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.

The cyanobacterium Dolichospermum circinale is a known producer of the neurotoxin saxitoxin and its analogues, collectively known as the paralytic shellfish toxins (PSTs). PSTs vary in potency, and the reported toxin profiles of D. circinale blooms vary in the quantities of individual PSTs, with the regulation of these profiles being poorly understood. In this study, we present the genomes of four D. circinale strains (ACBU01, ACMB03, ACMB13 and FSS-124) with unique PST profiles and perform genome-wide comparisons and specific analysis of the PST-producing biosynthetic gene cluster (sxt) to understand the variability in PST quotas. A reassessment of the previously published D. circinale AWQC131C genome was also performed to collate genomic variation between all strains. Analysis at the nucleotide and amino acid sequence level revealed that toxic strains maintain high genome-wide similarities, corroborated by the analysis of the pan- and variable genomes of each strain. Specifically, the sxt gene sequences were 99-100% identical across all strains. Novel tailoring (sxtSUL, sxtDIOX) and transport (sxtM4) genes were identified within the sxt cluster that were not reported previously in D. circinale. Taken together, these results indicate that the genetic machinery involved in PST production is conserved in this species, suggesting that the regulation of PST biosynthesis in D. circinale does not occur at the genomic level.

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.

The preclinical safety of human-equivalent lactoferrin alpha (heqLF; effera(R)), produced by Komagataella phaffii, was evaluated to support its use as a food ingredient in infant formula and products for young children. Genotoxicity was assessed using a bacterial reverse mutation (Ames) assay in five strains of Salmonella typhimurium and Escherichia coli, and an in vitro micronucleus assay in TK6 cells. Both studies followed OECD guidelines and were conducted up to the recommended limit concentrations (5000 {micro}g/plate and 2000 {micro}g/mL, respectively). A 14-day non-GLP juvenile dose range-finding study and a 13-week GLP juvenile rat toxicity study with a 4-week recovery phase were conducted under ICH S11 guidance. Neonatal Sprague-Dawley rats received heqLF at 0, 1500, 3000, or 5000 mg/kg body weight/day by twice-daily oral gavage from postnatal day 7 to 98. Bovine lactoferrin (bLF) and whey protein at 5000 mg/kg body weight/day served as reference controls. heqLF was non-mutagenic and non-clastogenic in both genotoxicity assays. In the 13-week juvenile rat study, no heqLF-related mortality, clinical signs, developmental, neurobehavioral, or immune toxicity effects were observed. Minor, non-adverse renal findings consistent with high protein intake were observed and largely reversed during recovery. Clinical pathology parameters fully resolved during recovery. Toxicokinetic evaluation showed no systemic accumulation. Based on the absence of toxicologically relevant adverse findings, heqLF is well tolerated at doses up to 5000 mg/kg body weight/day, establishing a no observed adverse effect level (NOAEL) of 5000 mg/kg/day, the highest dose tested, and supporting its safety across intended populations from birth to adulthood.

IntroductionSickle cell disease is an inherited hemoglobinopathy with defective red cell deformability. The defective deformability promotes microvascular occlusion and subsequent vaso-occlusion in sickle cell disease patients. Previous studies have demonstrated that thiol isomerases, an endoplasmic reticulum-resident oxidoreductase that is released from vascular cells into the bloodstream, are present on red cell membrane and contribute to cellular dehydration and sickling. However, the role of membrane-bound thiol isomerases on sickled red blood cells is unclear. MethodsUsing red blood cells from Townes humanized sickle cell or non-sickled mice, we performed ektacytometry assay under shear using laser assisted optical rotational cell analyzer (LORRCA) to assess the effects of antagonizing thiol isomerases with isoquercetin and a functional blocking monoclonal antibody. The densitometric properties of sickled red blood cells in the presence of isoquercetin was also tested using magnetic levitation. ResultsThiol isomerase antagonism increased sickled red cell elongation, cellular dehydration and the diamagnetic signature compared to control treatment. ConclusionThiol isomerases may be involved in regulating sickled red blood cells mechanical properties through mechanisms that require further investigation.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

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.

Coevolution proceeds through the evolution of traits that mediate ecological interactions and evolutionary outcomes. In the arms race between toxic Pacific newts (Taricha) and their garter snake predators (Thamnophis), this interface involves tetrodotoxin (TTX), an antipredator defense that inhibits nerve and muscle function by blocking voltage-gated sodium channels. In response, snakes have evolved TTX-resistant channels, in some cases leading to snake populations that are nearly invulnerable to TTX. For decades, newt TTX has been treated as a single defensive trait, yet TTX occurs as a family of structurally related analogs that may represent alternative defenses against snakes. Here, we characterize TTX analog diversity in all four species of Taricha and evaluate how these compounds interact with the sodium channels in coevolved garter snakes. Using LC-MS analysis of newt skin secretions, we detected a diverse suite of TTX analogs previously unrecognized in Pacific newts. We then used molecular docking models to evaluate interactions between various TTX analogs and variants of the skeletal muscle channel (Nav1.4) that span the range of TTX resistance in garter snakes. We found that some TTX analogs docked better than canonical TTX in resistant snake channels. Notably, we show that 11-deoxy-4-epi-TTX and 11-deoxy-TTX have favorable interactions with hydrophobic amino-acid substitutions in extremely resistant garter snake sodium channels, potentially circumventing predator resistance to canonical TTX. Our results suggest a complex arms race involving multiple newt TTX analogs and multiple snake sodium channel variants. As such, newts may keep pace with snakes by diversifying their arsenal of chemical weapons.

Crush syndrome (CS) is a serious medical condition characterized by damage to the muscle cells due to pressure and is associated with high mortality, even in patients receiving fluid therapy. We focused on adrenaline (Adr), a standard medication administered by medical teams dispatched during disasters. Adr is readily available for use in disaster scenarios owing to its inclusion in standard emergency kits. The effectiveness of Adr in the treatment of CS remains a subject of ongoing debate. This study aimed to evaluate the impact of Adr on acute complications, such as heart failure, shock, and renal failure, and explore whether its influence on inflammatory pathways is correlated with improved survival in rats with CS. The CS model involved subjecting anesthetized rats to bilateral hindlimb compression using a rubber tourniquet for 5 h. Subsequently, the rats were randomly divided into eight groups. Under continuous monitoring and recording of the arterial blood pressure, blood and tissue samples were collected for biochemical analyses at designated time points before and after reperfusion. The survival rate, vital signs, and blood gas parameters were higher in the CS group than in the sham group. They were improved in the Adr-treated group (0.01 or 0.01 mg/kg), which was not significantly different from that in the CS group, despite the improvement in shock and kidney dysfunction. In conclusion, intramuscular Adr provides immediate hemodynamic stabilization and renal protection during the early stages of CS. However, its use requires careful dose titration; low doses may promote the systemic release of lethal toxins, whereas high doses may worsen metabolic acidosis. These findings highlight the importance of combining Adr with other therapies, such as fluid resuscitation, to manage systemic toxemia inherent in CS.

Metabolic dysfunction-associated steatotic liver disease (MASLD) and Clostridioides difficile (C. difficile) infection (CDI) are clinically associated, yet there is limited effective treatment for both diseases. Bile salt analogs (BSAs) have demonstrated potential in treating either MASLD or CDI. We screened a library of BSAs (n=112) previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells as candidates for prevention and treatment of both MASLD and CDI. The screening was based on an in vitro model established by incubating HepG2 cells with free fatty acids, with obeticholic acid (OCA), a known BSA with anti-MASLD activity as a control. Gene and protein expressions were quantified to validate the treatment effect. We found that compounds C13, C24, C25, C74, C98, and C101 demonstrated significant effectiveness in both preventing the intracellular accumulation of lipids and removing pre-loaded cellular lipids. Gene expression analysis showed that C24, C25, and C74 produced a similar pattern characterized by a robust induction of FGF21 expression, while C13, C98, and C101 produced a transcription pattern that mirrors the effect of OCA. Structurally, while C13, C24, and C25 do not display drug-like properties, C74, C98, and C101 are drug-like and share a similar structure. Interestingly, C101 is a potent inhibitor of C. difficile spore germination. OCA shows a weak anti-gemination effect. Our study identified lead compound candidates for the development of novel therapeutics capable of treating both MASLD and CDI. Significance statementThe clinical association between MASLD and CDI remains an unmet need for dual acting therapeutic strategies. Given the reported potential of BSA, we screened 112 previously synthesized as potential inhibitors of C. difficile spore germination, for their therapeutic potential in reducing intracellular accumulation of fatty acids in HepG2 cells. Our study identified compounds that effectively reduce intracellular lipid accumulation and inhibit C. difficile spore germination. These results nominate lead candidates for developing dual-acting therapeutics targeting both MASLD and CDI.

Among venomous animals, cnidarians represent the oldest metazoan lineage in which venom production and a specialized delivery system are defining synapomorphies. Cnidarians also represent the only venomous lineage for which mutualistic symbioses have evolved resulting in scenarios where mutualistic symbionts may also be targets of their venom. The most iconic example of this relationship is the mutualism between clownfish and their venomous sea anemone hosts. To investigate how symbiont presence and establishment influence toxin gene expression, we used a comparative TagSeq and RNA-Seq approach to quantify venom gene dynamics during the first 48 hours of clownfish-anemone symbiosis establishment in five anemone species. Our taxonomic sampling included three typical hosting species (Entacmaea quadricolor, Radianthus crispa, and Stichodactyla haddoni), each representing distinct evolutionary lineages of clownfish hosts, and two atypical Caribbean species (Condylactis gigantea and Stichodactyla helianthus) that do not host clownfish in nature, but have reported to host within the aquarium trade. Tentacle samples were collected prior to hosting, approximately 12 hours after initial symbiont establishment, and again 48 hours after symbiosis establishment. Our analyses revealed that overall toxin assemblages remained relatively stable during the early establishment phase, with no significant changes in the most highly expressed toxin gene candidates. However, subtle transcript-level shifts occurred within multi-copy toxin gene families, including cytolytic actinoporins and Sea Anemone 8 (SA8)-like toxins. Notably, one C. gigantea actinoporin transcript exhibited a [~]600-fold increase in expression in a single individual, which coincided with two clownfish mortalities prior to successful association, which subsequently decreased after establishment. Comparative sequence alignments suggest that amino acid substitutions in this transcript may be functionally relevant to symbiosis intolerance, as the amino acid substitutions were unique to this transcript, and not found in any other previously described cytolytic actinoporin. Together, these findings reveal that early toxin gene expression in clownfish-hosting sea anemones is largely stable, yet subtly dynamic at the transcript level. This study provides the first comparative transcriptomic insights into the molecular processes shaping symbiosis establishment in clownfish-anemone mutualisms, offering a framework for understanding venom evolution in the context of co-evolutionary interactions. HighlightsO_LIComparative gene expression survey reveals relatively stable toxin assemblages throughout the first 48 hours of establishing clownfish-anemone symbiosis. C_LIO_LISubtle shifts were observed among transcript variants in multi-gene copy variants, with potential implications for barriers to establishing symbiosis. C_LIO_LIAlthough toxin assemblages varied among species, sea anemone 8 (SA8) toxin-like transcripts were highly abundant in four of the focal taxa. C_LIO_LIThis is the first comparative gene expression analysis investigating molecular processes surrounding symbiosis establishment between clownfish and sea anemones. C_LIO_LIThese results provide insight into toxin dynamics surrounding the establishment of symbiosis, with particular insights into key evolutionary transitions resulting in symbiosis among atypical clownfish hosting species. C_LI

Animal venoms represent a major source of chemical novelty, yet how venom compounds originate, diversify, and are maintained across deep evolutionary timescales remains poorly understood. This gap is especially pronounced in cephalopods, which evolved venom systems used in predation, defense, and sexual competition, but whose venom genetic architectures, secretory cell types, and venom-producing glands remain largely unexplored. To date, only a single cephalopod venom compound with confirmed paralytic activity and a known primary sequence, SE-CTX from the golden cuttlefish Acanthosepion esculentum, has been described. Here, we reconstruct the evolutionary history, molecular diversity, and glandular localization of SE-CTX-like proteins using a multimodal approach. We identify 29 homologs across 20 squid and cuttlefish species and define a previously unrecognized venom gene family, which we name deca-ctx, specific to decapodiform cephalopods (squids and cuttlefish). Phylogenetic analyses reveal a single origin of deca-ctx followed by gene duplication and lineage-specific diversification, indicating long-term retention of this venom gene. Predicted DECA-CTX protein structures were separated into two clusters and 20 singletons highlighting potentially extensive structural diversity within a single cephalopod venom gene family. Proteomic analysis confirms expression of five DECA-CTX proteins across three species. Our imaging and histological analyses localize deca-ctx expression to specialized secretory cells within squid and cuttlefish venom glands. Together, these findings reposition SE-CTX as part of an evolutionarily and chemically diverse venom system, rather than an isolated venom protein, and establish cephalopods as a key lineage for investigating how new venom genes arise, diversify, and are integrated into functional venom arsenals.

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.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

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.

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

One major aftermath of COVID-19 pandemic is cardiovascular consequences. SARS-CoV-2 binds to ACE2 and downregulates vasodilation. Dengue favors hypotension by weakening endothelial glycocalyx leading to plasma leakage. C1q levels, immune complexes (ICs), and proteomic profiles in serum samples from 52 COVID-19 and 19 pre-pandemic Dengue cases were studied. Unlike Dengue, COVID-19 serums showed elevated coagulation proteins promoting vaso-occlusion and peripheral artery diseases. The stress-induced chaperone and atherosclerosis marker, GRP78 (gene/ protein) was found upregulated upon SARS-CoV-2 spike expression in cardiac/ lung cell lines. Elevated GRP78 levels were also observed in serum samples from COVID-19-diagnosed individuals and subjects with myocardial infarction (MI) in post COVID-era. Surprisingly, spike antibodies (Abs) showed cross-binding to GRP78 and possibly contributed to the observed higher-level ICs in COVID-19 serums (cardiovascular embolism?). Co-localization studies showed that spike Abs (analogous to pro-atherosclerotic GRP78 auto-Abs) could directly bind to upregulated cellular GRP78 (type II hypersensitivity?). Both pathways could worsen vascular injury and atherosclerosis, leading to cardiac complications in COVID-19 cases with narrowed vessels.

Venoms are powerful weapons that shape ecological interactions across the animal kingdom. They also have high medical importance, causing thousands of human fatalities annually, while offering a rich resource for drug discovery. Despite this, considerable logistical, safety and ethical challenges mean only a fraction of the worlds venoms have been profiled quantitatively. Natural history collections offer opportunities to greatly expand our knowledge of venom systems. We used quantitative proteomic mass spectrometry on preserved venom glands, spanning 0-57 years in age, from 37 venomous snake species (32 elapids and 5 viperids), alongside fresh venom samples from most of the same taxa. Preserved glands and fresh venoms from the same species showed strong concordance in venom composition across multiple metrics. Critically, specimen age did not degrade data quality, indicating that decades-old material yields reliable quantitative profiles, opening the possibility for vast quantities of existing museum specimens to be used. When we integrate our data with published profiles, we confirm known elapid vs viper broad diversity patterns while revealing substantial Australasian elapid venom diversity. Our findings demonstrate the potential of natural history collections as a vast, largely untapped biochemical archive for high-throughput "museum venomics," enabling evolutionary and temporal analyses of venom diversity at unprecedented scale.

We report the isolation and identification of a Paenibacillus polymyxa strain from the citizen science project; Swab and Send. Through whole genome sequencing we are able to describe the biosynthetic gene cluster of polymyxin A produced by P. polymyxa 1G (NCBI accession no. JBVPZV000000000), compare the pmxA, pmxB and pmxE genes to five other polymyxin genes encoding known polymyxin variants, and provide mass spectrometry data that supports the production of polymyxin A1 (1157 m/z) and A2 (1143 m/z). Polymyxins are ranked in the highest priority critically important antimicrobials classification by the WHO and are of particular importance for treating gram-negative multidrug resistant pathogens. Due to the discovery of polymyxins occurring in the 1940s, there is little genetic research around polymyxins, and the literature focusses primarily on clinically used polymyxin E (colistin) and polymyxin B. Previous literature suggests that polymyxin A1 has similar/lower toxicity to clinically used polymyxins E and B. To test if polymyxin A was able to overcome current resistance mechanisms to clinically used polymyxins, the cell free supernatant from P. polymyxa 1G was tested against a panel of clinical isolates with various resistance genes. We found that resistance genes mcr-1 and mcr-4 confer resistance to polymyxin A produced by our isolate meaning that, while polymyxin A has good antimicrobial activity, clinical resistance mechanisms already confer resistance to this variant of polymyxin.