Toxins

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..

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.

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

Bacillus cereus biovar anthracis (Bcbva) strains, which cause fatal anthrax-like disease, have been isolated from animals in Central and West Africa. Like B. anthracis, Bcbva produces a poly-{gamma}-D-glutamic acid (PDGA) capsule, as well as edema factor (EF), lethal factor (LF) and protective antigen (PA). Bcbva also possesses a hyaluronic acid capsule that is not expressed by B. anthracis. The pathogenesis of Bcbva has recently been characterized in inhalational infection models, in rabbits and guinea pigs. Here, we characterized the ki-netics of Bcbva infection and toxin production in a murine model of cutaneous infection using the Bcbva CA strain isolated in Cameroon. We found that spore germination oc-curred rapidly at the inoculation site, followed by bacterial dissemination to the draining cervical lymph nodes. Germination was associated with rapid production and diffusion of LF and EF. Bacteria rapidly disseminated to lymphoid organs and other compartments, with distal germination observed 18 hours post-infection. Infected mice developed septi-cemia within 24 hours, accompanied by high circulating LF levels and signs of cardiac and renal injury. These findings may contribute to the fulminant course of Bcbva infection observed in animals and highlight its potential risk to humans.

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

Background: Global oral cholera vaccine (OCV) supply shortages and the retirement of Shanchol necessitate absolute reliance on alternative formulations. We conducted a systematic review and meta-analysis to evaluate the immunogenicity and safety of Euvichol variants compared with the historical Shanchol benchmark. Methodology/Principal Findings: A searched PubMed, Embase, MEDLINE, Scopus, and Cochrane CENTRAL up to May 2026 for randomized controlled trials (RCTs) comparing any Euvichol variant against Shanchol in healthy populations aged [≥]1 year. Primary outcomes were vibriocidal seroconversion rates at 14 days post-dose 2 pooled as Risk Ratios (RRs) via Mantel-Haenszel fixed-effects models. Five RCTs comprising 7,253 participants were included. Euvichol formulations demonstrated non-inferior, equivalent seroconversion rates compared with Shanchol for both Vibrio cholerae O1 Inaba (RR 1.00, 95% CI 0.98-1.02; I2=0%) and O1 Ogawa (RR 1.01, 95% CI 0.99-1.04; I2=26%). Log-transformed Geometric Mean Titers confirmed clinical equivalence for Inaba (MD 0.02, 95% CI -0.01 to 0.05) and Ogawa (MD 0.00, 95% CI -0.03 to 0.03). Overall safety was balanced between arms, showing no significant variation in the risk of any solicited adverse event (RR 0.91, 95% CI 0.75-1.11) or specific symptoms including fever (RR 0.84, 95% CI 0.52-1.35). Serious adverse events were rare and uniformly unrelated to vaccination. Conclusions/Significance: Euvichol variants demonstrate immunogenic non-inferiority and interchangeable safety thresholds compared with Shanchol. These findings robustly support the large-scale integration of modern Euvichol-Plus and streamlined Euvichol-S formulations to stabilize global OCV stockpiles.

Adherent Invasive Escherichia coli (AIEC) strains are associated with ileal Crohn's disease and may contribute to intestinal dysbiosis through antibacterial competition. The AIEC reference strain LF82 encodes two type VI secretion systems (T6SSs), a contractile machine that injects an effector-loaded needle made of Hcp, VgrG and PAAR into neighbouring rivals, favoring niche colonization. LF82 T6SSs harbour several putative antibacterial toxin-immunity modules. Here we characterize the T6SS-1 encoded TmeAIEC, a PAAR-fused T6SS effector carrying a C-terminal Tme domain, hallmark of Tme (Type VI membrane disrupting effector) toxins, and its DUF1240-like immunity protein TmiAIEC. When heterologously produced in E. coli K-12 as well as in E. coli and Citrobacter clinical isolates, TmeAIEC is toxic only upon periplasmic targeting, and its toxicity is neutralized by TmiAIEC. Periplasmically-targeted TmeAIEC inserts into the inner membrane, collapses the proton motive force and increases cell permeability, consistent with a membrane-disrupting activity. Mutational analyses coupled to functional assays identified a predicted hydrophobic transmembrane segment and a conserved aspartate residue within the Tme domain as key determinants for TmeAIEC toxicity. These findings establish TmeAIEC/TmiAIEC as a functional T6SS toxin-immunity pair and reveal conserved determinants underlying the activity of Tme-family membrane-disrupting effectors.

Invasive candidiasis has become more common in recent decades, particularly within ICUs. Identifying specific Candida spp., and testing their sensitivity to antifungal drugs is crucial for effective treatment that helps healthcare providers to detect potential treatment challenges early. This study aimed to identify Candida spp. at their spp. level present in blood, assess their susceptibility to antifungal agents, and apply a random forest classifier to predict the best antifungal drug for a given species. The isolates were classified at the species complex level through MALDI-TOF MS. The strains' susceptibility to antifungal agents such as AMB, FLZ, VOZ, ITZ, POZ, CAS, MFN, and ANF was assessed using CLSI guidelines. Out of the 354 Candida spp. isolated from blood, C. utilis was found to be the predominant species, followed by C. tropicalis, and C. albicans. This study demonstrated that MFN and ANF exhibited high potency, with an impressive MIC90 of 0.12 {micro}g/mL against all isolates, surpassing CAS, which had a slightly higher overall MIC90 of 0.25 {micro}g/mL. FLZ exhibited the highest MIC90 value among the azoles at 16 {micro}g/mL, while VOZ, ITZ, and POZ all had MIC90 values of 0.5 {micro}g/mL. AMB MIC90 values were 2 {micro}g/mL against all isolated Candida species. The Random Forest AI/ML model showed perfect overall prediction, with the actual and predicted best antifungal drugs matching for all fungal species. This study is particularly essential because rare Candida spp. becoming more common and exhibiting higher MICs to AMB and FLZ and required suitable remedies for controlling the infection.

Abstract Background: Chronic subdural hematoma (cSDH) remains a significant and increasingly prevalent cause of morbidity in older adults. Disease progression is sustained by de novo formed highly vascularized pseudomembranes and a persistent cycle of fluid accumulation and hematoma expansion. Although inflammatory and angiogenic pathways have been implicated in cSDH pathogenesis, the endothelial mechanisms underlying persistent vascular leak remain poorly understood. We investigated the cSDH vascular microenvironment in human patients and examined the role of vascular endothelial cadherin (VECAD) remodeling in cSDH endothelial dysfunction. Methods: Human cSDH membranes and fluid were evaluated by multiplex profiling, histology, immunofluorescence, immunoprecipitation, western blotting and exploratory bulk RNA sequencing. A human brain endothelial cell (HBEC) line was exposed to patient-derived cSDH fluid to assess VECAD remodeling, endothelial barrier permeability, and proliferation. Results: Multiplex profiling of patient-derived cSDH fluid detected mediators associated with endothelial cell (EC) activation, extracellular matrix remodeling, inflammation, and angiogenesis. In patient pseudomembranes, discontinuous VECAD coincided with erythrocyte extravasation. Two hyperdense (high-risk) specimens showed more extensive disruption than the hypodense (low-risk) specimen while vessel counts appeared similar. Bulk RNA sequencing identified an angiogenic environment and enrichment in cadherin regulation. A soluble VECAD (sVECAD) isoform was detected in cSDH fluid. Exposure of HBECs to patient-derived cSDH fluid increased EC proliferation, cytoplasmic VECAD (cVECAD) accumulation, and EC barrier permeability. Concurrent dexamethasone or minocycline treatment attenuated VECAD cleavage. Conclusions: VECAD disruption is a prominent feature of cSDH and is associated with EC barrier dysfunction. These findings support further evaluation of soluble VECAD as a candidate biomarker and of junction-preserving pathways as potential therapeutic targets.

Venom systems are key evolutionary innovations that have contributed to the ecological success of numerous animal lineages. In ants (Formicidae), venom exhibits an exceptional diversity in both chemical composition and delivery mechanisms. Here, we combine a synthesis of current knowledge on the evolution of ant venoms with new phylogenetic comparative analyses, integrating chemical, morphological, and phylogenetic data at the genus level. Comparative analyses across Formicidae support the hypothesis that peptide-rich venoms represent the ancestral condition in ants, as they are widely distributed across basal and derived lineages. Despite this ancestral state, we identify seven independent evolutionary transitions toward non-peptidic venom, occurring in major clades including Dolichoderinae, Formicinae, Stenammini, Solenopsidini, Crematogaster, Pheidole, and fungus-growing ants. Ancestral state reconstruction and tests of correlated evolution across 114 genera show that these seven shifts away from peptide-based venom were almost irreversible and tightly coupled with the loss of stinging capacity and a dietary shift away from predation. These transitions are consistently associated with profound morphological modifications of the venom apparatus, such as reduction or complete loss of the sting, or its transformation into non-injecting structures. Chemically, these systems are dominated by alkaloids, iridoids, formic acid, esters, and terpenoids. These repeated transitions reflect major ecological and evolutionary shifts. Lineages that rely less on individual predation and more on collective foraging or plant-based diets tend to exhibit reduced reliance on injectable peptide toxins. Instead, they favor metabolite-based venoms that can be co-opted from existing biochemical pathways and deployed in diverse ecological contexts. Conversely, peptide-rich venoms are retained in lineages subjected to strong selective pressures for rapid prey immobilization or defense against vertebrate predators. Overall, ants represent a unique model for understanding venom evolution, illustrating how shifts in chemistry, morphology, and ecological strategy can drive repeated and convergent innovations. Future integrative studies combining genomics, metabolomics, and functional assays will be essential to unravel the mechanisms underlying these transitions and their role in the extraordinary diversification of ants.

Hypertriglyceridemia is commonly managed with lipid-lowering drugs such as statins; however, adverse effects, dose intolerance, and financial barriers may limit their use. Therefore, affordable and potentially safer plant-derived alternatives warrant investigation. This study evaluated the lipid-lowering effects of ethanol extracts of Fucus vesiculosus and Phytolacca berry, administered individually or in combination, in mice fed a butter-enriched diet for 12 weeks. Forty-eight mice were randomly assigned to six groups: normal control (Group 1), butter control (Group 2), statin control (Group 3), Fucus vesiculosus extract (Ext-1; Group 4), Phytolacca berry extract (Ext-2; Group 5), and the combined extracts (Group 6). Body weight, organ-to-body-weight ratios, serum lipid parameters, and histopathological changes were assessed. Group 3 showed the lowest final body weight, while Group 6 demonstrated a comparable body-weight trajectory. Organ-to-body-weight ratios varied among groups, with Group 3 generally showing the highest values and Group 6 comparatively lower ratios for several organs. Group 5 had the lowest total cholesterol concentration, followed by Group 3, whereas Group 6 showed cholesterol levels comparable to the normal control and statin groups. Notably, Group 6 exhibited the lowest serum triglyceride concentration (129.9 mg/dL). Histopathological examination revealed relatively mild hepatic alterations and limited vascular plaque formation in Group 6. The combined extracts demonstrated favorable effects on body-weight gain and serum lipid profiles, particularly triglycerides, with comparatively mild histopathological changes. Further mechanistic, dose-response, toxicity, and long-term studies are warranted before clinical application can be considered.

Ogerin, a positive regulator of GPR68, reduces infarct volume and attenuates blood-brain barrier (BBB) leakage post transient middle cerebral artery occlusion (tMCAO). These data support the potential use of Ogerin for ischemic protection. For therapeutic interventions, tissue distribution, kinetics, and brain penetration are important characteristics for a compound. For these reasons, we performed liquid chromatography-mass spectrometry (LC-MS) analysis of Ogerin in mice. At 3 hours following intraperitoneal (IP) injection, liver exhibited highest level of Ogerin among all major organs assessed. We further analyzed time-dependent changes in Ogerin concentration in serum following intravenous delivery. Brain Ogerin level was ~140 ng/g at 2 hour and ~0.06ng/g at 24 hours. The brain-to-serum ratio of Ogerin was ~0.6 at 5 hr. To determine the effect of Ogerin on signaling, we collected samples 24 hours after tMCAO with or without Ogerin delivered and performed Western blotting. Consistent with the diminished level of Ogerin in brain at 24 hour, tMCAO increased similar changes in phosphorylation of PKA substrates and PKC substrates in vehicle- and Ogerin-treated groups. The pharmacokinetic data suggests that Ogerin is effective mainly during acute hours after injection. This finding generates important insights on the design of effective protocol for Ogerin delivery to potentiate GPR68 function in vivo.

Cytosolic phospholipase A2 (cPLA2), encoded by PLA2G4A plays a crucial role in platelet activation through thromboxane A2 biosynthesis. Defects in this pathway can lead to bleeding disorders; however, the precise role of cPLA2 in Glanzmann thrombasthenia (GT) or GT-like syndromes remains poorly understood. The current study emerged from a fortuitous discovery of novel PLA2G4A variants, p.Asp455Val (D455V) and p.Glu119Lys (E119K), among 3 GT patients from two unrelated families in south India. We envisaged a hypothesis to identify a hitherto unknown role of cPLA2 that could be attributed to GT-like bleeding symptoms in these patients. An extensive computational workflow, including variant classification, conservation analysis, stability prediction, molecular docking, molecular dynamics simulations, principal component analysis, and binding free energy (MMPBSA) calculations, were employed to assess mutation-induced alterations in the cPLA2 protein. The current findings depicted that both variants affected conformational dynamics, ligand recognition patterns, and local interaction networks, without causing any drastic global structural destabilization. In particular, the D455V partially disrupted the catalytic domain, potentially reducing substrate (PAPC) stabilization and hampering interactions with catalytic products. Likewise, the E119K variant partially affected the C2 domain, potentially altered electrostatic interactions and enhanced regional flexibility, which may compromise product (AA) stabilization. Further analyses revealed mutation-induced changes in collective motions, protein-ligand interactions, solvent accessibility, and secondary structural elements. Overall, our findings promote the hypothesis that both E119K and D455V variants may partially alter phospholipid-associated interactions, and cPLA2-mediated downstream signaling. Moreover, this study underscores cPLA2 as a potential modifier of platelet function and bleeding severity.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Inflammatory bowel disease challenges modern gastroenterology due to high relapse rates. This study evaluated the effects of medicinal fungal sclerotium Fu Ling (Wolfiporia cocos), its derived {beta}-1,3-1,6-Glucan, Bacillus coagulans GBI-30, 6086, and their combinations in dextran sulfate sodium-induced colitis in male BALB/c mice. The {beta}-glucan-B. coagulans combination significantly improved body weight, disease activity index, colon length and architecture, mucus secretion, and cecal fermentation over single ingredient. Consistently, this combination increased total short-chain fatty acids production, particularly acetate, improved gut tight-junction organization and reduced myeloperoxidase accumulation. Shotgun metagenomic sequencing indicated that this combination reshaped the gut microbiota, notably enriching Anaeromassilibacillus sp., which associated with improved clinical feature. These findings highlight the potential of fungal-derived {beta}-glucan and probiotic combinations as functional food ingredients for mitigating intestinal inflammation and supporting barrier repair.

Traumatic brain injury (TBI) is one of the world's leading causes of death and disability and a major risk factor for dementias. The primary dementia associated with TBI is chronic traumatic encephalopathy (CTE), a neurodegenerative disease classified as a tauopathy, in which toxic tau molecules lead to disease pathologies and degeneration. The processes that lead to tauopathy and subsequent dementia after TBI remain unclear. Here, we built upon the finding that seizures after TBI may be a mechanism leading to tauopathy, by dissecting the functions of the metabotropic glutamate receptor 5 - cellular prion protein (mGluR5-PrPC) pathway. We delivered TBI to larval in a blast paradigm, and quantified aggregation of Tau via a genetically-encoded Tau-GFP fusion reporter. Zebrafish larvae lacking prp2 (homolog of mammalian cellular Prion Protein, PrPC) displayed a 168% increase in post-traumatic seizures activity after TBI. An mGluR5 agonist (CHPG) reduced post-traumatic seizures, whereas an mGluR5 antagonist (MPEP) increased post-traumatic seizures. Moreover, agonizing mGluR5 reduced tau aggregation and antagonizing mGluR5 increased tau burden. Larvae seizing from convulsants, rather than TBI, were treated with CHPG/MPEP and provided a similar pattern of outcomes, suggesting seizures may be a factor needed for mGluR5 activity to influence tau aggregation. The PrPC-mGluR5 pathway is proposed as one candidate pathomechanism linking TBI to subsequent seizures and tauopathy, and thus it warrants investigation as a target for prophylactic interventions.

Chemobrain, characterized by cognitive dysfunction, has emerged as a significant health concern following cancer treatment. Doxorubicin, an FDA-approved chemotherapeutic agent, is widely used to treat various cancers. However, its adverse effects raise concerns regarding toxicity in non-cancerous cell types. Microglia, the resident immune cells of the brain, play a central role in maintaining neural homeostasis. Despite this, the mechanisms by which microglia become dysfunctional following doxorubicin exposure remain poorly understood. Understanding the transcriptomic alterations induced by doxorubicin in microglia could provide critical insights into mechanisms underlying its neurotoxic effects. In this study, the transcriptomic response of human microglia to doxorubicin using mRNA sequencing combined with bioinformatic analysis was investigated. Exposure to doxorubicin significantly altered microglial morphology, promoting a shift toward a process-bearing phenotype in human HMC3 cells. This phenotypic change was accompanied by elevated levels of DNA damage and was associated with the induction of cellular senescence. At the gene regulation level, the Illumina-based transcriptomic analysis provided 25,834 gene expression profiling in which 5,669 were differentially expressed. The future analyses revealed that 3,431 (upregulated) and 2,238 (downregulated) were differentially expressed under the treatment of doxorubicin. Genes associated with the p53 signaling pathway and inflammatory responses were upregulated, whereas genes involved in translation, DNA replication, and cell-cycle progression were downregulated. This transcriptional dysregulation was accompanied by differential expression of key transcription factors. This study provides transcriptomic evidence elucidating the genotoxic response to doxorubicin in microglial cells, offering mechanistic insights into chemobrain and highlighting key molecular pathways as potential targets for therapeutic intervention.

A native entomopathogenic nematode isolate, Heterorhabditis indica Che4.1, was recovered from soils in Valencia (Spain), and its symbiotic bacterium, Photorhabdus akhurstii Che4.1, was isolated and characterized. Nematode identification was confirmed by ITS and 28S rDNA sequencing, while genome-based analyses supported the taxonomic assignment of the bacterial symbiont. The nematode-bacterium complex exhibited insecticidal activity against Spodoptera exigua and Spodoptera littoralis. H. indica Che4.1 showed an LC of 53.7 infective juveniles (IJs)/mL against S. exigua, comparable to that of the commercial reference Larvanem (54.3 IJs/mL). Against S. littoralis, Che4.1 exhibited an approximately twofold lower LC than Larvanem (77.3 versus 155.9 IJs/mL), with non-overlapping 95% confidence intervals. The complete genome of P. akhurstii Che4.1 consists of a single circular chromosome of 5.66 Mb encoding a diverse repertoire of putative virulence factors, including toxin complex (Tc) components, YenTc-like proteins, Mcf toxins, App-family proteins, Pra/Prb proteins, and chitinases, together with several predicted secondary metabolite biosynthetic gene clusters. LC-MS/MS analyses of bacterial culture supernatants and infected insect cadavers detected multiple predicted virulence-associated proteins, including Tc-related, YenTc-like, App-family, and Pra/Prb proteins. In addition, bacterial cell-free supernatants caused 68.8-81.3% mortality in S. exigua neonates under laboratory conditions. Together, these findings reveal a diverse repertoire of potential virulence determinants in P. akhurstii Che4.1 and demonstrate the insecticidal potential of its native nematodebacterium complex. The performance of H. indica Che4.1 relative to a commercial reference supports further evaluation of this isolate for biological control of Spodoptera spp., particularly under greenhouse and field conditions.

The role of aberrant glycosylation in pancreatitis remains an under-investigated area of research. Here, we determined that the ST6GAL1 sialyltransferase, which adds 2-6-linked sialic acids to N-glycoproteins, was upregulated in pancreatic tissues from patients with acute (AP) and chronic (CP) pancreatitis, and in mice with experimental AP. Within these tissues, ST6GAL1 was selectively expressed in acinar cells undergoing acinar to ductal metaplasia (ADM), a process whereby injured acinar cells de-differentiate and re-enter the cell cycle to enable tissue repair. To study the functional role of ST6GAL1 in pancreatitis, we used HPNE pancreatic epithelial cells with modulated ST6GAL1 expression, along with pancreatic organoids from mice with transgenic expression of ST6GAL1 (SC mice). In these models, ST6GAL1 activity promoted the activation of EGFR (a well-known ADM-driver), ERK, and AKT. ST6GAL1-expressing cells also displayed an ERK-dependent upregulation of the anti-apoptotic proteins, Mcl-1 and Bcl-xL, and impaired apoptotic signaling by the TNFR1 and Fas death receptors. Unbiased kinomics profiling revealed that ST6GAL1 induced the activation of many receptor tyrosine kinases associated with cell survival and proliferation (e.g., EGFR, PDGFR, MET). Furthermore, ST6GAL1 enhanced the survival and proliferation of cells exposed to stress-inducing conditions such as serum withdrawal. Based on these findings, we hypothesized that the pro-survival phenotype imparted by ST6GAL1 would facilitate tissue healing following a bout of pancreatitis. Accordingly, we induced AP in wild type and SC mice via L-arginine injection and found that SC mice had more rapid and efficient tissue repair, evidenced by accelerated restoration of the acini, diminished acinar apoptosis, and reduced immune cell infiltration. Together, these findings highlight a novel glycosylation-dependent mechanism that drives cell survival, positioning ST6GAL1 as a key adaptive mediator of pancreatic regeneration.