The detection of a deletion in the type B neurotoxin gene of A(B) strains by a two-step PCR. and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain name, a BoNT/F5 light-chain domain name, and a novel translocation domain name most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that this toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The mouse bioassay indicated a specific activity of this toxin of 3.8 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A MG-262 to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is usually early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a treatment-resistant and highly potent toxin. However, the toxins chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons. is usually widespread in nature and forms spores that can survive many standard antibacterial treatments, such that botulism remains a perennial concern in the food industry. Third, there is no cure for botulism, and treatment is restricted to supportive care, including mechanical ventilation in severe cases, and administration of antitoxin, which is usually most effective if given within the first 72?h after ingestion of MG-262 neurotoxins in contaminated food. The antitoxin contains equine-derived polyclonal antibodies to the seven known BoNT serotypes (3). Administration of MG-262 antitoxin results in neutralization of circulating toxin but is usually ineffective against toxin that has already joined neuronal cells. After neuronal cell entry, which occurs mainly during the first 3? days after the toxin enters the body, BoNTs can have an extraordinarily long duration of action inside neuronal cells, leading to the prolonged flaccid paralysis characteristic of botulism (2). During this time, intensive supportive care is required, and the antitoxin is usually ineffective. In infant botulism (and in MG-262 very rare cases in adults), colonization of occurs in the immature or immunocompromised intestine, leading to continuous toxin production (4). Because toxin continues to be produced by in the intestine, neutralizing antibodies in the circulation systems of these patients can continue to reduce the severity and duration of symptoms, and a human IgG (BabyBIG) for treatment of these cases has been developed to avoid the side effects associated with equine antitoxin (5). Due to the high potency of BoNTs, the lack of a cure, the potentially high mortality rate in the absence of treatment, and the high cost and limited availability of the required intensive supportive LFA3 antibody care, these toxins are considered a potential bioterrorism weapon and remain of high national and international significance (6, 7). BoNTs are produced by a diverse group of anaerobic bacteria designated and a few strains of otherwise atoxic species, including (8, 9). BoNTs have been classified into seven serotypes, designated BoNT/A through BoNT/G, on the basis of their specific neutralization by antiserum raised against the homologous serotype (10, 11), and are further subdivided into subtypes/variants (12, 13). Most strains of produce only a single serotype of BoNT. However, a number of strains are known to produce two or even three different BoNTs, with one of MG-262 the toxins usually dominant as determined by activity in mice (10, 12,C21). One such bivalent strain was reported in 2014 from an infant botulism case. This strain, designated strain IBCA10-7060, was isolated and cultured, and neutralization experiments and immunological.
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- Rats received either automobile (0
- Abbreviations: CON, control; CANA, canagliflozin; AMPK, AMP-activated proteins kinase; ACC, acetyl-CoA carboxylase
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