Botulinum Neurotoxin Type Research Articles

Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E.

Effects of botulinum toxin type A facial injection on monoamines and their metabolites in sensory, limbic and motor brain regions in rats

Despite its toxicity, botulinum neurotoxin type A (BTX-A) is a valuable therapeutic agent for several motor, autonomic and pain disorders. Numerous studies have described its peripheral as well as central effects. Using reversed-phase High Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED) and gradient elution, we quantified the concentrations of dopamine (DA), noradrenaline (NA), serotonin (5-HT) and their metabolites in 10 brain regions, ipsilateral and contralateral from the site of unilateral BTX-A administration (5U/kg) into the rat whisker pad. In regions associated with nociception and pain processing we also examined possible BTX-A effects in combination with formalin-induced inflammatory orofacial pain. The dominant BTX-A effects on the monoamines and their metabolites were insignificant. The only significant increase caused by BTX-A alone was that of NA in striatum and serotonin in hypothalamus. While antinociceptive effects of BTX-A are most probably not related to central monoamine concentrations, the localized increased NA and 5-HT concentrations might play a role in reported BTX-A efficacy for the treatment of depression.

Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5.

Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L54-E55, of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20–65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2′, P1′, P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development.

Botulism (type A) in a horse - case report

This paper presents the case of a six year-old, male, thoroughbred horse with clinical signs of inappetence, weakness, and incoordination when walking. Clinical examination showed that the horse staggered and leaned to the left side. Feedstuff was present inside and around its mouth. Salivation was increased and there was no reflex at the palpebrae and tongue. The horse had difficulty swallowing and the tone of its tail was reduced. Botulism was diagnosed based on the clinical signs. Antibiotic (ceftiofur) and fluid-electrolyte treatment was commenced. Next day, neostigmin was added to the horse’s treatment, and it became recumbent. The horse’s palpebral, tongue and tail reflexes returned partially after neostigmine methylsulphate treatment on the same day and it stood up on day four. However, it could not swallow anything during the whole week, so after getting permission from the owner, the horse was euthanized on day 10. Samples of the colonic content and blood serum were sent by courier to the laboratory for toxin neutralization, however, botulinum neurotoxins could not be detected. After that, serum samples from days 6 and 10 were sent to another laboratory for testing for botulinum neurotoxin antibodies by ELISA. Specific antibodies against botulinum neurotoxin type A were measured, indicating a previous, immuno-relevant contact with the toxin. This seroconversion for type A supports the clinical botulism diagnosis. Type A botulism is rarely seen in Europe and has been detected in a horse in Turkey for the first time.

COMPLEX REHABILITATION PATIENTS WITH POST-STROKE SPASTICITY OF THE LOWER LIMB: THE EFFICACY OF BOTULINUM TOXIN (ONABOTULINUMTOXIN A)

The purpose of the study was to examine the effectiveness of the drug of onabotulinumtoxin A (Botox) in complex rehabilitation of patients with post-stroke spasticity of the legs in the early rehabilitation period of stroke. Materials and methods. The study group was comprised 31 patients in the early recovery period after stroke with the presence of equino-varus deformity of the foot with the level of spasticity grade of 3 or more (MAS). All patients received as part of a comprehensive rehabilitation program injections onabotulinumtoxin a (Botox) in a total dose 300 ED. In the process of treatment was evaluated dynamics of neurological status, spasticity, pain, function of walking and everyday activity. Results and conclusion. Proven high efficacy of botulinum neurotoxin type A: reduces the severity of spasticity, the severity of the discomfort in the leg and foot; facilitated the possibility of independent movement of the patient, increases walking speed.

Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization

Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron–like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell–derived neurons as well as the mouse lethality assay.

Comparative characterization of botulinum neurotoxin subtypes F1 and F7 featuring differential substrate recognition and cleavage mechanisms

BoNT/F7, one of the seven subtypes of botulinum neurotoxin type F (F1 to F7), is the second-most divergent subtype of this group. Despite sharing >60% identity with BoNT/F1 at both holotoxin and enzymatic domain levels, it requires an N-terminal extended peptide substrate for efficient substrate cleavage, suggesting its unique substrate recognition and specificity mechanism. Substrate mapping and saturation mutagenesis analysis revealed that VAMP2 (20–65) was likely a minimally effective substrate for LC/F7 (light chain of BoNT/F7), and in addition, LC/F7 recognized VAMP2 in a unique way, which differed significantly from that of LC/F1, although both of them share similar substrate binding and hydrolysis mode. LC/F7 utilizes distinct pockets for specific substrate binding and recognition in particular for the B1, B2 and S2 sites recognitions. Our findings provide insights into the distinct substrate recognition features of BoNT subtypes and useful information for therapy development for BoNT/F.

Frequency and characteristics of goal attainment following BoNT-A injection for management of spasticity

Purpose: To determine which Goal Attainment Scale (GAS) goals are commonly achieved in patients with upper limb and/or lower limb spasticity following Botulinum Neurotoxin Type A (BoNT-A) injection.Method: Adults who attended a Spasticity Management Clinic for upper and/or lower limb BoNT-A injection were included in this prospective cohort study. Goals were set by participants and/or carers in conjunction with the therapist using the GAS, prior to injection and reviewed at one month following the injection. Three out of the five categories of goals were passive. Goals were categorised into: mobility/transfers, pain/comfort, upper limb use, hygiene, and cosmesis. The number of responders for the GAS total score, and in each of the GAS categories, was calculated.Results: Sixty-seven participants were recruited (mean age 51 ± 16 years; range 18–85), 70% had a stroke. Responders for mobility and transfer goals were further post injury or disease onset than non-responders (median 5.9 vs. 1.2 years, p = 0.03). Clients with stroke were less likely than other participants to achieve mobility and transfer goals (p = 0.02). There was a trend for those who achieved mobility and transfer goals to be younger (mean 49 years vs. 55 years, p = 0.06). Although active goals are more commonly identified, passive goals were more likely to be achieved.Conclusions: Although active goals are commonly identified by people with spasticity, passive goals were more likely to be achieved following BoNT-A injection. A long duration of spasticity does not preclude patients from achieving mobility and transfer goals. Non-stroke participants were more likely to achieve mobility and transfer goals.Implications for RehabilitationPatients with chronic spasticity should be considered for BoNT-A as clinically meaningful outcomes can be achieved.When spasticity is present in multiple muscles, the GAS can be an assistive tool to guide clinicians in determining which muscles are a priority for injection, because the client will be more motivated to improve those specific goals.Although carers and patients are more willing to set active goals, these are more difficult to achieve possibly because follow up intervention or independent practise is required.

Cost-Effectiveness of Incobotulinumtoxin-A with Flexible Treatment Intervals Compared to Onabotulinumtoxin-A in the Management of Blepharospasm and Cervical Dystonia

BackgroundIncobotulinumtoxin-A (Xeomin®, Merz Pharmaceuticals, Sydney, New South Wales) is a formulation of botulinum neurotoxin type A that is free of complexing proteins. ObjectiveTo assess the cost-effectiveness of incobotulinumtoxin-A administered with flexible treatment intervals compared to onabotulinumtoxin-A (Botox®, Sydney, New South Wales) in blepharospasm and cervical dystonia from the perspective of Australian health care providers. MethodsA Markov state transition model was developed to perform a cost-utility analysis to compare the cost and health benefits of incobotulinumtoxin-A to that of onabotulinumtoxin-A. The cost-utility analysis compared incobotulinumtoxin-A treatment, given at minimum intervals of 6 weeks and maximum intervals of 20 weeks, with onabotulinumtoxin-A treatment, given at minimum intervals of 12 weeks and maximum intervals of 20 weeks. The Markov model consisted of three health states and followed patients in weekly cycles for 5 years. Only direct health care costs associated with the acquisition and administration of type A botulinum neurotoxins were included. Utility values were derived from a prospective, open-labeled cohort study. The primary outcome measure was the incremental cost per quality-adjusted life-year. Univariate and probabilistic sensitivity analyses were conducted. ResultsIncobotulinumtoxin-A was cost-effective compared to onabotulinumtoxin-A in both blepharospasm and cervical dystonia, with an incremental cost/quality-adjusted life-year gained of A$ 25,588 and A$ 23,794, respectively. ConclusionsIncobotulinumtoxin-A administered at flexible treatment intervals determined by the needs of the patient was found to be a cost-effective treatment option when compared to the administration of onabotulinumtoxin-A in the Australian health care system. The option to administer incobotulinumtoxin-A according to the needs of the patient resulted in patients experiencing symptoms for a fewer number of weeks compared to onabotulinumtoxin-A given at minimum 12-week intervals.

TAT-BoNT/A(₁₋₄₄₈), a novel fusion protein as a therapeutic agent: analysis of transcutaneous delivery and enzyme activity.

Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent for the treatment of some abnormal muscle contractions. In this study, TAT(47-57) peptide, a cell-penetrating peptide, was fused with the catalytic domain of BoNT/A for therapeutic purposes. HeLa and BE(2)-C cell lines were treated separately with purified TAT-BoNT/A(1-448) recombinant protein, and transduction of protein was analyzed by western blotting. Also, transcutaneous delivery through mouse skin surface was evaluated by immunohistochemistry. The in vitro catalytic activity of TAT-BoNT/A(1-448) was evaluated by HPLC. The presence of recombinant protein was detected in both of the cell lines as well as mouse skin cryosections after 60 and 120min of incubation. The concentration of intracellular proteins was increased over time. HPLC analysis showed that this fusion protein has a biological activity 1.5 times as much as the full-length BoNT/A(1-448) protein. TAT-BoNT/A(1-448) fusion protein is biologically active and can transmit through living cells in vitro and in vivo successfully and more effectively compared with BoNT/A(1-448) protein as control.

An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E.

The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.

Submolecular recognition of the C-terminal domain of the heavy chain of botulinum neurotoxin type A by T cells from toxin-treated cervical dystonia patients

We determined the T-cell proliferative responses of the peripheral blood lymphocytes (PBL) from 25 botulinum neurotoxin (BoNT)-treated patients to 31 overlapping synthetic peptides encompassing the C-terminal half (residues 855–1296) of BoNT/A heavy chain. Responses of PBL to HC peptides varied among patients. Samples from 14 patients treated solely with BoNT/A recognized 2–13 (average 6.4) peptides/sample at Z>3.0 level. Six peptide regions representing residues 855–873, 1023–1041, 1051–1069, 1093–1111, 1135–1153 and 1247–1265 were frequently recognized by 36–57% of these PBLs. Influence of treatment parameters on T-cell recognition of the peptides was also investigated.

Botulinum neurotoxin type A in the treatment of classical Trigeminal Neuralgia (BoTN): study protocol for a randomized controlled trial.

BackgroundTrigeminal neuralgia is characterized by paroxysmal facial pain attacks. Adequate prophylactic drug therapy is often limited by the lack of efficacy and intolerance due to central nervous system side effects.Subcutaneous injections of botulinum toxin type A are a promising treatment option for patients with unsatisfactory response to drug therapy or neurosurgical intervention. Its effects are expected to last for at least 3 months, so it could be a potential long-term treatment.This is the study protocol of a prospective, placebo-controlled, double blind clinical trial investigating the add-on therapy of subcutaneous administration of botulinum toxin type A injections to standard treatment in therapy-refractory classical trigeminal neuralgia.Methods and designBoTN is a prospective, double blind, placebo-controlled trial with a randomized withdrawal design in which a single blind phase is followed by a double blind phase (see also Methods and design). Eligible patients with classical trigeminal neuralgia who are otherwise refractory to medical and neurosurgical treatment will receive subcutaneous injections of botulinum toxin type A into injection sites of the affected trigeminal branch.In the first phase all patients will receive botulinum toxin type A in a single blinded intervention. Twelve weeks later therapy responders will be allocated to the verum or placebo (saline) arm in a double blind, randomized manner. These injections will be performed at the same sites as the first injections.This trial will be conducted in a tertiary outpatient clinic specialized in the treatment of headache and facial pain. There will be three investigators performing the injections who are experienced in the treatment of headache and facial pain and trained in botulinum toxin type A injections.DiscussionBoTN is designed to assess the efficacy and safety of subcutaneous botulinum toxin type A injections in addition to standard prophylactic treatment in therapy-refractory trigeminal neuralgia.Trial registration numberEU Clinical Trials Register: EudraCT-No: 2014-001959-24 https://www.clinicaltrialsregister.eu/ctr-search/rest/download/trial/2014-001959-24/DEDate of trial registration26 August 2014Electronic supplementary materialThe online version of this article (doi:10.1186/s13063-015-1052-z) contains supplementary material, which is available to authorized users.

In Vivo Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A.

A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT. We determined lethality (LD50) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0μg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model. drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin. Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.

Systematic Literature Review of AbobotulinumtoxinA in Clinical Trials for Blepharospasm and Hemifacial Spasm.

BackgroundThe aim was to elucidate clinical trial efficacy, safety, and dosing practices of abobotulinumtoxinA (ABO) treatment in adult patients with blepharospasm and hemifacial spasm. To date, most literature reviews for blepharospasm and hemifacial spasm have examined the effectiveness of all botulinum neurotoxin type A products as a class. However, differences in dosing units and recommended schemes provide a clear rationale for reviewing each product separately.MethodsA systematic literature review was performed to identify randomized controlled trials and other comparative clinical studies of ABO in the treatment of blepharospasm and hemifacial spasm published in English between January 1991 and March 2015. Medical literature databases (PubMed, Cochrane library, EMBASE) were searched. A total of five primary publications that evaluated ABO for the management of blepharospasm and hemifacial spasm were identified and summarized.ResultsData included 374 subjects with blepharospasm and 172 subjects with hemifacial spasm treated with ABO. Total ABO doses ranged between 80 and 340 U for blepharospasm and 25 and 85 U for hemifacial spasm, depending on the severity of the clinical condition. All studies showed statistically significant benefits for the treatment of blepharospasm and hemifacial spasm. ABO was generally well tolerated across the individual studies. Adverse events considered to be associated with ABO treatment included: ptosis, tearing, blurred vision, double vision, dry eyes, and facial weakness.DiscussionThese data from 5 randomized clinical studies represents the available evidence base of ABO in blepharospasm and hemifacial spasm. Future studies in this area will add to this evidence base.

Evidence for a natural humoral response in dairy cattle affected by persistent botulism sustained by non-chimeric type C strains

Bovine botulism is a sporadic acute disease that usually causes catastrophic losses in the herds. The unusual clinical evolution of a persistent mild outbreak in a dairy herd, prompted us to characterize the neurotoxin gene profile of the strain involved and to evaluate whether seroconversion had occurred.Diagnosis was based on mild classical symptoms and was supported by PCR and bacteriological findings, which revealed the involvement of a non-mosaic type C strain.An in-house ELISA was developed to detect antibodies to botulinum neurotoxin type C and its performance was evaluated in a vaccination study.Fifty days after the index case, fecal and serum samples were collected from the 14 animals of the herd and screened for Clostridium botulinum and anti-botulinum neurotoxin antibodies type C, respectively. The in-house developed ELISA was also used to test 100 sera samples randomly collected from 20 herds.Strong ELISA reactions were observed in 3 convalescent and 5 asymptomatic animals involved in the studied outbreak. The ELISA-positive cows all tested positive for non-mosaic C. botulinum type C in the feces and the same strain was also detected in the alfalfa hay, suspected to be the carrier source. Ten out of the 100 randomly collected sera tested positive for anti-botulinum neurotoxin type C antibodies: 7 had borderline values and 3 from the same herd showed titers three times higher than the cut-off.We concluded that type C botulism in cattle may occur with variable severity and that prolonged exposure to sublethal doses of botulinum neurotoxin C may occur, resulting in detectable antibodies.

Systematic Literature Review of AbobotulinumtoxinA in Clinical Trials for Blepharospasm and Hemifacial Spasm

<strong>Background:</strong> The aim was to elucidate clinical trial efficacy, safety, and dosing practices of abobotulinumtoxinA (ABO) treatment in adult patients with blepharospasm and hemifacial spasm. To date, most literature reviews for blepharospasm and hemifacial spasm have examined the effectiveness of all botulinum neurotoxin type A products as a class. However, differences in dosing units and recommended schemes provide a clear rationale for reviewing each product separately. <strong>Methods:</strong> A systematic literature review was performed to identify randomized controlled trials and other comparative clinical studies of ABO in the treatment of blepharospasm and hemifacial spasm published in English between January 1991 and March 2015. Medical literature databases (PubMed, Cochrane library, EMBASE) were searched. A total of five primary publications that evaluated ABO for the management of blepharospasm and hemifacial spasm were identified and summarized. <strong>Results:</strong> Data included 374 subjects with blepharospasm and 172 subjects with hemifacial spasm treated with ABO. Total ABO doses ranged between 80 and 340 U for blepharospasm and 25 and 85 U for hemifacial spasm, depending on the severity of the clinical condition. All studies showed statistically significant benefits for the treatment of blepharospasm and hemifacial spasm. ABO was generally well tolerated across the individual studies. Adverse events considered to be associated with ABO treatment included: ptosis, tearing, blurred vision, double vision, dry eyes, and facial weakness. <strong>Discussion:</strong> These data from 5 randomized clinical studies represents the available evidence base of ABO in blepharospasm and hemifacial spasm. Future studies in this area will add to this evidence base.

Characterizing the Lateral Border of the Frontalis for Safe and Effective Injection of Botulinum Toxin.

The forehead is a common site for injection of botulinum neurotoxin type A (BoNT-A) to treat hyperactive facial muscles. Unexpected side effects of BoNT-A injection may occur because the anatomy of the forehead musculature is not fully characterized. The authors described the lateral border of the frontalis in terms of facial landmarks and reference lines to determine the safest and most effective forehead injection sites for BoNT-A. The hemifaces of 49 embalmed adult Korean cadavers were dissected in a morphometric analysis of the frontalis. L2 was defined in terms of FT (the most protruding point of the frontotemporal region), L0 (the line connecting the infraorbital margin with the tragus), and L1 (the line parallel to L0 and passing through FT) such that L2 was positioned 45° from L1 and passed through FT. The distance from FT to the superior margin of the orbicularis oculi was 12.3 ± 3.3 mm. The frontalis extended more than 5 cm along L2 in 49 of 49 cases (100%), more than 6 cm in 47 cases (95.9%), more than 7 cm in 34 cases (69.4%), more than 8 cm in 11 cases (22.4%), and more than 9 cm in 3 cases (6.1%). The lateral border of the frontalis ran parallel to and within 1 cm of the medial side of L2. Surface anatomy mapping can assist with predicting the lateral border of the frontalis to minimize the side effects and maximize the efficiency of BoNT-A injections into the forehead.

Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.

BackgroundThe disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found.MethodsComponents of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS.ResultsGel bands were identified with sequence coverages of 91 % for BoNT/G, 91 % for NTNH, 89 % for HA-70, and 88 % for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93 % sequence coverage.ConclusionsThe BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.