Toxins

Bacillus thuringiensis (Bt) is currently the most commercialized biopesticide worldwide. Despite the success of Bt application in biological control programs, diverse strategies have been developed to enhance the toxicity of Bt toxins and delay the emergence of insect resistance, including the addition of cadherin fragments as a synergistic agent. In this study, the synergistic effect of two different cadherin fragments, one from a lepidopteran species, Spodoptera exigua (rSeCad1bp), and the other from a coleopteran species, Tenebrio molitor (rTmCad1p), was evaluated on the toxicity of Cry1Aa, Cry1Ab, and Cry1Ia against S. exigua and Grapholita molesta. Our results show that while the toxicity of Cry1 proteins to S. exigua was not affected by rSeCad1bp, the toxicity of Cry1Aa against G. molesta increased about 2.6-fold. No other synergistic or antagonist effects were observed. The potential mechanisms for the detected toxicity enhancement in G. molesta were studied, including protection against proteolysis, promotion of oligomerization, and increased binding to receptors on midgut brush border membrane vesicles (BBMV). Results revealed a slight increase in Cry1Aa binding to BBMV. These data highlight the great potential of using rSeCad1bp in combination with Cry1Aa for control of G. molesta. Key ContributionOur results show that the cadherin fragment from Spodoptera exigua synergized Cry1Aa toxicity against Grapholita molesta, but not to S. exigua. The synergistic effect observed might be partly associated with an increase in Cry1Aa binding to the target midgut membrane.

Symptoms of Clostridioides difficile infection (CDI) are primarily caused by two major protein toxins, toxin A (TcdA) and toxin B (TcdB). In addition, approximately 5-30% of C. dif[fi]cile strains produce a third toxin, C. difficile binary toxin (CDT), which is has been associated with enhanced virulence and severe disease. CDT consists of an enzymatic component CDTa, and a binding and translocation component CDTb, which mediates the delivery of CDTa into host cells. CDTb contains two receptor-binding domains, RBD1 and RBD2. Recent structural studies suggest that RBD2 plays a critical role in the formation and stabilization of the di-heptameric CDTb assembly required for efficient intoxication of host cells. In this study, we evaluated the immunogenicity and protective potential of RBD1 and RBD2 using in silico, in vitro and in vivo approaches. Sequence analysis demonstrated that RBD2 is highly conserved among diverse CDT-producing C. difficile ribotypes and toxinotypes. Immunization of mice with RBD2, but not RBD1 conferred effective protection against direct CDT challenge. Moreover, RBD2 immunization protected hamsters against infection with a CDT-only-producing C. difficile strain (DSM 101085; TcdA-TcdB-CDT). Mechanistically, anti-RBD2 serum, but not anti-RBD1 serum, effectively neutralized CDT-mediated cytotoxicity, as demonstrated by inhibition of cell rounding in Vero cells. Collectively, these findings identify RBD2 as a promising vaccine antigen targeting CDT and provide functional evidence supporting its critical role in CDT-mediated host-cell intoxication. Incorporation of RBD2 into multivalent C. difficile vaccines may broaden protection against hypervirulent, CDT-producing strains.

Disintegrins are snake venom toxins that bind integrins and modulate platelet aggregation and cell migration. Isolation from venom often yields insufficient quantities for comprehensive study, making recombinant expression essential for detailed toxin characterisation. Here, we produced and characterised four disintegrins from Echis coloratus and Echis ocellatus in Escherichia coli. These homologous proteins contain distinct integrin-binding motifs: EcDis_RGD, EoDis_RGD, EcDis_KGD, and EcDis_VGD. Functional activities were evaluated using human platelet aggregation and endothelial cell scratch wound-healing assays. RGD-containing disintegrins exhibited the strongest inhibition of platelet aggregation, with EcDis_RGD showing the highest potency. The EcDis_KGD disintegrin inhibited platelet aggregation less potently, whereas EcDis_VGD showed no detectable activity in our assays. In endothelial cells, EcDis_RGD disintegrin markedly impaired cell migration, while EoDis_RGD displayed weaker anti-migratory activity. Neither KGD- nor VGD-containing disintegrins significantly affected wound closure. EoDis_RGD exhibited similar antiplatelet activity to the native, venom-purified disintegrin ocellatusin, validating our recombinant expression strategy. However, the corresponding PII-SVMP preparation displayed reduced inhibitory activity, consistent with incomplete generation of fully mature disintegrins species. Our findings demonstrate the importance of both integrin-binding motif identity and surrounding sequence context in determining disintegrin function, and support further use of recombinant toxins in toxinology and as therapeutic lead molecules.

Clostridioides difficile is a major cause of healthcare associated infections. C. difficile infection (CDI) is a toxin-mediated disease driven by the secretion of two large protein toxins, Toxin A (TcdA) and Toxin B (TcdB). TcdA and TcdB cause neurogenic inflammation that can contribute to intestinal inflammation in rat and mouse models. Substance P (SP), a potent vasoactive neuropeptide, is a major contributor to C. difficile toxin induced inflammation in animal models. SP acts on multiple receptors including the high affinity receptor NK1-R, and lower affinity receptors (Mrgprb2 and NK2-R). Through in vivo mouse infection models, we confirmed the effects of SP in hypervirulent C. difficile are largely mediated by the neurokinin-1 receptor (NK1-R), the high affinity receptor for SP. Through genetic mice that lack mast cells (cKitW-sh) we determined that mast cells which respond to SP in certain kinds of neurogenic inflammation appear dispensable for inflammation caused by C. difficile infection. We explored the role of NK1-R antagonists in a retrospective study of a large clinical cohort. We determined that, after propensity matching, NK1-R antagonists were associated with a significant survival benefit and that patients treated with NK1-R antagonists showed evidence of reduced vascular inflammation (increased serum albumin). Together these data suggest blocking SP activity through antagonism of NK1-R with small molecule inhibitors may be a useful strategy for adjunctive treatment during C. difficile infection.

The muscle-relaxing effects of botulinum neurotoxin (BoNT) serotypes A and B are widely utilized in clinical and aesthetic medicine. While the mouse bioassay remains the gold standard for evaluating the potency of pharmaceutical BoNT preparations, a non-animal alternative would be preferable to comply with European Directive 2010/63/EU. The Binding and Cleavage (BINACLE) assay, which measures BoNT activity based on the receptor-binding and proteolytic properties of the toxin, represents a promising alternative. Previous studies showed that this method allows reliable and sensitive activity determination. Here, we describe a collaborative study involving ten international laboratories, which aimed to characterize the applicability of the BINACLE assay for the potency testing of pharmaceutical BoNT products. Our results demonstrate that the BINACLE assay enables reproducible relative potency determinations for BoNT/A, the most pharmaceutically relevant serotype, with variability within acceptable limits. Reproducible relative potency calculations were also demonstrated for BoNT/B, while the data highlighted opportunities for refinement of the BoNT/B BINACLE protocol to further enhance the robustness of the method. Overall, this work confirms the utility of the BINACLE assay as a reliable and sensitive method for determining BoNT product potency, thereby substantiating its applicability as an alternative to the mouse bioassay.

Pore forming toxins (PFTs) are cytotoxins secreted in water-soluble form by pathogenic bacteria. They have the ability to form pores in the membrane of host cells, ultimately leading to cell death by lytic activity. Staphylococcus aureus produces a variety of bi-component PFTs, the leukocidins, which target and lyse particular leukocytes, erythrocytes and endothelial cells through specific interactions with membrane receptors. Most of these receptors belong to the family of complement or chemokine receptors that are G protein-coupled receptors (GPCRs). Gamma-hemolysins (Hlgs) are the major leukocidins secreted by S. aureus, and form receptor-dependent hetero-octameric pores through mechanisms that are not fully elucidated. Studying these molecular mechanisms is technically challenging due to the requirement of specific receptors in a lipid bilayer environment. In the present article, we developed a simple and highly sensitive method allowing cell surface expression of a large diversity of target receptors and recording in real-time, currents generated by neo-formed pores. This method is based on the heterologous expression of receptors in Xenopus oocytes and on the two-electrode voltage-clamp technique with electrophysiological robots. Using this approach, we characterized the concentration dependent-kinetics of pore formation, determined the receptor density as a limiting factor, showed specific response to non-cognate pairing of PFTs, observed cell surface binding of F subunits preceding pore formation and propose a hybrid model of subunit oligomerization. This method could be easily implemented for the in vitro characterization of various PFTs on a wide diversity of membrane receptors, to decipher early mechanisms of pore formation or to screen therapeutic agents blocking the cytotoxicity of receptor-dependent PFTs. Author SummaryStaphylococcus aureus is a bacterial species naturally present in our external flora and environment, but it is also one of the main pathogens responsible for nosocomial infections in hospital, with strains having highly problematic multi-resistance to antibiotics. S. aureus is able to secrete various virulence factors, some of which can specifically target and lyse our immune cells, making us more vulnerable to this pathogen. Thus, leukotoxins bind to receptors on the cell surface, drastically change their conformation and form cytotoxic pores in the membrane. Studying the molecular mechanisms underlying the formation of these pores is technically challenging due to their requirement for specific receptors. Here, we tested a simple electrophysiological method enabling the real-time measurement of pore formation on model cells (Xenopus oocytes), which express the receptors of interest. We were thus able to elucidate the kinetics of pore formation, the limiting role of receptors in this process, and propose a complementary model to the standard model. We also demonstrated the ability of this method to detect pore formation of non-cognate pairs of subunits and suggest further applications to characterize pore-forming properties of other toxins, to identify new target receptors, or to screen therapeutic agents inhibiting the formation of pores.

The discovery of distinctive function-phenotype relationships in monogenetic channelopathies has turned out to be critical for the development of precision medicine approaches. However, the best prediction of clinical phenotypes depends on neuronal function, where existing models lack tools to isolate currents of individual voltage-gated potassium channel subunits and differentiate variant effects in complex systems. Ideally, one should be able to overexpress subunit variants with an additional mutation that confers resistance against the tool to isolate the variant effect. Therefore, we solid-phase synthesized the KV1.2 specific scorpion toxin peptide BMK86-P1 and oxidized it with modest efficacy. In mammalian cells this BMK86-P1 selectively inhibited KV1.2 homomers, but not heteromers with KV1.1. Critically, the KCNA2 p.Val381Tyr mutation, which reverses BMK86-P1's selective inhibition of KV1.2, also altered the activation of KV1.2 homomers to resemble those of KV1.1. In addition, BMK86-P1 in murine neurons did not alter passive membrane properties, single action potential properties, or action potential firing. Surprisingly, it induced only minimal changes in spontaneous excitatory postsynaptic currents. In summary, this KV1.2 subunit selective toxin peptide asserts its effects primarily on homomeric channels, while only weakly inhibiting KV1.2-heteromeric channels and consequently preventing any meaningful impact on neuronal function. This highlights the limits of peptide synthesis together with the need for testing specific compounds on complex systems.

Vibrio parahaemolyticus (Vp) is the major foodborne pathogen transmitted via shellfish products, which has posed significant threats to modern public health and resulted in significant economic damage to the seafood industry. Numerous studies have documented diverse aspects of Vp pathogenicity, among which thermostable direct haemolysin (TDH) and TDH related haemolysin (TRH) are considered as the major virulence biomarkers of Vp. Despite their established roles as key biomarkers, a systematic understanding of the divergence of TDH and TRH across sequence, structure, and function remains limited. In this study, a multi scale analysis of TDH and TRH was performed using publicly available (2131 and 99 records from NCBI and Uniprot database, respectively) amino acid sequence data combined with representation learning and structure prediction. Global alignment of curated TDH and TRH sequences revealed extensive, distributed mutations and clear separation between TDH and TRH at the amino acid level (percentage identity of between TDH and TRH ranging from 56.1 to 67.4%). In contrast, protein language model derived embeddings showed high global functional similarity while preserving distinct clustering patterns, indicating conserved core functionality alongside nuanced divergence echoed with the structural inference by AlphaFold. Importantly, modeling of mutation trajectories demonstrated that the transition from TDH to TRH is driven by accumulated, genome-wide residue changes rather than a small set of key mutations. Together, these results suggested that TDH and TRH represent functionally conserved yet evolutionarily diverged toxins driven by accumulated sequence variation throughout the full-length amino acid sequence. These accumulated point mutations lead to major structural difference: TRH forms an alpha-helical tail that TDH lacks, which suggests that TDH and TRH disrupt host membranes by different mechanisms despite their conserved core function, such as pore-forming and ion flux induction capability. These methods provided unprecedented detailed insights into the functional and structural properties of Vp haemolysin, offering critical information on how multi-dimensional variations in sequence might influence their role in Vp pathogenicity. Insights from this study reinforce the rationale for using Vp strains harboring tdh and trh genes in experimental design for environmental fitness investigation.

The type VII secretion system (T7SS) is a membrane-embedded protein export pathway found in mycobacteria and Gram-positive bacteria. Recently it was shown that Mycobacterium abscessus uses its ESX-4 variant of the T7SS to secrete a toxin, EatA, which targets arabinogalactan present in the mycobacterial cell envelope. Prior to its export, EatA forms a complex with a pair of small proteins from the WXG100 family, TapA1 and TapA2. Here we investigated a structural model of the EatA N-terminal domain in complex with TapA1 and TapA2 using site-directed mutagenesis and bacterial 2-hybrid assays. Our results are consistent with the three proteins forming a stacked bundle of alpha-helices. Structural modelling also predicted an interaction of the EatA-TapA1-TapA2 complex with EsxT-EsxU, a second pair of WXG100-family proteins that are likely required for the mechanistic operation of ESX-4. Whilst we could demonstrate a potential interaction between TapA2 and EsxT by bacterial 2-hybrid analysis, we were not able to purify a complex of all five proteins.

Ivermectin is an important broad-spectrum anthelmintic used to treat nematode parasites including gastro-intestinal infections of humans and animals. The mode of action for Ivermectin is understood to involve activation of inhibitory glutamate-gated chloride channels (GluCls). Ivermectin has also been reported to inhibit the release of extracellular vesicles (EVs). We found that EVs are released from the whole intestine of the gastro-intestinal parasite, Ascaris suum. Proteomic analysis identified 1,574 proteins within these intestinal EVs, including 96 nematode proteins with putative immune-associated functions based on homology to proteins involved in host immune processes and 130 proteins with predicted digestive functions. Comparative analysis following ivermectin exposure revealed 38 differentially abundant proteins that included the putative immune-related proteins: transthyretin-like proteins, a small heat-shock antigen, a phospholipase A2, and the NF-{kappa}B subunit p105. Thus, ivermectin modulated the potential immune-related cargo of intestinal EVs. The ivermectin inhibition of intestinal EV release was concentration-dependent with an IC50 of 64 nM. We also identified the expression of GluCl subunit receptor genes in the Ascaris intestine. The potent inhibitory effect of ivermectin on the release of these EVs from the nematode intestine and the expression of GluCl channel subunits sheds further light on the site and mechanisms of action of this important anthelmintic.

Background and aims Model systems for bile duct disorders are needed for testing therapeutic interventions. Current models have poor human relevance or limited potential for recreating the complex bile duct microenvironment at scale. We aimed to generate a humanized microphysiological system to model and treat cholangiopathies. Methods An in vitro bile duct was created using 3D printed microfluidic chips containing a collagen-embedded canal seeded with patient-derived primary human cholangiocytes. Barrier permeability and compound transport across the epithelium was measured, and disruption of the barrier was performed with lipopolysaccharide treatment. The duct was challenged with the known hepatotoxicant Chlorpromazine. Biliatresone was used to model biliary-atresia and treated using N-acetyl-L-cysteine. Results Cholangiocytes in the bile duct chip established a tight, polarized epithelial barrier. Verapamil and Linerixibat inhibited transport of rhodamine 123 and cholyl-lys-fluorescein respectively with 66 % (p = 0.0004) and 57 % (p = 0.03) reduction. 10 g/mL lipopolysaccharide led to a loss of epithelial barrier integrity, measured by an increase of over 1000 % in leakage of both 3 kDa (p = 0.0002) and 10 kDa dextran (p = 0.0001) along with upregulation of cytokines. Chlorpromazine displayed dose-dependent toxicity with EC50 values of 84, 140 and 96 M for three patient lines. Biliatresone induced a dose-dependent abnormal phenotype with loss of viability. The induced phenotype could be treated with N-acetyl-L-cysteine, improving viability from 23 % to 59 % (p < 0.0001) with treatment of 2 g/mL Biliatresone. Conclusions Our novel platform allows complex studies of bile duct biology, testing of off-target effects from drugs and treatment of a disease phenotype.

The eukaryotic pathogen Leishmania major causes disfiguring cutaneous lesions, whose resolution can be complicated by secondary bacterial infections. Bacteria, including Aeromonas spp., also interact with L. major promastigotes in the sandfly midgut. The mechanisms by which L. major competes with bacteria and resists their toxins are poorly defined. Prior work proposed that L. major resists the Aeromonas-produced pore-forming toxin aerolysin using an altered GPI-anchor. However, we found that L. major is sensitive to aerolysin. Here, we determined the mechanism by which L. major promastigotes are sensitive to aerolysin, using flow cytometry and biochemical approaches to analyze promastigotes genetically deficient in enzymes that produce key membrane components. The virulence factor lipophosphoglycan protected L. major from aerolysin cytotoxicity. The metalloproteinase GP63 exerted the necessary furin-like protease activity to activate aerolysin. Leishmanial GPI-anchored proteins were necessary for aerolysin heptamerization and killing of L. major promastigotes. Finally, mutation of the GPI-anchor binding domain of aerolysin crippled its cytotoxicity, consistent with its reliance on the GPI-anchor binding site to engage GPI anchors on the surface of L. major promastigotes. Taken together, we propose the L. major virulence factor lipophosphoglycan defends against pore-forming toxins made by bacterial competitors, while the GP63 metalloproteinase activates pro-aerolysin like furin. Overall, this study highlights approaches microbes use to compete with each other. Graphical AbstractAerolysin cytotoxicity depends on gp63 and LPG in Leishmania major promastigotes. (A) Wild type Leishmania major promastigotes are sensitive to aerolysin, which forms lethal heptameric pore complexes in the plasma membrane (B) L. major lpg1-- promastigotes are highly sensitive to aerolysin challenge because they lack LPG. (C) L. major gp63-- promastigotes have wild type sensitivity to aerolysin challenge but resist pro-aerolysin. (D) L. major gpi8-- knockout promastigotes are resistant to aerolysin and show no heptameric pore complexes in the plasma membrane. Created in BioRender.

Cinnamaldehyde (CA) is extensively used as flavorant and in traditional medicine. CA is com-monly used in pain research as specific agonist of TRPA1, a polymodal cation channel expressed in nociceptive primary sensory neurons. However, we previously showed that CA inhibits the L-type Ca2+ channel in cardiac and smooth muscle cells, raising the possibility that other ion channels can be modulated by this compound as well. Here, we investigated whether CA exhibits affects the activity on the cardiac NaV1.5 channel. We used the whole-cell patch-clamp technique to record Na+ currents in HEK293T cells expressing the human NaV1.5 channel, as well as in cells expressing hNaV1.5 channels baring mutation in the binding pocket of local anesthetics (LA). Our results show that CA exhibit LA-like actions on hNaV1.5 channels: CA blocks hNaV1.5 currents in a tonic and voltage-dependent fashion. Residues F1760 and Y1767 are important for the blockade of NaV1.5 by CA and a double point mutation F1760A/Y1767A abolishes the blockade of INa by CA. We conclude that CA and LID share common structural determinants for the inhibition of Na+ channels and that CA has LA-like actions.

BackgroundThe variants G1 and G2 within the APOL1 gene confer a higher risk of APOL1-mediated kidney disease (AMKD) whilst associated with an evolutionary advantage against trypanosome-mediated sleeping sickness. MethodsIn this study, we analysed transcriptome data of kidney biopsies from FSGS patients with the APOL1 high-risk (HR) and low-risk (LR) variants and compared it to cellular models based on patient-specific podocytes, HEK cells with engineered over-expressing HR variants and HR variant single-cell-RNA-seq data from kidney organoids. ResultsWe identified a signature of up- and down-regulated genes between biopsies from FSGS patients with APOL1 HR and LR variants. The up-regulated genes are functionally annotated to be associated with Calcium and mTOR signaling, whilst the down-regulated genes with inflammatory and immune response pathways. These pathways were confirmed by comparing with cellular models. Analysis of small molecules reverting the IFN-{gamma} stimulated gene expression to the non-stimulated gene expression in genome-edited APOL-G1 kidney organoids revealed several putative candidates such as the mTOR inhibitor AZD-2014. ConclusionWe have unveiled a signature of up- and down-regulated genes between APOL1 HR and LR kidney biopsies which could be assigned as associated with Calcium and mTOR signaling and down-regulated immune response.

Advanced in vitro models that better recapitulate human intestinal physiology are needed to improve translational relevance and reduce reliance on animal experimentation. Microfluidic gut-on-chip systems provide controlled architectural and mechanical microenvironments that more closely resemble native tissue than conventional static cultures. However, how these features influence epithelial responses to microbial stimuli remains incompletely understood. In this study, we compared conventional two-dimensional (2D) Caco-2 monolayers with a perfused three-dimensional (3D) gut-on-chip model exhibiting crypt-like epithelial organization. Microbial challenge was employed as a biologically relevant perturbation to investigate barrier-associated responses under distinct culture conditions. Following exposure to the probiotic Escherichia coli Nissle 1917 (EcN), epithelial morphology, barrier-associated gene expression, inflammatory signaling, and temporal response patterns were assessed over time. In a complementary approach, susceptibility to bacterial stress was examined using increasing doses of Citrobacter rodentium (C. rodentium). Both culture systems responded dynamically to microbial exposure; however, differences in response patterns were observed between static and perfused cultures. EcN induced transient alterations in epithelial organization followed by recovery-associated responses, while C. rodentium caused dose-dependent structural and transcriptional changes. Notably, epithelial damage became apparent at lower bacterial loads in conventional 2D cultures, whereas the chip-based model maintained tissue integrity under comparable conditions. Together, these findings indicate that epithelial architecture and the surrounding culture environment influence barrier-associated responses to microbial challenge. The study highlights the utility of gut-on-chip systems for investigating host-microbe interactions and provides a framework for functional comparison of advanced intestinal in vitro models.

Successful treatment of primary Clostridioides difficile infection (CDI) while preventing its recurrence remains a major clinical challenge, with recurrent CDI (R-CDI) affecting 20-30% of treated patients. Persistence of C. difficile spores within the intestinal mucosa is thought to drive R-CDI following antibiotic treatment. Here, we show that intracellular C. difficile spores remain protected from taurocholate-triggered germination, and that nystatin, an FDA-approved antifungal that blocks spore entry into host cells, reduces this protected intracellular population and redirects spores toward an extracellular, germinating population accessible to antibiotics. In a murine model of R-CDI, oral co-administration of taurocholate with nystatin and antibiotics during primary CDI reduced R-CDI incidence from 100% to 44%. When administered during an active R-CDI episode, the same formulation reduced a second R-CDI from 100% to 62%. To improve bioavailability, we encapsulated nystatin and vancomycin in chitosan nanoparticles. Oral administration of nanoparticle-encapsulated vancomycin, alone or combined with nanoparticle-encapsulated nystatin, reduced R-CDI colonization to 33% and 44%, respectively, compared with 100% in mice receiving free vancomycin. Together, these findings support a therapeutic strategy that repurposes nystatin and taurocholate, in combination with antibiotics and nanoparticle-based delivery, to target intracellular spore persistence and reduce the incidence of C. difficile recurrence..

Kinema is a traditional fermented soybean food indigenous to the eastern Himalayan regions of Nepal and India. The fermentation process is primarily mediated by the bacterium Bacillus subtilis, which produces several bioactive compounds and enzymes with potential therapeutic applications. Considering the growing burden of cardiovascular diseases and the need for effective fibrinolytic agents for thrombolytic therapy, this study aimed to extract, partially purify, and evaluate the thrombolytic potential of kinemakinase derived from kinema prepared from white soybeans. Partial purification of the enzyme was achieved using ammonium sulfate precipitation. Thrombolytic activity was assessed in vitro using human blood clots, where three enzyme dilutions demonstrated clot lysis ranging from 66% to 68%, indicating considerable fibrinolytic potential. In silico analyses were also performed to investigate the structural and functional characteristics of the enzyme. The tertiary structure obtained from UniProt was modeled using the Robetta server and refined with GalaxyRefine. Docking with fibrin using ClusPro 2.0 and molecular dynamics simulations using iMODS confirmed favorable interaction and structural stability, while disulfide engineering enhanced protein stability. The findings suggest that kinema-derived kinemakinase may serve as a promising alternative thrombolytic agent, warranting further biochemical characterization and dosage optimization.

Severe dengue can damage the liver through unestablished mechanisms. We investigated the role of myeloperoxidase (MPO), a neutrophil enzyme, in dengue through patients, fatal liver samples, and mouse infection models. Observations from two independent clinical cohorts revealed elevated plasma MPO levels in dengue and, in one cohort, MPO was further linked to liver injury markers during the critical phase of disease, whereas livers from dengue fatal cases revealed MPO build-up in the vicinity of CD177+ activated neutrophils. In mice, dengue led to MPO overexpression, oxidative damage, and broad activation of innate and systemic inflammatory pathways in livers. Blocking MPO activity alleviated these and improved survival in one model and delayed disease progression without preventing death in another. These findings establish MPO as a functional mediator of severe dengue-associated liver injury and inflammation, which warrants further preclinical investigation into its hepatic pathogenic mechanism and its validity as target for therapeutic intervention.

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

Melittin, the principal bioactive peptide of bee venom, exhibits promising antitumor activity, whereas its molecular mechanisms in cervical cancer remain incompletely understood. In this study, the biological effects and molecular responses of melittin in U14 cervical cancer cells were investigated using Astral data-independent acquisition (Astral-DIA)-based quantitative proteomics combined with molecular validation. The effects of melittin on cell migration, invasion, and cell death were evaluated by Transwell assays and PI/Hoechst staining. Differentially expressed proteins (DEPs) were screened and subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) analyses. Representative oxidative stress-related genes and proteins were further validated by RT-qPCR and Western blotting. Melittin significantly inhibited the migration and invasion of U14 cervical cancer cells and increased cell death. Quantitative proteomics identified 9,782 protein groups and 187 DEPs, including 71 up- and 116 down-regulated proteins. KEGG pathway enrichment analysis revealed oxidative phosphorylation (OXPHOS) as the most significantly enriched pathway, together with glutathione metabolism, ferroptosis-related pathways, reactive oxygen species signaling, and mitophagy. GO term enrichment analysis indicated that DEGs were mainly engaged in mitochondrial function, electron transport, oxidoreductase activity, and energy metabolism. RT-qPCR assay demonstrated altered expression of Duox1, Gpx4, Gsx2, Nfe2l2, and Gstp2. Additionally, PPI analysis identified Gstp2 and ODC1 as representative hub proteins involved in redox regulation and metabolic adaptation. Furthermore, Western blotting confirmed increased GSTP2 expression following melittin treatment. Overall, these findings provide a comprehensive proteomic landscape of melittin-treated U14 cervical cancer cells and suggest that mitochondrial OXPHOS remodeling and redox-associated pathways may contribute to the antitumor activity of melittin.