Botulinum neurotoxins (BoNTs) are the most potent biological toxins known and widely used as biopharmaceuticals. Yet pharmaceutical potency testing still relies largely on the mouse bioassay, raising ethical concerns and conflicting with 3 R principles. Sensitive, serotype‑independent human cell‑based alternatives are particularly needed for the emerging serotypes BoNT/E and BoNT/F, for which no validated in vitro potency assay currently has been reported. In this study, human iPSC‑derived motor neurons were generated, cryopreserved as motor neuron progenitors, matured to day 30, and exposed to serial dilutions of BoNT/E or BoNT/F. Toxin activity was quantified via Western blot detection of SNAP25 (BoNT/E) using a full-length antibody, whereas for VAMP2 (BoNT/F), both full-length and neoepitope-specific antibodies were used to quantify substrate cleavage. IC₅₀ values were calculated by non‑linear regression and converted to LD₅₀/mL equivalents for comparison with mouse bioassay data. Measured IC₅₀ values were 0.769 pM for BoNT/E and 0.802 pM (full‑length) or 7.645 pM (neoepitope) for BoNT/F, corresponding to 4.54 LD₅₀/mL for BoNT/E and 2.46 LD₅₀/mL (full‑length) for BoNT/F, closely matching mouse bioassay potencies. Combining these data with prior results for BoNT/A and BoNT/B demonstrates a potency order: BoNT/A ≫ BoNT/B ≈ BoNT/F ≈ BoNT/E. These results confirm human iPSC‑derived motor neurons as a sensitive, physiologically relevant model capable of detecting four medically important BoNT serotypes. While Western blotting provides robust determination of potency, this cellular model well suited for adaptation to serotype-independent high‑throughput formats, paving the way for animal‑free BoNT potency testing.

Fast-acting botulinum neurotoxins (BoNTs) are highly desirable for both medical and aesthetic indications, but the underlying mechanism for the differing onset of BoNTs' action remains unknown. Here, we demonstrate that the "belt" of BoNTs, a largely unstructured loop wrapping around their catalytic light chain (LC), is key to onset of intoxication. The more flexible BoNT/E belt promotes quicker LC translocation into the neuronal cytosol, leading to faster onset of action compared to BoNT/A. Furthermore, we discover a "belt-buckle" checkpoint that regulates this process. By loosening the BoNT/A belt-buckle via protein engineering, we enhance its sensitivity to acidic pH, leading to an accelerated onset of action. Conversely, locking the belt-buckle with an antibody neutralizes BoNT/A. Our findings open avenues for developing fast-acting BoNTs and effective countermeasures.

Staphylococcal enterotoxins (SEs) are major contributors to foodborne intoxications. Reliable detection methods for SEs are essential to maintain food safety and protect public health. Since the heat-stable toxins also exert their toxic effect in the absence of the bacterium, reliance on DNA detection alone can be misleading: it does not allow for determining which specific toxins encoded by a given strain are produced and epidemiologically linked with a given outbreak. Commercially available diagnostic assays for SE detection are so far limited in sensitivity and specificity as well as in the range of targeted toxins (SEA-SEE), thus non-targeted SEs linked to foodborne illness remain undetected at the protein level. This study aimed to develop a highly sensitive and specific multiplex suspension immunoassay (SIA) for SEA to SEI. To this end, high-affinity monoclonal antibodies (mAbs) for the specific detection of the individual SEs were generated. When implemented in sandwich ELISAs and multiplex SIA, these mAbs demonstrated exceptional sensitivity with detection limits in the low picogram per millilitre range. When applied for the analysis of SE production in liquid cultures of a panel of 145 whole-genome sequenced strains of Staphylococcus spp. and Enterococcus faecalis, the novel multiplex SIA detected and differentiated the eight SEs with assay accuracies of 86.9-100%. Notably, the multiplex SIA covered one to four sequence variants for each of the individual SEs. Validation confirmed high recovery rates and reliable performance in three representative complex food matrices. The implementation of the novel mAbs in a multiplex SIA enabled, for the first time, simultaneous detection, differentiation, and quantification of multiple SEs from minimal sample volumes using Luminex® technology. As a result, the multiplex SIA will help strengthen food safety protocols and public health response capabilities.

Botulinum neurotoxin serotype A (BoNT/A) is naturally produced by bacteria along with four nontoxic neurotoxin-associated proteins (NTNH, HA70, HA33, and HA17), forming a bimodular large progenitor toxin complex (L-PTC). The BoNT/A-NTNH complex protects the toxin from adverse environment, while the complex consisting of HA proteins facilitates toxin absorption during oral intoxication. How these two independent modules assemble into the L-PTC remains unclear. Here, we report the crystal structure of the BoNT/A-NTNH-HA70 complex at ~2.9-Å resolution. The structure reveals that the BoNT/A-NTNH complex is anchored into a concentric double β-barrel channel of trimeric HA70 through a short β-hairpin of NTNH (termed nLoop), resembling a nut-and-bolt attachment. We find that the nLoop of NTNH is strictly conserved across HA-containing BoNT complexes and that NTNH-HA70 binding is interchangeable among them. Furthermore, we demonstrate that the nLoop functions as a minimal motif enabling attachment of a protein-of-interest to the HA complex, with potential applications in oral biologics delivery.

Abrin is a highly toxic plant protein encompassing four isoforms, abrin-a, -b, -c and -d. An abrin reference material was isolated from Abrus precatorius and certified (EURM-113) by the EuroBioTox consortium. Here, we present a detailed characterisation of the N-glycosylation profile of EURM-113. The monosaccharide composition of the N-glycans was determined and quantified. Release of the N-glycans yielded 13 different partially xylosylated, oligomannosidic and paucimannosidic glycan structures. Two N-glycans were found at N82 and N110 of the abrin-b A-chain and another two at N100 and N140 of the B-chains. The N-glycosylation sites N200 in the A-chain and N141 in the B-chain were non-glycosylated. Whereas N82 and N110 of abrin-b comprised paucimannosidic glycans, N100 and N140 of the B-chains revealed oligomannosidic N-glycans. Xylose was absent in the glycans at N100 but was present in about half of the glycans at N140. Hence, this study revealed substantially different types of glycan structures within the B-chains compared to the abrin-b A-chain. Furthermore, the most C-terminal N-glycosylation site in the A-chain was found to be non-glycosylated in all abrin isoforms detected. Additionally, the establishment of the N-glycosylation profile of the abrin reference material led to the identification of the abrin isoforms -a, -b and -c. In conclusion, the abrin N-glycosylation profile is highly similar to the one of ricin and yields high analytical value to be further exploited as a fingerprint in forensic investigations to uncover toxin production or toxin provenance.

Botulinum neurotoxins (BoNTs) rank among the most potent toxins and many of them are produced by bacteria carrying the orfX gene cluster that also encodes four nontoxic proteins (OrfX1, OrfX2, OrfX3 and P47). The orfX gene cluster is also found in the genomes of many non-BoNT-producing bacteria, often alongside genes encoding oral insecticidal toxins. However, the functions of these OrfXs and P47 remain elusive. Here, we demonstrate that the combined action of all four components (OrfXs and P47) drastically boosts the oral toxicity of BoNT in mice, following proteolytic activation by digestive proteases that oral toxins regularly confront. In particular, OrfX2 adopts a self-inhibiting state, engaging with BoNT through another clostridial protein, nontoxic non-hemagglutinin (NTNH), only after proteolytic activation. Cryo-electron microscopy studies unveil that two molecules of protease-activated OrfX2 simultaneously associate with NTNH, a binding mode crucial for boosting BoNT oral toxicity. Collectively, these studies offer novel insights into the physiological functions and regulatory mechanisms of OrfXs and P47 of BoNTs, shedding light on the pathogenesis of other bacterial toxins associated with homologous OrfXs and P47. Using complementary cryo-electron microscopy and functional studies, the authors demonstrate that botulinum neurotoxins exploit the accompanying nontoxic bacterial proteins, OrfXs and P47, activated by host digestive proteases, to greatly enhance their oral toxicity.

Botulism is a potentially life-threatening disease caused by botulinum neurotoxin (BoNT)-producing bacteria of the genus Clostridium. Laboratory detection of BoNTs in patients’ samples is essential to confirm clinical diagnoses and to identify the causative BoNT serotype. The current ’gold standard’ method for BoNT detection is the mouse bioassay (MBA), a highly stressful animal experiment. A viable animal experiment replacement method must demonstrate high sensitivity, specificity, reproducibility and robustness, as well as comprehensive BoNT subtype detection, and be widely accepted in the field, necessitating rigorous validation. Here, we report on the validation of a previously established in vitro endopeptidase suspension immunoassay (Endopep-SIA) for the simultaneous detection, differentiation and quantification of BoNT serotypes A and B, the most frequent serotypes associated with human botulism. This assay uses monoclonal antibodies for BoNT extraction, followed by detection of the catalytic activity using neoepitope-specific monoclonal antibodies and suspension array technology. The Endopep-SIA showed high reproducibility with intra- and inter-assay variabilities between 7 and 22%, it demonstrated a sensitivity two- to twenty-fold higher than the MBA for BoNT in buffer samples and was equally sensitive for human serum samples with a limit of detection of 0.4 MLD/mL for BoNT/A and 1.0 MLD/mL for BoNT/B. Importantly, it reliably detected all six BoNT/A and six BoNT/B subtypes tested, including clinically relevant and bivalent strains, hereby proving high diagnostic safety. Based on the results obtained, we expect the Endopep-SIA to be instrumental in markedly reducing the number of animals used in botulism diagnostics.

Botulinum neurotoxins are the causative agents of botulism, a lethal paralytic disease, but are also one of the most commonly used therapeutics for the treatment of numerous neuromuscular conditions. These toxins recognise motor nerve terminals with high specificity and affinity by using a dual binding mechanism involving gangliosides and protein receptors. The initial recognition of gangliosides is crucial for the toxins' potency. In this study, we employed a synaptosome-binding screening strategy to identify BoNT/A mutants with enhanced ganglioside-binding which translated into improved potency. X-ray crystallography and receptor-binding assays were used to elucidate the molecular mechanisms underlying the increased affinity or altered ganglioside selectivity of these mutants. Our findings provide a basis for the development of BoNT/A variants with enhanced therapeutic potential.

Abstract The effects of sample prep with a Ga+-ion Focused Ion Beam (Ga-FIB) on measurements of electron beam induced current (EBIC) were studied. Concerns have been occasionally raised about amorphization from the beam, or even Ga+ implantation ruining the ability to make useful measurements for purposes of either failure analysis or device tailoring. To understand the magnitude of any deleterious effects, two different lamellae from a 5 nm SRAM sample were prepared with different areas of increasingly improved polish, as indicated by decreasing, cumulative, FIB beam energy, followed by EBIC measurements at 1 or 2 kV beam landing energy. A first experiment looked at the ability to generate EBIC measurements from depletion zones and found no difference across the various beam polish cells. A second experiment considered leakage and/or shorts and found little problematic currents, within standard deviations.

An experiment to understand possible detrimental effects from processing SRAM samples with gallium focused ion beams (Ga-FIB) prior to electron beam induced current (EBIC) characterization is presented. To understand the magnitude of sample surface amorphization upon the ability to collect EBIC measurements, a lamella was prepared with different areas of increasingly improved polish, followed by EBIC measurements at 1 and 2 kV beam landing energy. The results indicate that EBIC measurements on a 5 nm SRAM lamella are minimally impacted by variations in final FIB polishing optimization, a reminder that these measurements are interacting with a volume in the sample, rather than merely the surface. A second experiment showed the ability to perform high-magnification EBIC measurements underneath gates.

Abstract This work employs an easy-to-use method to quickly find and characterize leakage currents on a semiconductor sample by combining electrical fault isolation and electrical measurements. By using a simple add-on for a probing system’s tip holders, a prober is transformed into a scanning device that measures currents through a sample’s surface and visualizes the currents in a 2D color map that can be superimposed onto the SE image. As a case study, an area of 1.5 µm x 1.5 µm of a 3 nm device was scanned while the current through the contacts was measured and visualized with Current Imaging (CI) and gate currents were characterized. One leaking gate could be identified and the position of the failure was localized using Electron Beam Induced Resistance CHange (EBIRCH) imaging. This technique also avoids any damage caused by electron beam irradiation as the beam can be switched off during scanning.