Toxins

Alternaria fungi produce various structurally diverse mycotoxins, several of which exhibit immunomodulatory properties. Among these, alternariol monomethyl ether (AME), alternariol (AOH), alterperylenol (ALTP), altertoxin I (ATX-I), and altersetin (AST) have been reported to suppress lipopolysaccharide (LPS)-induced inflammatory responses. However, the precise molecular mechanisms underlying these effects remain unclear. The present study aimed to elucidate how these selected Alternaria mycotoxins (0.1-50 M) target the NF-{kappa}B signaling pathway in THP-1 monocytes. Key components of the NF-{kappa}B cascade were analyzed by immunofluorescence microscopy, Western blotting and qRT-PCR. Nuclear translocation of NF-{kappa}B p65 and its phosphorylated form (p- NF-{kappa}B p65) was assessed by Western blot, while cytokine responses were determined at transcript (qRT-PCR) and protein (ELISA) levels. Moreover, in silico docking analyses were performed to investigate potential interactions of the toxins with IKK{beta}, and receptor-mediated crosstalk was studied using the glucocorticoid receptor (GR) antagonist RU486. Co-treatment with RU486 attenuated the immunosuppressive effects of 1 and 5 M AOH, indicating partial involvement of GR-dependent mechanisms. AME, AOH, ALTP, ATX-I, and AST increased total I{kappa}B levels while reducing its phosphorylated form. Additionally, AST and ALTP decreased the protein levels of Toll-like receptor 4 (TLR4), the I{kappa}B kinase (IKK) complex, NF-{kappa}B p65, and p- NF-{kappa}B p65. While AOH (5 M) and AST (25 M) reduced nuclear translocation of p65 and p-p65, ALTP (2 M) enhanced nuclear localization despite decreasing cytokine expression. Together, these findings suggest toxin-specific interference at multiple regulatory levels of NF-{kappa}B signaling and provide novel mechanistic insight into the immunomodulatory effects of Alternaria mycotoxins.

Botulinum neurotoxin (BoNT) is an etiologic agent of food poisoning caused by Clostridium botulinum. The large progenitor toxin complex (L-PTC) crosses the intestinal epithelial barrier to deliver BoNT to target neurons; however, it is not clearly understood how BoNT enters the host. Here, we identified low-density lipoprotein receptor-related protein 1 (LRP1) as a major enterocyte transcytosis receptor for the hyper-oral-toxic L-PTC serotype B-Okra (L-PTC/BOkra). We found that hemagglutinin (HA), a neurotoxin-associated protein within the L-PTC/BOkra complex, binds to LRP1 via N-glycans. HA/BOkra co-localized with LRP1 within the internalized vesicles in cultured cells and enterocytes. LRP1 deletion inhibited the apical-to-basal transcytosis of L-PTC/BOkra in an intestinal epithelial cell line, and this effect was rescued by LRP1 re-expression. Finally, intestinal epithelial cell-specific LRP1-deficient mice displayed reduced susceptibility to toxicity caused by oral administration of L-PTC/BOkra. Taken together, these results indicate that N-glycosylated LRP1 mediates L-PTC/BOkra transcytosis via enterocytes, enabling BoNT to traverse the intestinal epithelial barrier.

Tenuazonic acid (TeA) is one of the most frequently detected Alternaria mycotoxins in contaminated food. Despite its frequent occurrence, its immunomodulatory effects remain insufficiently characterized. Therefore, the present study investigated the impact of TeA on inflammatory signaling and cytokine regulation in monocytes and intestinal epithelial cell (IEC) models. NF-{kappa}B activity was assessed using a reporter gene assay in THP1-Lucia monocytes, while cytokine mRNA expression and protein secretion were quantified in Caco-2 and HCEC-1CT cells by qRT-PCR and ELISA, respectively. In THP-1 monocytes, TeA significantly suppressed lipopolysaccharide (LPS)-induced NF-{kappa}B activation in a concentration-dependent manner starting at 25 M, while cytotoxicity occurred only at concentrations [≥]100 M. In HCEC-1CT and differentiated Caco-2 cells, TeA increased IL-6, IL-8, and TNF- mRNA levels at non-cytotoxic concentrations ([≥]10 M). In Caco-2 cells, these transcriptional changes were accompanied by increased cytokine secretion, whereas HCEC-1CT cells showed only partial effects on the protein level after short-term exposure. Following prolonged incubation, TNF- secretion was increased and IL-6 and IL-8 secretion were slightly reduced. IL-10 remained unaffected under all conditions. Overall, TeA exerted cell type-dependent immunomodulatory effects characterized by immunoinhibitory activity in monocytes and pro-inflammatory responses in IECs, highlighting the complex immunotoxic potential of this Alternaria mycotoxin.

An acetaminophen (APAP) overdose is the leading cause of drug-induced hepatotoxicity and acute liver failure (ALF) in the United States. While N-acetylcysteine (NAC), is highly effective when administered early after an overdose, its efficacy decreases with delayed administration. Since most patients present late to the clinic, there is an urgent need for novel late-acting therapeutic options to prevent progression to ALF. We previously demonstrated the benefit of delayed activation of the Adenosine A2B Receptor (A2BAR) in attenuating APAP-induced hepatotoxicity and this study focuses on its effects on liver recovery after injury. Fasted male C57BL/6J mice were treated with 300 mg/kg APAP, followed by activation of A2BAR 6 or 9 h later and sacrifice 24, 48 or 72 h post-APAP with evaluation of liver injury, the innate immune response and liver regeneration. Delayed activation of A2BAR significantly enhanced liver recovery, with accelerated repopulation of the liver by Kupffer cells, increased macrophage migration to the necrotic areas and their faster resolution. A2BAR activation also upregulated lipid metabolism related genes in non-parenchymal cells and cell proliferation and metabolism genes in hepatocytes. Remarkably, genes such as Cidec and Plin2, crucial for lipid droplet formation, were upregulated, indicating that A2ABR activation enhances lipid metabolism which plays a key role in providing energy for liver regeneration. Overall, these findings highlight the potential of A2BAR activation not only in protecting against liver injury, but also in promoting and accelerating liver regeneration by modulating the innate immune responses and metabolic pathways.

Anthelmintic drugs are used to control soil-transmitted helminths that infect a third of the worlds human population. There is increasing concern about the development of resistance to anthelmintic drugs because of the limited number of compounds available and there is an unmet need for new resistance-busting drugs. Here we describe the presence of a previously unrecognized endogenous acetylcholine analogue, {beta}-alanine betaine, which may serve as an endogenous ligand for an alternate subfamily of nicotinic receptors (DEG-3/DES-2) that could be developed as novel drug targets because their analogues are not present in their human or animal hosts. We collected peri-enteric fluid from female Ascaris suum (a model for the human parasite, Ascaris lumbricoides) and subjected it to chromatography and MS/MS to reveal signals consistent with acetylcholine, choline, and {beta}-alanine betaine but we did not recover betaine. We injected betaine into female Ascaris suum which produced no effect. However, injection of {beta}-alanine betaine, produced characteristic pretzel coiling and injection of levamisole produced a rod-like spastic paralysis. The differences between {beta}-alanine betaine and levamisole suggested that they activate different nAChRs subfamilies. PCR showed that messages of the DEG-3 subfamily of nAChR channels, which are betaine targets and were present in the intestine and body wall of A. suum. Calcium signaling experiments showed that {beta}-alanine betaine increased intracellular calcium of the intestine enterocytes and electrophysiology of the body muscle cells demonstrated that {beta}-alanine betaine produced membrane potential depolarization. In N2 elegans, application of {beta}-alanine betaine produced gradual inhibition of motility, which was reduced in acr-20, acr-23, des-2, deg-3 and lgc-41 null-mutants. These observations suggest that, in addition to acetylcholine, {beta}-alanine betaine - an anaerobic analog of betaine - may function as an endogenous ligand in anaerobic nematodes such as A. suum. An expanded repertoire of nicotinic acetylcholine receptor subfamilies in nematodes relative to mammals may reflect a corresponding need for diversification of cholinergic endogenous ligands in these organisms. This repertoire could allow their simpler neuronal system to perform more complex controls and be exploited for development of different and novel subfamily selective cholinergic anthelmintics.

The type VII secretion system (T7SS) is found in many Gram-positive bacteria and secretes toxins with antibacterial activity. Most characterised substrates have an N-terminal LXG domain that interacts with other helical partner proteins to form a composite T7SS targeting signal. Here we describe only the second substrate family to have a reverse domain arrangement. We show that TslM has a C-terminal LXG-like domain and an N-terminal lipase domain that has phospholipase activity. Secretion of TslM requires a single helical partner protein that binds to the TslM C-terminus, and its toxic activity is neutralised by a distinct family of membrane proteins. Genome analysis reveals that Staphylococcus aureus strains have the capacity to encode up to seven paralogous copies of this toxin family. Taken together our findings show that lipases are an important component of the staphylococcal T7SS toxin arsenal, and that toxins with a reverse domain arrangement are more widespread than previously appreciated.

Thymosin {beta}4 (T{beta}4) is a conserved acidic polypeptide with 43-amino acids participating in multiple pathophysiological processes. In this study in vivo effects of T{beta}4 on liver regeneration are investigated in carbon-tetrachloride (CCL4) induced rodent animal liver jury models. Results illustrate that exogenous T{beta}4 treatment significantly reduced CCL4-rendered liver necrosis around central vein. At 48 hours after CCL4 insults hepatocytes proliferation occur mainly around the periportal area, while hepatocytes proliferation around the necrosis area is prominently increased by exogenous T{beta}4 treatment. The holistic proliferation level of liver tissues are also enhanced by exogenous T{beta}4. Hepatocyte proliferation activities negatively correlate with the necrosis extent of the liver tissue. These results suggested firstly exogenous T{beta}4 treatment could enhance liver regeneration and exhibit prosperous potential for application in clinical conditions such as liver transplantation.

Introduction: Thromboinflammation, which represents the pathological interplay between inflammation and thrombosis, is a leading cause of global mortality. Current therapies are frequently associated with an increased risk of bleeding and do not adequately address the inflammatory component of the disease. The African tree Guibourtia tessmannii represents a promising source of natural anti-inflammatory compounds. This study aimed to synthesize and characterize silver nanoparticles using an aqueous bark extract of G. tessmannii (GT-AgNPs) and to evaluate their anti-inflammatory and anticoagulant properties. Methods: GT-AgNPs were synthesized by reducing silver nitrate with an aqueous extract of G. tessmannii bark. The nanoparticles were comprehensively characterized using UV-Vis spectroscopy, FTIR spectroscopy, powder X-ray diffraction, and scanning electron microscopy. In vitro anti-inflammatory activity was evaluated through inhibition of bovine serum albumin denaturation, whereas in vivo anti-inflammatory activity was assessed using the carrageenan-induced rat paw edema model. Anticoagulant activity was investigated by measuring activated partial thromboplastin time (aPTT) and prothrombin time (PT), corresponding to the intrinsic and extrinsic coagulation pathways, respectively. Results: The synthesis successfully produced GT-AgNPs with an average particle size of approximately 20 nm. Both the aqueous extract and GT-AgNPs exhibited marked anti-inflammatory activity. The nanoparticles achieved 95% inhibition of protein denaturation in vitro and 95% inhibition of carrageenan-induced paw edema in vivo at a dose of 0.4 mg/kg body weight after 5 h. Furthermore, both the extract and GT-AgNPs demonstrated dose-dependent anticoagulant activity. Conclusion: The study demonstrated that GT-AgNPs, synthesized from the bark of G. tessmannii, possess significant anti-inflammatory and anticoagulant properties. These findings highlight the potential of GT-AgNPs as nanotherapeutic candidates for the management of thrombo-inflammatory disorders.

BackgroundNeurulation is a fundamental process in the formation of the central nervous system (CNS). The process begins with the folding and fusion of the neural plate to form the neural tube which subsequently gives rise to the development of the brain and spinal cord. Environmental and genetic factors that disrupt neurulation can induce neural tube defects (NTDs) and consequently cause additional developmental complications, including motor impairments. Purinergic signaling is a conserved form of extracellular communication (i.e. paracrine, synaptic signaling) that plays a role in early development. This signaling is mediated by purine nucleotides and nucleosides, which activate metabotropic P2Y and ionotropic P2X purinoceptors, respectively. Distinct patterns of intracellular calcium dynamics are observed throughout vertebrate development, from fertilization through organogenesis, including neurulation. Among P2X receptors, P2X4 is an ATP-modulated, Ca2+-permeable, ligand-gated ion channel characterized by having the highest Ca2+ permeability and is known to be modulated by ivermectin (IVM). ObjectiveOur investigation focuses on assessing the effects of IVM treatment during neurulation and evaluating the impact of this drug on phenotype, motor behavior and neuromuscular junction (NMJ) structure at tadpole stage. These results were compared with those obtained following separate treatments with compounds that specifically block glycine, GABA(A) and nACh receptors, all which have been described as IVM targets. ResultsIn this study we demonstrate the transcriptional expression for both P2X and P2Y purinergic receptors during neurulation, as well as the expression of P2X4. Following IVM neurula-treatments, we observed neural tube defects (NTDs), pigmentation changes, motor paralysis and alterations in neuromuscular junction (NMJ) structure, particularly affecting axonal branching. In contrast, treatment with the blockers strychnine, bicuculline and -bungarotoxin, used to assess the involvement of GlyR, GABA(A)R and 7nAChR, respectively, failed to show similar outcomes. ConclusionsIn summary, our results highlight the critical role of purinergic signaling during early development, particularly P2X4 receptor mediated signaling during neurulation which may account for the pharmacological effects induced by the positive allosteric modulator ivermectin.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, poses a growing global public health threat due to the rapid emergence of multidrug-resistant strains and the limited availability of effective treatments. Since there are no known lytic gonophages, we explored prophages present in the genome of N. gonorrhoeae FA1090, with a particular focus on prophage-encoded endolysins. In this study, we evaluate antigonococcal properties of prophage-encoded endopeptidases with the NlpC/P60 enzymatic domain. Recombinant endolysin Phi1gp518 exhibits intrinsic bactericidal activity against non-permeabilized N. gonorrhoeae FA1090 cells. Furthermore, it shows an expanded host range against clinical gonococcal isolates. The gonolysin remains stable across all human body temperatures, a pH range of 5-10, and shows no cytotoxic effects toward human cervical epithelial cells, supporting its potential safety for therapeutic applications. Additionally, Phi1gp518 impairs the formation of gonococcal microcolonies and prevents proper biofilm establishment. The antigonococcal properties of Phi1gp518 endopeptidase make it a good candidate for further protein engineering and development as an alternative treatment strategy for drug-resistant N. gonorrhoeae infections.

Key PointsO_LIPerfused human kidney MPS revealed CsA-associated sublethal tubular stress that was not detected by conventional 2D viability assays or by KIM-1 release in 3D MPS. C_LIO_LIAt matched exposure, VCS preserved mitochondria and activated ER chaperones and iron detoxification, with no p21 arrest compared to CsA. C_LIO_LIMechanistic separation supports VCSs nephroprotection potential and early mechanism-based biomarkers to guide CNI choice. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/737071v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@e5e01dorg.highwire.dtl.DTLVardef@1dc9167org.highwire.dtl.DTLVardef@1ce22f8org.highwire.dtl.DTLVardef@5a053a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG BackgroundCalcineurin inhibitors (CNIs) are indispensable for transplantation immunosuppression, yet cyclosporine A (CsA) produces renal toxicity. Voclosporin (VCS), a CsA analog, is proposed to be less nephrotoxic, but mechanisms remain unclear. MethodsPrimary human proximal tubule epithelial cells (PTECs) were exposed to CsA or VCS in 2D monolayers and perfused 3D kidney microphysiological system (MPS). Viability was assessed in 2D cultures by MTS, mitochondrial membrane potential ({Delta}{Psi}m) by TMRM flow cytometry, and soluble injury and inflammatory biomarkers in MPS effluents by ELISA and MSD multiplex assays. RNA sequencing of 3D-cultured PTECs was used to identify differentially expressed genes and pathways. ResultsIn 2D PTECs, neither drug reduced viability. In 3D MPS effluents, KIM-1 did not distinguish CsA from VCS, whereas the MSD biomarker panel showed larger aggregate deviation with CsA. Confocal tomography showed CsA-associated mitochondrial fragmentation, whereas VCS preserved reticular mitochondrial architecture. TMRM flow cytometry showed a treatment-dependent difference in TMRM-positive cells, with VCS yielding the highest TMRM-positive fraction and exceeding CsA, supporting preservation of {Delta}{Psi}m relative to CsA. RNA-seq identified 1188 CsA-specific and 185 VCS-specific differentially expressed genes, with 304 shared. Pathway analysis indicated CsA enrichment of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress, p21-associated G2/M checkpoint arrest, and transcriptional signatures consistent with ferroptosis priming, while VCS mainly induced ER chaperone and ER-associated degradation gene programs without activating canonical UPR sensors and showed limited cell-cycle suppression. ConclusionsA physiologically relevant 3D kidney MPS revealed sublethal tubular stress from CsA that is masked in 2D culture, including mitochondrial depolarization, proteostatic stress, and ferroptosis priming. At matched exposure, VCS preserved mitochondrial function and proteostasis while eliciting a narrower, adaptive ER quality control response. These data support VCS as a nephron-sparing immunosuppressant and 3D MPS as a mechanism-based platform for evaluating renal safety of drugs and nominating early sub-lethal tubular injury biomarkers.

Tenuazonic acid (TeA) is an emerging Alternaria mycotoxin frequently detected in food and feed commodities, raising concerns about its toxicological relevance. Chronic oral exposure to TeA has been reported to induce dysplastic alterations in the esophageal mucosa of mice, while human biomonitoring data indicate an association between TeA exposure and esophageal cancer, although a causal relationship has not yet been established. At a mechanistic level, the effects of TeA in esophageal cells remain poorly characterized. Therefore, this study investigated the impact of TeA on cytotoxicity, oxidative stress, DNA damage, mitochondrial homeostasis, cell-cycle distribution and transcriptomic stress responses in human esophageal KYSE-510 cells. TeA induced a concentration-dependent reduction in metabolic activity and total protein content after 24 h exposure to 0.1-100 M. Significant cytotoxicity was measured starting from 20 M. At sub-cytotoxic concentrations, TeA triggered rapid ROS formation within 5-30 min exposure and induced formamidopyrimidine-DNA glycosylase (FPG) sensitive DNA damage after 1 h exposure (5-7.5 M), indicating oxidative DNA lesions. In addition, TeA altered mitochondrial morphology after 4 h exposure at 7.5 M, manifested by shrinkage of the mitochondrial network area and perinuclear redistribution, while mitochondrial respiration showed only a non-significant tendency towards reduced respiratory capacity. RNA sequencing after 6 h exposure to 10 M TeA revealed oxidative stress-associated transcriptional changes, impaired antioxidant and stress-adaptive responses, and p53-associated stress signaling. Furthermore, TeA induced significant G2/M phase accumulation after 24 h exposure to 1-10 M.

IntroductionLomentospora prolificans is a pathogenic filamentous fungus that causes disease primarily in people with severely compromised immune systems. It is pan-resistant to antifungal drugs, but the mechanism of its resistance to amphotericin B (AMB) is unknown. ObjectivesWe aimed to investigate the mechanism of resistance to AMB of L. prolificans. MethodsThe AMB susceptibility of L. prolificans protoplasts was measured using broth microdilution. L. prolificans, either intact, homogenized or fractionated was incubated with AMB in broth. The same activity was carried out with Aspergillus fumigatus as a control. This broth was then used to prepare microdilution plates with Saccharomyces cerevisiae to determine the activity of the conditioned AMB. ResultsAMB was 16-fold more effective in inhibiting the growth of L. prolificans protoplasts than conidia, but only two-fold more effective against A. fumigatus protoplasts than conidia. Incubation of L. prolificans hyphae with AMB in media diminished drug activity to a much greater extent than A. fumigatus, with 8-fold greater fungal mass of the latter required to achieve the effect of the former. Homogenization and fractionization of L. prolificans revealed that the factor inhibiting AMB activity was soluble with a mass >100 kda. DNase, trypsin, proteinase K, amyloglucosidase, SDS and 0.22 m had no effect on the AMB resistance factor, while treatment with urea, acetonitrile inactivated it. ConclusionWe report a different mechanism for AMB resistance based on the existence of a substance residing in the L. prolificans cell wall that can eliminate the antifungal activity of AMB.

The pathogenic fungus Trichosporon asahii causes severe mycoses in immunocompromised hosts, such as neutropenic patients. In Cryptococcus neoformans, the unfolded protein response (UPR) sensor Ire1 induces hxl1 mRNA splicing and contributes to stress responses and virulence. The function of Ire1-triggered hxl1 mRNA splicing in stress tolerance and virulence of T. asahii, however, remains unclear. Here, we demonstrated that ire1- and hxl1 gene-deficient T. asahii mutants are sensitive to dithiothreitol (DTT), an inducer of endoplasmic reticulum stress, and exhibit reduced virulence in a silkworm infection model. DTT treatment induced hxl1 mRNA splicing in the wild-type strain, whereas ire1 gene-deficient mutants did not undergo hxl1 mRNA splicing. The ire1 gene-deficient mutants were more sensitive than the parent strain to DTT, H2O2, Congo red, and SDS, and showed reduced virulence in silkworms. Similarly, hxl1 gene-deficient mutants exhibited increased sensitivity to these stressors and reduced virulence. Both the ire1 gene-deficient and hxl1 gene-deficient mutants showed decreased expression of reactive oxygen species-detoxifying related genes CAT2, SOD1, and SOD2, compared with the parent strain. Together, these findings suggest that Ire1-triggered hxl1 mRNA splicing contributes to stress resistance and virulence in T. asahii.

Probiotics produce antimicrobial peptides and small molecules that are secreted into the medium. Antimicrobial activity of cell-free supernatants can be tested using various qualitative and quantitative methods. Many of these techniques employ methods which introduce uncontrolled variables, impacting reproducibility and making comparison of reported results difficult. Here, we present a simple procedure for quantitative estimation of antimicrobial activity of cell-free supernatants which overcomes drawbacks of commonly used methods.

Background: Snakebite envenoming causes an estimated 138,000 deaths and more than 400,000 cases of permanent disability each year, disproportionately affecting low-resource regions. Antivenom is the only treatment but requires intravenous administration. We evaluated intravenous followed by oral varespladib as adjunctive therapy to antivenom in hospitalized patients with snakebite envenoming. Methods and findings: In this multicenter, randomized, double-blind, placebo-controlled phase II trial in India (CTRI/2023/10/058782) and the USA (NCT05717062), patients with snakebite envenoming were randomly assigned (1:1) to receive varespladib or placebo in addition to standard of care. For elapid envenoming, the primary endpoint was time to recovery of 5-second head-lift. For viper envenoming, the primary endpoint was area under the curve (AUC) from baseline to Day 14 of a 3-item Snakebite Severity Score (SSS). Between June 3rd, 2023 and October 19th, 2024, 140 patients were randomized and 139 were analyzed (73 varespladib; 66 placebo). Study drug was initiated a mean of 7.3 hours after the bite and 3.3 hours after antivenom. Among elapid-bite patients, mean time to head-lift recovery was 27.6 hours with varespladib vs. 36.2 hours with placebo (p = 0.6). Among viper-bite patients, SSS AUC to Day 14 was 660 vs. 629 in varespladib vs. placebo (p = 0.7). No serious adverse event occurred with varespladib. Conclusions: In this trial in which varespladib was initiated on average 7 hours after snakebite and several hours after antivenom, adjunctive varespladib did not improve the primary endpoints. Other study designs and settings will be needed to assess the effect of varespladib when initiated prior to antivenom.

Background & Aims: Activation of apoptosis signal-regulating kinase 1 (ASK1), a ubiquitous redox-sensitive kinase, results in inflammation, apoptosis, and fibrosis, key common pathways in human liver disease. SRT-015 is a novel, small molecule inhibitor of ASK1. This study evaluated the in vitro efficacy of SRT-015, compared it to other ASK1 inhibitors, and determined the in vivo efficacy of SRT-015 across multiple acute and chronic liver disease models. Methods: In vitro studies determined the kinase potency and selectivity of SRT-015, and cellular studies were used to demonstrate direct mechanisms of action. The cardiac hERG channel inhibition was assessed and PK determined in rodents and nonhuman primates. In vivo studies evaluated SRT-015 efficacy in rodent models of drug-induced hepatotoxicity (acetaminophen (APAP) overdose), alcohol-associated liver disease (ALD), metabolic-disease associated steatohepatitis (MASH) and cholestatic disease (bile duct ligation, BDL). Results: SRT-015, was demonstrated a selective ASK1 kinase, and SRT-015 treatment directly inhibited fibrosis, apoptosis and inflammation in activated human fibroblasts, hepatocytes and PBMCs, respectively without safety signals or hERG inhibition. Other ASK1 inhibitors had safety concerns or limited functional activity. Liver and kidney selective PK were observed for SRT-015 in all species evaluated. In vivo, SRT-015 treatment was efficacious in the acute mouse APAP overdose and ALD model significantly (P<0.05) decreasing serum ALT. Using a therapeutic diet-induced obesity (DIO)-MASH model with biopsy-verified fibrosis, SRT-015 treatment significantly (P<0.05) inhibited DIO-induced liver enzymes, hepatomegaly, fibrosis, inflammation, and apoptosis independent of body weight loss whereas treatment with selonsertib was ineffective. In a rat cholestatic model, SRT-015 treatment significantly (P<0.05) decreased fibrosis and stellate cell activation. Conclusions: These findings support SRT-015 as a potential therapeutic for human liver diseases of any etiology.

BackgroundLyme borreliosis is a common tick-borne disease in Europe caused by spirochetes of the Borrelia burgdorferi sensu lato complex, including Borrelia afzelii, which is maintained in nature through interactions with rodent reservoir hosts. These spirochetes have evolved several surface proteins to manipulate rodent host immunity, some of which remain polymorphic in Borrelia populations. Among these proteins, OspE, which binds the host complement-regulating factor CFH to evade destruction by complement, is one of the most variable. Yet, what evolutionary forces maintain this polymorphism is not well understood. Motivated by a recent discovery of CFH polymorphism in the bank vole (Clethrionomys glareolus), the main reservoir host of B. afzelii, we hypothesized that the polymorphism is maintained by host-parasite coevolution involving specific associations between host and parasite genetic variants. MethodsWe analyzed associations between bank vole CFH alleles and B. afzelii OspE variants across three datasets sampled in Poland. Selection acting on OspE was evaluated using omegaMap. Host-pathogen genotype associations were tested using partial redundancy analysis (RDA), and co-structure was assessed using co-correspondence analysis (CoCA). ResultsWe found that OspE evolves under positive selection, however, we found no evidence for an association between OspE and host CFH variants at the individual level based on RDA or at the population level based on CoCA. ConclusionsDespite evidence of positive selection acting on OspE, we found no support for specific genetic matching between B. afzelii and its bank vole host at the CFH-OspE interface. These results suggest that the evolution of CFH and OspE may be shaped by broader selective pressures, potentially including interactions with multiple host species.

Alternaria toxins are naturally occurring food contaminants with limited and often inconsistent genotoxicity and mutagenicity data. Within the European Partnership for the Assessment of Risks from Chemicals (PARC), an OECD-aligned in vitro testing strategy was applied to fill existing data gaps and to characterize the genotoxic potential of major Alternaria toxins using high-purity test materials. Mutagenicity was assessed using bacterial reverse mutation test (OECD TG 471) and SOS/umu assay, while chromosomal damage was assessed using the in vitro micronucleus (MN) assay (OECD TG 487) in TK6 and HepG2 cells, complemented by fluorescence in situ hybridization (FISH) and {gamma}H2AX assay in HepaRG cells. Alternariol (AOH), alternariol monomethyl ether (AME), and altertoxin-I (ATX-I) showed clear mutagenicity in bacteria, whereas altenuene (ALT), tenuazonic acid (TeA), and tentoxin (TEN) were negative under the tested conditions. In mammalian cells, AOH, AME, and ATX-I induced MN formation in TK6 cells at concentrations [&ge;]5.5 {micro}M, [&ge;]2.5 {micro}M, and [&ge;]0.21 {micro}M, respectively, with FISH analysis supporting a clastogenic mode of action. In HepG2 cells, all tested toxins induced chromosomal damage, with effect threshold ranging from [&ge;]6.25 {micro}M (AOH) to [&ge;]50 {micro}M (TeA). {gamma}H2AX induction confirmed DNA damage for AOH and ATX-I, and at higher concentrations for TeA (1000 {micro}M). Overall, the data indicate clear in vitro genotoxic potential for AOH, AME, and ATX-I and provide evidence of chromosomal damage for ALT, TEN, and TeA, thereby reducing critical data gaps for hazard assessment.