Abrin and ricin, both type II ribosome-inactivating proteins, are toxins of significant concern and are under international restriction by the Chemical Weapons Convention and the Biological and Toxin Weapons Convention. The development of a rapid and sensitive detection method for these toxins is of the utmost importance for the first emergency response. Emerging rapid detection techniques, such as surface-enhanced Raman spectroscopy (SERS) and lateral flow assay (LFA), have garnered attention due to their high sensitivity, good selectivity, ease of operation, low cost, and disposability. In this work, we generated stable and high-affinity nanotags, via an efficient freezing method, to serve as the capture module for SERS-LFA. We then constructed a sandwich-style lateral flow test strip using a pair of glycoproteins, asialofetuin and concanavalin A, as the core affinity recognition molecules, capable of trace measurement for both abrin and ricin. The limit of detection for abrin and ricin was 0.1 and 0.3 ng/mL, respectively. This method was applied to analyze eight spiked white powder samples, one juice sample, and three actual botanic samples, aligning well with cytotoxicity assay outcomes. It demonstrated good inter-batch and intra-batch reproducibility among the test strips, and the detection could be completed within 15 min, indicating the suitability of this SERS-LFA method for the on-site rapid detection of abrin and ricin toxins.

Sensitively detecting hazardous and suspected bioaerosols is crucial for safeguarding public health. The potential impact of pollen on identifying bacterial species through fluorescence spectra should not be overlooked. Before the analysis, the spectrum underwent preprocessing steps, including normalization, multivariate scattering correction, and Savitzky-Golay smoothing. Additionally, the spectrum was transformed using difference, standard normal variable, and fast Fourier transform techniques. A random forest algorithm was employed for the classification and identification of 31 different types of samples. The fast Fourier transform improved the classification accuracy of the sample excitation-emission matrix fluorescence spectrum data by 9.2%, resulting in an accuracy of 89.24%. The harmful substances, including Staphylococcus aureus, ricin, beta-bungarotoxin, and Staphylococcal enterotoxin B, were clearly distinguished. The spectral data transformation and classification algorithm effectively eliminated the interference of pollen on other components. Furthermore, a classification and recognition model based on spectral feature transformation was established, demonstrating excellent application potential in detecting hazardous substances and protecting public health. This study provided a solid foundation for the application of rapid detection methods for harmful bioaerosols.

Ricin and abrin are highly potent plant-derived toxins, categorized as type II ribosome-inactivating proteins. High toxicity, accessibility, and the lack of effective countermeasures make them potential agents in bioterrorism and biowarfare, posing significant threats to public safety. Despite the existence of many effective analytical strategies for detecting these two lethal toxins, current methods are often hindered by limitations such as insufficient sensitivity, complex sample preparation, and most importantly, the inability to distinguish between biologically active and inactive toxin. In this study, a cytotoxicity assay was developed to detect active ricin and abrin based on their potent cell-killing capability. Among nine human cell lines derived from various organs, HeLa cells exhibited exceptional sensitivity, with limits of detection reaching 0.3 ng/mL and 0.03 ng/mL for ricin and abrin, respectively. Subsequently, toxin-specific neutralizing monoclonal antibodies MIL50 and 10D8 were used to facilitate the precise identification and differentiation of ricin and abrin. The method provides straightforward and sensitive detection in complex matrices including milk, plasma, coffee, orange juice, and tea via a simple serial-dilution procedure without any complex purification and enrichment steps. Furthermore, this assay was successfully applied in the unambiguous identification of active ricin and abrin in samples from OPCW biotoxin exercises.

Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.

The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is mainly due to its ricin toxin A chain (RTA), which has become an important target for drug development. Previous studies have identified two essential binding pockets in the active site of RTA, but most existing inhibitors only target one of these pockets. In this study, we used computer-aided virtual screening to identify a compound called RSMI-29, which potentially interacts with both active pockets of RTA. We found that RSMI-29 can directly bind to RTA and effectively attenuate protein synthesis inhibition and rRNA depurination induced by RTA or ricin, thereby inhibiting their cytotoxic effects on cells in vitro. Moreover, RSMI-29 significantly reduced ricin-mediated damage to the liver, spleen, intestine, and lungs in mice, demonstrating its detoxification effect against ricin in vivo. RSMI-29 also exhibited excellent drug-like properties, featuring a typical structural moiety of known sulfonamides and barbiturates. These findings suggest that RSMI-29 is a novel small-molecule inhibitor that specifically targets ricin toxin A chain, providing a potential therapeutic option for ricin intoxication.

Ricin (ricin toxin, RT) has the potential to cause damage to multiple organs and systems. Currently, there are no existing antidotes, vaccinations, or effective therapies to prevent or treat RT intoxication. Apart from halting protein synthesis, RT also induces oxidative stress, inflammation and autophagy. To explore the mechanisms of RT-induced inflammatory injury and specific targets of prevention and treatment for RT poisoning, we characterized the role of cross-talk between autophagy and NLRP3 inflammasome in RT-induced damage and elucidated the underlying mechanisms. We showed that RT-induced inflammation was attributed to activation of the TLR4/MyD88/NLRP3 signaling and ROS production, evidenced by increased ASC speck formation and attenuated TXNIP/TRX-1 interaction, as well as pre-treatment with MCC950, MyD88 knockdown and NAC significantly reduced IL-1β, IL-6 and TNF-α mRNA expression. In addition, autophagy is also enhanced in RT-triggered MLE-12 cells. RT elevated the levels of ATG5, p62 and Beclin1 protein, provoked the accumulation of LC3 puncta detected by immunofluorescence staining. Treatment with rapamycin (Rapa) reversed the RT-caused TLR4/MyD88/NLRP3 signaling activation, ASC specks formation as well as the levels of IL-1β, IL-6 and TNF-α mRNA. In conclusion, RT promoted NLRP3 inflammasome activation and autophgay. Inflammation induced by RT was attenuated by autophagy activation, which suppressed the NLRP3 inflammasome. These findings suggest Rapa as a potential therapeutic drug for the treatment of RT-induced inflammation-related diseases.

Ricin is a highly toxic plant toxin that can cause multi-organ failure, especially liver dysfunction, and is a potential bioterrorism agent. Despite the serious public health challenge posed by ricin, effective therapeutic for ricin-induced poisoning is currently unavailable. Therefore, it is important to explore the mechanism of ricin poisoning and develop appropriate treatment protocols accordingly. Previous studies have shown that lipid peroxidation and iron accumulation are associated with ricin poisoning. Ferroptosis is an iron-dependent form of cell death caused by excessive accumulation of lipid peroxide. The role and mechanism of ferroptosis in ricin poisoning are unclear and require further study. We investigated the effect of ferroptosis on ricin-induced liver injury and further elucidated the mechanism. The results showed that ferroptosis occurred in the liver of ricin-intoxicated rats, and Ferrostatin‑1 could ameliorate hepatic ferroptosis and thus liver injury. Ricin induced liver injury by decreasing hepatic reduced glutathione and the protein level of glutathione peroxidase 4 and Solute Carrier Family 7 Member 11, increasing iron, malondialdehyde and reactive oxygen species, and mitochondrial damage, whereas Ferrostatin‑1 pretreatment increased hepatic reduced glutathione and the protein level of glutathione peroxidase 4 and Solute Carrier Family 7 Member 11, decreased iron, malondialdehyde, and reactive oxygen species, and ameliorated mitochondrial damage, thereby alleviated liver injury. These results suggested that ferroptosis exacerbated liver injury after ricin poisoning and that inhibition of ferroptosis may be a novel strategy for the treatment of ricin poisoning.

BACKGROUND: M-type phospholipase A2 receptor (PLA2R) is the major autoantigen in adult idiopathic membranous nephropathy (IMN). Although reactive epitopes in the PLA2R domains have been identified, the clinical value of these domains recognized by anti-PLA2R antibodies remains controversial. Accordingly, this study aimed to quantitatively detect changes in the concentrations of different antibodies against epitopes of PLA2R in patients with IMN before and after treatment to evaluate the clinical value of epitope spreading.
METHODS: Highly sensitive time-resolved fluorescence immunoassay was used to quantitatively analyze the concentrations of specific IgG and IgG4 antibodies against PLA2R and its epitopes (CysR, CTLD1, CTLD6-7-8) in a cohort of 25 patients with PLA2R-associated membranous nephropathy (13 and 12 in the remission and non-remission groups, respectively) before and after treatment, and the results were analyzed in conjunction with clinical biochemical indicators.
RESULTS: The concentration of specific IgG (IgG4) antibodies against PLA2R and its epitopes (CysR, CTLD1 and CTLD6-7-8) in non-remission group was higher than that in remission group. The multipliers of elevation of IgG (IgG4) antibody were 5.6(6.2) fold, 3.0(24.3) fold, 1.6(9.0) fold, and 4.2(2.6) fold in the non-remission/remission group, respectively. However, the difference in antibody concentrations between the two groups at the end of follow-up was 5.6 (85.2), 1.7 (13.1), 1.0 (5.1), and 1.5 (22.3) times higher, respectively. When detecting concentrations of specific IgG antibodies against PLA2R and its different epitopes, the remission rate was 66.67% for only one epitope at M0 and 36.36% for three epitopes at M0. When detecting concentrations of specific IgG4 antibodies against PLA2R and its different epitopes, the remission rate was 100.00% for only one epitope at M0 and 50.00% for three epitopes at M0. A trivariate logistic regression model for the combined detection of eGFR, anti-CTLD678 IgG4, and urinary protein had an AUC of 100.00%.
CONCLUSIONS: Low concentrations of anti-CysR-IgG4, anti-CTLD1-IgG4, and anti-CTLD6-7-8-IgG4 at initial diagnosis predict rapid remission after treatment. The use of specific IgG4 against PLA2R and its different epitopes combined with eGFR and urinary protein provides a better assessment of the prognostic outcome of IMN.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.