS system, which is primary due to the generation of 1O2. The removal efficiencies of these four pollutants were significantly improved under alkaline conditions. In addition, the TOC removal rates were 21.88 % for MB and 26.53 % for OTC within 180 min in the CO32–/PMS system. The presence of Cl− and SO42− can greatly enhance the OTC removal efficiency both in the unactivated and CO32–/PMS systems. Through the high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis, OTC degradation products in the unactivated PMS and CO32–/PMS systems were identified, revealing six different reaction mechanisms, including hydroxylation (+16 Da), decarbonylation (−28 Da), demethylation (−14 Da), secondary alcohol oxidation (−2 Da), deamination (−15 Da), and dehydration (−18 Da). This study provides insight into the reaction mechanism of catalyst-free PMS systems and may promote the application of unactivated PMS and CO32–/PMS systems for the remediation of organic contaminated water.

AKT-mediated phosphorylation of TSC2 controls stimulus- and tissue-specific mTORC1 signaling and organ growth.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates diverse intracellular and extracellular growth signals to regulate cell and tissue growth. How the molecular mechanisms regulating mTORC1 signaling established through biochemical and cell biological studies function under physiological states in specific mammalian tissues are unknown. Here, we characterize a genetic mouse model lacking the 5 phosphorylation sites on the tuberous sclerosis complex 2 (TSC2) protein through which the growth factor-stimulated protein kinase AKT can active mTORC1 signaling in cell culture models. These phospho-mutant mice (TSC2-5A) are developmentally normal but exhibit reduced body weight and the weight of specific organs, such as brain and skeletal muscle, associated with cell intrinsic decreases in growth factor-stimulated mTORC1 signaling. The TSC2-5A mouse model demonstrates that TSC2 phosphorylation is a primary mechanism of mTORC1 activation in some, but not all, tissues and provides a genetic tool to facilitate studies on the physiological regulation of mTORC1.

Selective Enhancer Gain of Function Deregulates MYC Expression in Multiple Myeloma

Abstract MYC deregulation occurs in the majority of multiple myeloma (MM) cases and is associated with progression and worse prognosis. Enhanced MYC expression occurs in about 70% of MM patients, but it is known to be driven by translocation or amplification events in only ~40% of myelomas. Here, we used CRISPR interference (CRISPRi) to uncover an epigenetic mechanism of MYC regulation whereby increased accessibility of a plasma cell-type specific enhancer leads to increased MYC expression. This native enhancer activity was not associated with enhancer hijacking events but led to specific binding of c-MAF, IRF4, and SPIB transcription factors that activated MYC expression in the absence of known genetic aberrations. In addition, focal amplification was another mechanism of activation of this enhancer in approximately 3.4% of MM patients. Together, these findings define an epigenetic mechanism of MYC deregulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

Activation Pathways of Murine Macrophages by Lipophosphoglycan from Strains of Leishmania major (FV1 and LV39).

Lipophosphoglycan (LPG) is an important Leishmania virulence factor. It is the most abundant surface glycoconjugate in promastigotes, playing an important role in the interaction with phagocytic cells. While LPG is known to modulate the macrophage immune response during infection, the activation mechanisms triggered by this glycoconjugate have not been fully elucidated. This work investigated the role that LPGs purified from two strains of Leishmania major (FV1 and LV39) play in macrophage activation, considering the differences in their biochemical structures. Bone marrow-derived macrophages from BALB/c mice were stimulated with 10 μg/mL purified LPG from the LV39 and FV1 strains. We then measured the production of nitric oxide (NO) and cytokines, the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and the activation of MAPK pathways. LPG from the LV39 strain, which has longer poly-galactosylated side chains, induced a more pro-inflammatory profile than that from the FV1 strain. This included higher production of NO, TNF-α, and PGE2, and increased expression of COX-2 and iNOS. Additionally, the phosphorylation of ERK-1/2 and JNK was elevated in macrophages exposed to LPG from the LV39 strain. No difference in IL-10 production was observed in cells stimulated by both LPG. Thus, intraspecific structural differences in LPG contribute to distinct innate immune responses in macrophages.

Allosteric mechanism of membrane fusion activation in a herpesvirus.

Herpesviridae infect nearly all humans for life, causing diseases that range from painful to life-threatening1. These viruses penetrate cells by employing a complex apparatus composed of separate receptor-binding, signal-transmitting, and membrane-fusing components2. But how these components coordinate their functions is unknown. Here, we determined the 4.19-angstrom cryoEM reconstruction of the central signal-transmitting component from herpes simplex virus 2, the gH/gL complex, in its elusive pre-activation state. Analysis of the continuum of conformational ensembles observed in cryoEM data revealed a series of structural rearrangements in gH/gL that allosterically transmit the fusion-triggering signal from the receptor-binding glycoprotein gD to the membrane fusogen gB. Furthermore, we identified a structural "switch" element in gH/gL that refolds and flips 180 degrees during the transition from pre-activation to activated form. Conservation of this "switch" in gH/gL homologs suggests that the proposed fusion triggering mechanism may apply to all Herpesviridae and points to a new target for subunit-based vaccines and treatment efforts.

Retraction: Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies

Retraction: Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies

Advanced electrochemical oxidation of EDTA-Ni via cobalt single-atom catalysts: Exploring indirect persulfate activation pathways

This study explores an innovative electrochemical strategy for the removal of the highly stable and toxic EDTA-Ni complex found in electroplating wastewater. Utilizing a cobalt-based single-atom catalyst (Co-NC) in an electro-enhanced system, we achieved significant activation of peroxydisulfate (PDS) for effective degradation of EDTA-Ni. Under optimal conditions of 40 mA current density and a pH range of 3–7, more than 97% of EDTA-Ni (1 mM) was successfully degraded within 90 min. Through detailed electrochemical experiments, we identified that atomic hydrogen (H*) played a crucial role in the indirect activation of PDS, facilitating the formation of reactive sulfate radicals (·SO4-). Computational analysis using density functional theory (DFT) confirmed that the H*-mediated reduction pathway had a notably low energy barrier (ΔGbs = 0.51 eV), making it the dominant activation mechanism. Gas chromatography-mass spectrometry (GC–MS) further revealed the primary degradation intermediates, providing insights into the breakdown process of EDTA-Ni. This research underscores the potential of Co-NC catalyst as a highly effective catalyst for treating persistent heavy metal complexes in advanced oxidation systems.

Purified CDT toxins and a clean deletion within the CDT locus provide novel insights into the contribution of binary toxin in cellular inflammation and Clostridioides difficile infection.

Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1β and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.