HKJ participated in the experimental design with SS and performed most of the experiments. SK and AK helped in some experiments. JBP contributed to new reagents. BAW performed mass spectrometry. PJ and LAH helped in Fedratinib in vitro iTRAQ data analysis. HKJ and SS analyzed the data and wrote the manuscript. All authors read and approved the manuscript.”
“Background

Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces botulinum neurotoxin (BoNT), the most potent toxin known [1–3]. BoNT is classified as a Category A biothreat agent by the Centers for Disease Control and Prevention (CDC) because of its lethality and ease of production, transport and dissemination [4, 5]. In addition, BoNT poses several threats to the public health: first, the possibility of foodborne botulism represents a major potential health hazard that requires continual monitoring by the food industry. Second, infant botulism has been the most common form of human botulism in the United States for more than 20 years and hospitalizes approximately 80-100 U.S. infants annually [6]. Third, cases of wound botulism due to intravenous drug use continue to increase [7, 8]. Botulism toxicity results from one

of seven serologically distinct neurotoxins (types A-G) that cause a severe neuroparalytic disease characterized by descending flaccid paralysis [9]. Rarely, unique strains of C. butyricum and C. baratii may also cause human botulism through MAPK Inhibitor Library nmr production and release of BoNT/E and F, respectively [10, 11]. The toxin acts by binding C1GALT1 to peripheral cholinergic nerve endings and inhibiting release of acetylcholine at the neuromuscular

junction. A part of the toxin is a zinc-dependent protease that cleaves target substrate proteins (SNAREs), located either on the plasma membrane or the synaptic vesicle, thereby preventing their binding, fusion and release of neurotransmitter. BoNTs cleave specific amino acids on the target proteins of the SNARE complex. BoNT/A and BoNT/E act on SNAP-25, while BoNT/C targets syntaxin as well as SNAP-25. The remaining toxin types (BoNT/B, BoNT/D, BoNT/E and BoNT/F) all act on synaptobrevin, but at different cleavage sites [12–15]. The potential severity and lethality of the disease warrants sensitive and specific detection and serotyping of toxin and its typing to enable correct administration of serotype-specific antitoxin in a timely manner. Although treatment with Human Botulism Immune Globulin (BabyBIG®) or equine antitoxin is based on clinical findings and should be instituted as rapidly as possible [5, 16, 17], definitive microbiological diagnosis may take several days or even longer. This extended time to diagnosis occurs because detection of the bacterium and its toxin relies on toxicity assessment in mice (the mouse protection bioassay) and lengthy culture assays, which, while sensitive and specific, may be time-consuming and difficult [18, 19].