Identification and Biological Evaluation of a Novel Small-Molecule Inhibitor of Ricin Toxin.

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.

Ferrostatin­1 alleviates liver injury via decreasing ferroptosis following ricin toxin poisoning in rat.

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 Ferrostatin­1 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 Ferrostatin­1 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.

Structure-Function Analysis of the A1 Subunit of Shiga Toxin 2 with Peptides That Target the P-Stalk Binding Site and Inhibit Activity.

Shiga toxin 2a (Stx2a) is the virulence factor of Escherichia coli (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin-ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the P-stalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.

Structure-based design and optimization of a new class of small molecule inhibitors targeting the P-stalk binding pocket of ricin.

Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.

Long-Term Pulmonary Damage in Surviving Antitoxin-Treated Mice following a Lethal Ricin Intoxication.

Ricin, a highly potent plant-derived toxin, is considered a potential bioterrorism weapon due to its pronounced toxicity, high availability, and ease of preparation. Acute damage following pulmonary ricinosis is characterized by local cytokine storm, massive neutrophil infiltration, and edema formation, resulting in respiratory insufficiency and death. A designated equine polyclonal antibody-based (antitoxin) treatment was developed in our laboratory and proved efficacious in alleviating lung injury and increasing survival rates. Although short-term pathogenesis was thoroughly characterized in antitoxin-treated mice, the long-term damage in surviving mice was never determined. In this study, long-term consequences of ricin intoxication were evaluated 30 days post-exposure in mice that survived antitoxin treatment. Significant pulmonary sequelae were demonstrated in surviving antitoxin-treated mice, as reflected by prominent histopathological changes, moderate fibrosis, increased lung hyperpermeability, and decreased lung compliance. The presented data highlight, for the first time to our knowledge, the possibility of long-term damage development in mice that survived lethal-dose pulmonary exposure to ricin due to antitoxin treatment.

Quantitative detection and prognostic value of antibodies against M-type phospholipase A2 receptor and its cysteine-rich ricin domain and C-type lectin domains 1 and 6-7-8 in patients with idiopathic membranous nephropathy.

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%. CONCLUSION: 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.

A Comprehensive Review on Euphorbiaceae lectins: Structural and Biological Perspectives.

Euphorbiaceae, also known as the spurge family, is a large group of flowering plants. Despite being tropical natives, they are now widespread. Due to its medicinal and commercial importance, this family of plants attracted a lot of attention in the scientific community. The distinctive characteristic of the family is production of milky latex, which is a rich source of several lectins, the proteins that bind carbohydrates. Although their function is unclear, they are believed to defend plants against damaging phytopathogenic microorganisms, insects, and predatory animals. Additionally, they serve as crucial metabolic regulators under a variety of stressors. Detection, separation, purification, and characterization of lectins from the Euphorbiaceae family - mostly from the latex of plants - began over 40 years ago. This effort produced over 35 original research papers that were published. However, no systematic review that compiles these published data has been presented yet. This review summarizes and describes several procedures and protocols employed for extraction and purification of lectins belonging to this family. Physicochemical properties and biological activities of the lectins, along with their medicinal and pharmacological properties, have also been analyzed. Additionally, using examples of ricin and ricin agglutinin, we have structurally analyzed characteristics of the lectin known as Ribosome Inactivating Protein Type II (RIP-Type II) that belongs to this family. We anticipate that this review article will offer a useful compendium of information on this important family of lectins, show the scientists involved in lectin research the gaps in our knowledge, and offer insights for future research.

The Biological Action and Structural Characterization of Eryngitin 3 and 4, Ribotoxin-like Proteins from Pleurotus eryngii Fruiting Bodies.

Ribotoxin-like proteins (RL-Ps) are specific ribonucleases found in mushrooms that are able to cleave a single phosphodiester bond located in the sarcin-ricin loop (SRL) of the large rRNA. The cleaved SRL interacts differently with some ribosomal proteins (P-stalk). This action blocks protein synthesis because the damaged ribosomes are unable to interact with elongation factors. Here, the amino acid sequences of eryngitin 3 and 4, RL-Ps isolated from Pleurotus eryngii fruiting bodies, were determined to (i) obtain structural information on this specific ribonuclease family from edible mushrooms and (ii) explore the structural determinants which justify their different biological and antipathogenic activities. Indeed, eryngitin 3 exhibited higher toxicity with respect to eryngitin 4 against tumoral cell lines and model fungi. Structurally, eryngitin 3 and 4 consist of 132 amino acids, most of them identical and exhibiting a single free cysteinyl residue. The amino acidic differences between the two toxins are (i) an additional phenylalanyl residue at the N-terminus of eryngitin 3, not retrieved in eryngitin 4, and (ii) an additional arginyl residue at the C-terminus of eryngitin 4, not retrieved in eryngitin 3. The 3D models of eryngitins show slight differences at the N- and C-terminal regions. In particular, the positive electrostatic surface at the C-terminal of eryngitin 4 is due to the additional arginyl residue not retrieved in eryngitin 3. This additional positive charge could interfere with the binding to the SRL (substrate) or with some ribosomal proteins (P-stalk structure) during substrate recognition.

Structural Basis of Antibody-Mediated Inhibition of Ricin Toxin Attachment to Host Cells.

Monoclonal antibodies, JB4 and SylH3, neutralize ricin toxin (RT) by inhibiting the galactose-specific lectin activity of the B subunit of the toxin (RTB), which is required for cell attachment and entry. It is not immediately apparent how the antibodies accomplish this feat, considering that RTB consists of two globular domains (D1, D2) each divided into three homologous subdomains (α, ß, γ) with the two functional galactosyl-specific carbohydrate recognition domains (CRDs) situated on opposite poles (subdomains 1α and 2γ). Here, we report the X-ray crystal structures of JB4 and SylH3 Fab fragments bound to RTB in the context of RT. The structures revealed that neither Fab obstructed nor induced detectable conformational alterations in subdomains 1α or 2γ. Rather, JB4 and SylH3 Fabs recognize nearly identical epitopes within an ancillary carbohydrate recognition pocket located in subdomain 1ß. Despite limited amino acid sequence similarity between SylH3 and JB4 Fabs, each paratope inserts a Phe side chain from the heavy (H) chain complementarity determining region (CDR3) into the 1ß CRD pocket, resulting in local aromatic stacking interactions that potentially mimic a ligand interaction. Reconciling the fact that stoichiometric amounts of SylH3 and JB4 are sufficient to disarm RTB's lectin activity without evidence of allostery, we propose that subdomain 1ß functions as a "coreceptor" required to stabilize glycan interactions principally mediated by subdomains 1α and 2γ. Further investigation into subdomain 1ß will yield fundamental insights into the large family of R-type lectins and open novel avenues for countermeasures aimed at preventing toxin uptake into vulnerable tissues and cells.

Reduction is sufficient for the disassembly of ricin and Shiga toxin 1 but not Escherichia coli heat-labile enterotoxin.

Many AB toxins contain an enzymatic A moiety that is anchored to a cell-binding B moiety by a disulfide bridge. After receptor-mediated endocytosis, some AB toxins undergo retrograde transport to the endoplasmic reticulum (ER) where reduction of the disulfide bond occurs. The reduced A subunit then dissociates from the holotoxin and enters the cytosol to alter its cellular target. Intoxication requires A chain separation from the holotoxin, but, for many toxins, it is unclear if reduction alone is sufficient for toxin disassembly. Here, we examined the link between reduction and disassembly for several ER-translocating toxins. We found disassembly of the reduced Escherichia coli heat-labile enterotoxin (Ltx) required an interaction with one specific ER-localized oxidoreductase: protein disulfide isomerase (PDI). In contrast, the reduction and disassembly of ricin toxin (Rtx) and Shiga toxin 1 (Stx1) were coupled events that did not require PDI and could be triggered by reductant alone. PDI-deficient cells accordingly exhibited high resistance to Ltx with continued sensitivity to Rtx and Stx1. The distinct structural organization of each AB toxin thus appears to determine whether holotoxin disassembly occurs spontaneously upon disulfide reduction or requires the additional input of PDI.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

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.

Easy, Robust, and Repeatable Online Acid Cleavage of Proteins in Mobile Phase for Fast Quantitative LC-MS Bottom-Up Protein Analysis─Application for Ricin Detection.

Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.

A novel EGFR-specific recombinant ricin-panitumumab (scFv) immunotoxin against breast and colorectal cancer cell lines; in silico and in vitro analyses.

The Epidermal Growth Factor Receptor (EGFR) has been of high importance as it is over expressed in a wide diversity of epithelial cancers, promoting cell proliferation and survival pathways. Recombinant immunotoxins (ITs) have emerged as a promising targeted therapy for cancer treatment. In this study, we aimed to investigate the antitumor activity of a novel recombinant immunotoxin designed against EGFR. Using an in silico approach, we confirmed the stability of the RTA-scFv fusion protein. The immunotoxin was successfully cloned and expressed in the pET32a vector, and the purified protein was analyzed by electrophoresis and western blotting. In vitro evaluations were conducted to assess the biological activities of the recombinant proteins (RTA-scFv, RTA, scFv). The novel immunotoxin demonstrated significant anti-proliferative and pro-apoptotic effects against cancer cell lines. The MTT cytotoxicity assay revealed a decrease in cell viability in the treated cancer cell lines. Additionally, Annexin V/Propidium iodide staining followed by flow cytometry analysis showed a significant induction of apoptosis in the cancer cell lines, with half maximal inhibitory concentration (IC50) values of 81.71 nM for MDA-MB-468 and 145.2 nM for HCT116 cells (P < 0.05). Furthermore, the EGFR-specific immunotoxin exhibited non-allergenic properties. The recombinant protein demonstrated high affinity binding to EGFR. Overall, this study presents a promising strategy for the development of recombinant immunotoxins as potential candidates for the treatment of EGFR-expressing cancers.

Cloning and Functional Verification of Endogenous U6 Promoters for the Establishment of Efficient CRISPR/Cas9-Based Genome Editing in Castor (Ricinus communis).

Castor (Ricinus communis) seeds are rich in a type of hydroxy fatty acid called ricinoleic acid, which is in high demand for the production of plant-based plastics, lubricants, and hydraulic oils. However, the high content of ricin, a toxic protein, in these seeds has restricted further expansion in the area of castor cultivation. Therefore, the development of ricin-free castor is needed. Genome editing technology, although successfully applied in several plant species, is still in the developing stages in castor and awaits the identification of an endogenous U6 promoter with robust function. Here, we searched for U6 small nuclear RNA (snRNA) genes in the castor genome. This led to the identification of six U6 snRNA genes. The promoters of these U6 snRNA genes were cloned, and their function was examined in castor cells using the particle delivery method. The results showed that a U6 promoter length of approximately 300 bp from the transcription start site was sufficient to activate gene expression. This study provides insights into the endogenous castor U6 promoter sequences and outlines a method for verifying the function of U6 promoters in plants using the particle delivery system.

Crystal structure of ricin toxin A chain complexed with a highly potent pterin-based small-molecular inhibitor.

Ricin toxin A chain (RTA), from Ricinus communis, is a deadly protein that inactivates ribosomes by degrading an adenine residue at position 4324 in 28S rRNA. Recently, we have demonstrated that pterin-7-carboxamides with peptide pendants were potent RTA inhibitors. Among these, N-(pterin-7-carbonyl)glycyl-L-tyrosine (7PCGY) is the most potent RTA inhibitor as a small organic molecule. However, despite this fascinating inhibitory activity, the mode of interaction of 7PCGY with RTA remains elusive. This study aimed to elucidate the factors responsible for the high RTA inhibitory activity of 7PCGY based on X-ray crystallographic analysis. Herein, we report the successfully resolved X-ray crystal structure of 7PCGY/RTA complexes, revealing that the interaction between the phenolic hydroxy group in 7PCGY and Asn78 of RTA through a hydrogen bonding and the conformational change of Tyr80 and Asn122 are responsible for the high RTA inhibitory activity of 7PCGY.

Ribosome inactivating proteins in insects: HGT, gene expression, and functional implications.

Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.

Anti-ricin toxin human neutralizing antibodies and DMAbs protection against ricin toxin poisoning.

DNA-encoded monoclonal antibodies (DMAbs) and in vivo expression of antibody therapeutics presents an innovative alternative to conventional delivery methods. Therefore, in order to prevent the lethal dose of ricin toxin (RT) and to avoid human anti-mouse antibody (HAMA) reaction, we developed the human neutralizing antibody 4-4E against RT and constructed DMAb-4-4E. The human neutralizing antibody 4-4E could neutralize RT in vitro and in vivo, while the mice in RT group all died. Using intramuscular electroporation (IM EP), antibodies were rapidly expressed in vivo within 7 days and were enriched in intestine and gastrocnemius muscle mostly. Besides, we found that DMAbs have shown a broad protective efficacy of RT poisoning prophylaxis. Driven by plasmids for IgG expression, mice were survived and the blood glucose level of mice in DMAb-IgG group returned to normal at 72 h post RT challenge, and the RT group died within 48 h. Furthermore, hindrance of protein disulfide isomerase (PDI) and accumulation of RT in endosomes were found in IgG-protected cells, revealing the possible mechanism of neutralization details. These data support the further study of RT-neutralizing monoclonal antibodies (mAbs) in the development.

[Forensic chemical and chemicotoxicological examination of ricin poisoning by the HPLC-MS/MS method]. / Sudebno-khimicheskoe i khimiko-toksikologicheskoe issledovanie metodom VEZhKh-MS/MS pri otravlenii ritsinom.

THE AIM OF THE STUDY: Is to suggest the method of ricin determination in biological liquids during forensic medical and chemicotoxicological examination. This research describes the optimal conditions of sample processing of biological liquids, allowing to extract the components (ricinine and ricinoleic acid) of castor seeds. The recommended analysis conditions allow to perform research for 15 minutes by high resolution mass spectrometry method combined with high-value liquid chromatography on a chromato-mass spectrometer to detect ricinine and ricinoleic acid. The chromatographic (retention time) and mass-spectrometric parameters (mass spectra) were established for the exact high-quality determination of ricinine and ricinoleic acid.

Autophagic Degradation Is Involved in Cell Protection against Ricin Toxin.

Autophagy is a complex and highly regulated degradative process, which acts as a survival pathway in response to cellular stress, starvation and pathogen infection. Ricin toxin is a plant toxin produced by the castor bean and classified as a category B biothreat agent. Ricin toxin inhibits cellular protein synthesis by catalytically inactivating ribosomes, leading to cell death. Currently, there is no licensed treatment for patients exposed to ricin. Ricin-induced apoptosis has been extensively studied; however, whether its intoxication via protein synthesis inhibition affects autophagy is not yet resolved. In this work, we demonstrated that ricin intoxication is accompanied by its own autophagic degradation in mammalian cells. Autophagy deficiency, by knocking down ATG5, attenuates ricin degradation, thus aggravating ricin-induced cytotoxicity. Additionally, the autophagy inducer SMER28 (Small Molecule Enhancer 28) partially protects cells against ricin cytotoxicity, an effect not observed in autophagy-deficient cells. These results demonstrate that autophagic degradation acts as a survival response of cells against ricin intoxication. This suggests that stimulation of autophagic degradation may be a strategy to counteract ricin intoxication.

SCX-tip-aided LC-MS detection of active ricin via oligonucleotide substrates for depurination kinetics.

The accurate and sensitive detection of active biotoxin proteins and the determination of their kinetics are vital for the upsurge of chemical attacks but still limited. Herein, we report a liquid chromatography-tunable ultraviolet spectroscopic-quadrupole mass spectrometric detection (LC-TUV-QDa) method of active ricin. This method has the advantage of the accurate quantification of active ricin in decreased oligonucleotide (oligo) substrates as well as the produced adenine, in which the QDa detection offers the confirmative evidence of oligo and adenine products. We invented a strong cation exchange (SCX)-tip sample pretreatment way to facilitate the requirement of clean product injection without any fouling proteins. After full-method validation, a wide linear range of 1-5000 ng mL-1 was obtained with a high sensitivity of 1 ng mL-1 active ricin based on the most preferable deoxynucleobase-hybrid RNA (Rd) substrate, Rd12, and without any enrichment. We also fully depicted the kinetic parameters of ricin and its six Rd or RNA substrates and evaluated 11 nucleobase-modified oligos as substrates based on Rd12. Further, we fulfilled an improved molecular docking analysis and revealed that the binding of Rd12 to ricin was more likely to occur at pH 7.4 (typical in vitro and in vivo conditions) than at pH 4.0 (typical ex vitro conditions). With the aid of SCX-tip as a microenzymatic reactor, we can exert the catalytic activity of ricin as N-glycosidase in pH 7.4 toward its Rd12 substrate, with a comparable catalytic efficiency at pH 4.0. This is the first successful implementation of an ex vitro experiment toward oligo substrates at neutral pH, standing on the shoulder of plenty of previously reported efforts all performed under acidic conditions. This method will provide a new and powerful way to detect active ricin when tackling relevant problems in public safety and security.