Neutralization of ricin toxin on building interior surfaces using liquid decontaminants.

Ricin is a highly toxic protein, capable of inhibiting protein synthesis within cells, and is produced from the beans of the Ricinus communis (castor bean) plant. Numerous recent incidents involving ricin have occurred, many in the form of mailed letters resulting in both building and mail sorting facility contamination. The goal of this study was to assess the decontamination efficacy of several commercial off-the-shelf (COTS) cleaners and decontaminants (solutions of sodium hypochlorite [bleach], quaternary ammonium, sodium percarbonate, peracetic acid, and hydrogen peroxide) against a crude preparation of ricin toxin. The ricin was inoculated onto four common building materials (pine wood, drywall joint tape, countertop laminate, and industrial carpet), and the decontaminants were applied to the test coupons using a handheld sprayer. Decontamination efficacy was quantified using an in-vitro cytotoxicity assay to measure the quantity of bioactive ricin toxin extracted from test coupons as compared to the corresponding positive controls (not sprayed with decontaminant). Results showed that decontamination efficacy varied by decontaminant and substrate material, and that efficacy generally improved as the number of spray applications or contact time increased. The solutions of 0.45% peracetic acid and the 20,000-parts per million (ppm) sodium hypochlorite provided the overall best decontamination efficacy. The 0.45% peracetic acid solution achieved 97.8 to 99.8% reduction with a 30-min contact time.

Short- and long-term outcomes of pulmonary exposure to a sublethal dose of ricin in mice.

Ricin, an extremely potent toxin produced from the seeds of castor plant, Ricinus communis, is ribosome-inactivating protein that blocks cell-protein synthesis. It is considered a biological threat due to worldwide availability of castor beans, massive quantities as a by-product of castor oil production, high stability and ease of production. The consequence of exposure to lethal dose of ricin was extensively described in various animal models. However, it is assumed that in case of aerosolized ricin bioterror attack, the majority of individuals would be exposed to sublethal doses rather than to lethal ones. Therefore, the purpose of current study was to assess short- and long-term effects on physiological parameters and function following sublethal pulmonary exposure. We show that in the short-term, sublethal exposure of mice to ricin resulted in acute lung injury, including interstitial pneumonia, cytokine storm, neutrophil influx, edema and cellular death. This damage was manifested in reduced lung performance and physiological function. Interestingly, although in the long-term, mice recovered from acute lung damage and restored pulmonary and physiological functionality, the reparative process was associated with lasting fibrotic lesions. Therefore, restriction of short-term acute phase of the disease and management of long-term pulmonary fibrosis by medical countermeasures is expected to facilitate the quality of life of exposed survivors.

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.

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit.

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

Caspase-3/Gasdermin E-mediated pyroptosis contributes to Ricin toxin-induced inflammation.

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.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis.

BACKGROUND: Ricin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. METHODS: Ricin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin-biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. RESULTS: The LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90-62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. CONCLUSION: The developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Crosstalk between autophagy and inflammasomes in ricin-induced inflammatory injury.

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-1ß, 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-1ß, 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.

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.

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.

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.

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.

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.