In the recent past about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons, but are also recognized pharmaceuticals for efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here we report the primary structure of a ninth subtype of BoNT/F. Its amino acid sequence diverges by at least 8.4% at the holotoxin and 13.4% on the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q⁵⁸/K⁵⁹ bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6 and vary by up to factor eight. K_M values increase in the order F1 > F9 > F6 ≈ F7, whereas k_cat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

All the available botulinum type A neurotoxins for clinical uses are of A1 subtype. We developed a subtype A2 low molecular weight (150kD) neurotoxin (A2NTX), with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed its efficacy superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (onabotulinumtoxinA) and low molecular weight (150kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n=90; 50-360 mouse LD50 units) or A1NTX (n=30; 50-580 units) were switched to A2NTX (n=120; 25-600 units) from 2010 till 2018 (number of sessions ~ 27, cumulative doses ~11,640 units per patient). Adverse events for A2NTX included weakness (n=1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n=2, A1NTX), dysphagia (8), ptosis (6), local weakness (7) and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had the clinical safety up to the dose of 500 units, and was well tolerated compared to A1 toxins.

Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation’s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay (CANARY^® Zephyr) detects BoNT/A in buffer and various food matrices rapidly in ≤ 40 min, in small volumes ≈ 50 μL, with minimal processing of samples, and is extremely portable (suitcase-sized equipment). BoNT/A was detected at limits of detection (LOD) < 0.075 ng ± 0.02 in assay buffer while milk matrices (non-fat, 2 %, whole milk) increased the LOD to < 0.175 – 0.314 ng. Limits of detection for the assay in complex foods were < 1 ng ± 0.0 (neutralized acidic juices-carrot, orange and apple); < 16.7 ng ± 7.7 (liquid egg); and varied from < 0. 39 – 3.125 ng for solid complex foods (ground beef, green bean baby puree, smoked salmon). These results show that the CANARY^® Zephyr assay can be a highly useful tool in clinical, environmental, and food safety surveillance programs.

Intradermal injection of botulinum neurotoxin is a frequently performed procedure in aesthetic dermatology to improve facial skin tone, texture, fine wrinkles, and enlarged pores. In practice, botulinum neurotoxin type A is also used to reduce skin oiliness of the face. There is increasing evidence that acetylcholine plays specific roles in sebum production, suggesting that botulinum neurotoxin type A may reduce sebum production by interfering with cholinergic transmission between sebaceous glands and autonomic nerve terminals. Botulinum neurotoxins can also inhibit several pathogenetic components of acne development, suggesting that botulinum neurotoxins can be used as a safe and effective treatment modality for acne and other skin disorders related to the overactivity of sebaceous glands. This review aims to explore the current evidence behind the treatment of oily skin and acne with botulinum neurotoxin type A.

Incobotulinumtoxin A and Yoga-like Isometric Exercise in Adolescent Idiopathic Lumbar Scoliosis – a randomized pilot study Abstract: Background: Approximately 90% of scoliosis is adolescent idiopathic (AIS). From its first appearance at 10-14 until age 18 it is most vulnerable to deterioration; young people are most susceptible to the condition worsening. An effective non-surgical means of remediation would be welcome. Design: Randomized control two-arm study, assessing the safety and efficacy of combining incobotulinum injections with yoga to reverse lumbar and thoracolumbar AIS. Methods: In a private clinic setting, non-pregnant healthy 12 – 18 year-olds were either taught a symmetrical “placebo” yoga pose (control sub-group 1), given the side plank (Vasisthasana) done thrice daily with placebo injection (control sub-group 2) or given the thrice-daily side-plank with botulinum injection (Intervention group 3). Injection: 33 IU of incobotulinum toxin type A (Xeomin) injected into concave-side lumbar paraspinals and quadratus lumborum at L2-3 and the psoas muscle at L3-4, or injected similarly with placebo. Randomization by Random.com. Objective: Assess whether muscular asymmetry treated with botulinum toxin injection and the side-plank are safe and effective in AIS.Results: Outcome: Twelve intervention and 12 placebo patients (Groups 1 + 2), 12 -18 years old completed the three-month study period. Mean daily side-plank time = 165 seconds. Mean initial lumbar curvature was 36.9 degrees (SD 14.36), (p<0.0001); mean curvature at 3 weeks was 29.5 degrees. (SD 14.23) (p<0.0001); mean curvature at 3 months was 26.0 degrees (SD 12.81) Onset vs 3-month value: p<0.0001. Two patients in Group 3 complained of shoulder pain, and 2 of wrist pain that resolved when the side-plank was done on the elbow. Conclusion: Muscle asymmetry appears relevant to AIS treatment. Incobotulinum injections combined with the side-plank done with the convex side downward may be safe and helpful in adolescent idiopathic lumbar scoliosis.

Incobotulinumtoxin A and Yoga-like Isometric Exercise in Adolescent Idiopathic Lumbar Scoliosis – a randomized pilot study Abstract: Background: Approximately 90% of scoliosis is adolescent idiopathic (AIS). From its first appearance at 10-14 until age 18 it is most vulnerable to deterioration; young people are most susceptible to the condition worsening. An effective non-surgical means of remediation would be welcome. Design: Randomized control two-arm study, assessing the safety and efficacy of combining incobotulinum injections with yoga to reverse lumbar and thoracolumbar AIS. Methods: In a private clinic setting, non-pregnant healthy 12 – 18 year-olds were either taught a symmetrical “placebo” yoga pose (control sub-group 1), given the side plank (Vasisthasana) done thrice daily with placebo injection (control sub-group 2) or given the thrice-daily side-plank with botulinum injection (Intervention group 3). Injection: 33 IU of incobotulinum toxin type A (Xeomin) injected into concave-side lumbar paraspinals and quadratus lumborum at L2-3 and the psoas muscle at L3-4, or injected similarly with placebo. Randomization by Random.com. Objective: Assess whether muscular asymmetry treated with botulinum toxin injection and the side-plank are safe and effective in AIS. Results: Outcome: Twelve intervention and 12 placebo patients (Groups 1 + 2), 12 -18 years old completed the three-month study period. Mean daily side-plank time = 165 seconds. Mean initial lumbar curvature was 36.9 degrees (SD 14.36), (p<0.0001); mean curvature at 3 weeks was 29.5 degrees. (SD 14.23) (p<0.0001); mean curvature at 3 months was 26.0 degrees (SD 12.81) Onset vs 3-month value: p<0.0001. Two patients in Group 3 complained of shoulder pain, and 2 of wrist pain that resolved when the side-plank was done on the elbow. Conclusion: Muscle asymmetry appears relevant to AIS treatment. Incobotulinum injections combined with the side-plank done with the convex side downward may be safe and helpful in adolescent idiopathic lumbar scoliosis.

Following spinal cord injury (SCI), pathological reflexes develop that result in altered bladder function and sphincter dis-coordination, with accompanying changes in the detrusor. Bladder chemodenervation is known to ablate the pathological reflexes, but the resultant effects on the bladder tissue are poorly defined. In a rodent model of contusion SCI, we examined the effect of early bladder chemodenervation with botulinum toxin A (BoNT-A) on bladder histopathology and collagen deposition. Adult female Long Evans rats were given a severe contusion SCI at spi-nal level T9. The SCI rats immediately underwent open laparotomy and received detrusor injec-tions of either BoNT-A (10 U/animal) or saline. At 8 weeks post injury, the bladders were col-lected, weighed, and examined histologically. BoNT-A injected bladders of SCI rats (SCI-BoNT-A) weighed significantly less than saline injected bladders of SCI rats (SCI-saline) (241 ± 25 mg vs. 183 ± 42 mg; p<0.05). Histological analyses showed that SCI resulted in significantly thicker bladder walls due to detrusor hypertrophy and fibrosis compared to bladders from uninjured animals (339 ± 89.0 m vs. 193 ± 47.9 m; p<0.0001). SCI-BoNT-A animals had significantly thinner bladder walls compared to SCI-saline animals (202 ± 55.4 m vs. 339 ± 89.0 m; p<0.0001). SCI-BoNT-A animals had collagen organization in the bladder walls similar to that of uninjured animals. Detrusor chemodenervation soon after SCI appears to preserve bladder tissue integrity, by reducing the development of detrusor fibrosis and hypertrophy associated with SCI.

Background: Chronic lateral epicondylitis challenges the therapeutical approach; underlying mechanisms are incompletely understood; neuropathic pain and central and peripheral sensitization may explain the fact that botulinum toxin was found to play a role in pain and function management. Methods: We searched the literature for MeSH terms: lateral epicondylitis or synonyms and botulinum toxin. Results: We found 14 papers containing trials on botulinum toxin injection into the tendon or into the extensor muscles (particularly extensor carpi radialis brevis and extensor communis digitorum). We followed the administration pathways, doses, timing and side effects. Conclusion: With chronic course, the focus of the therapy shifts from the afflicted tendon to the inserting muscles, as muscle contracture may create a vicious loop to perpetuate and aggravate the disease. Doses, timing and side effects are discussed.

To demonstrate if botulinum toxin type A (BoNT-A) improves mandibular range of motion and muscle sensibility to palpation in refractory myofascial pain (MFP) patients. METHODS: Eighty consecutive female subjects with refractory MFP, were randomly divided into four equal groups (n=20): BoNT-A low (BoNTA-L/10 U in each temporalis and 30 U in each masseter), BoNT-A medium (BoNTA-M/20 U in each temporalis and 50 U in each masseter), BoNT-A high (BoNTA-H/25 U in each temporalis and 75 U in each masseter) and saline solution 0.9% (SS, placebo control group/0.4 mL in each temporalis and 0.6 mL in each masseter). Clinical measurements of the mandibular movements included: pain-free opening, maximum unassisted and assisted opening, and right and left lateral movements. Palpation tests were performed bilaterally in masseter and temporalis muscle. Results were expressed as median, minimum, maximum, and means ± standard deviation (SD). Chi-Square Test was used to compare differences among groups. A 5% probability level was considered significant in all tests RESULTS: Regardless of dose, all parameters of mandibular range of motion significantly improve after 180 days in BoNT- A groups, when compared to the control group. Pain to palpation on muscles, significantly reduced in all BoNT- A groups regardless of dose, when compared to the control group, after 28 and 180 days of treatment. CONCLUSIONS: Independent of doses, BoNT-A improved mandibular range of motion and muscle sensibility to palpation in refractory MFP patients when compared to SS injections.

The aim of this study was to evaluate the usefulness of cystoscopic injection of Botulinum-A toxin (BTX) in the detrusor wall in the treatment of children with decreased bladder capacity due to neurogenic bladder. The prospective, randomized non placebo controlled trial is conducted in our institution since year 2006 with the approval of the local Ethics Committee. 556 cystoscopic injections of BTX were performed in 141 children with neurogenic bladder in age 1 to 18 years. In all cases decreased bladder capacity and bladder overactivity with urine incontinence were estimated. The pre-and post-treatment evaluations included determination of urinary continence status, bladder function in frequency/volume chart of catheterized urine and in urodynamic studies. Parameters measured in urodynamic investigations included maximal cystometric capacity, detrusor reflex volume, maximal detrusor pressure. Parameters were analyzed before the cystoscopy and during the follow-up examinations in 5 age groups. Values of all measured parameters improved significantly and equally after therapy in every from 5 age groups. The results obtained from the study confirmed that endoscopic administration of BTX improves function of urinary bladder in children with neurogenic bladder, and the method represents an alternative approach to conservative treatment and surgical augmentation.

AB toxins have historically been associated with significant morbidity, mortality through infections such as botulinum, anthrax, cholera, and diphtheria. These AB toxin-mediated diseases remain prevalent in low and middle income countries, with intermittent outbreaks of Shiga toxin-producing Escherichia coli (STEC) or whooping cough by Bordetella pertussis in high-income countries. These reports warrant an investigation to better understand the distinct characteristics of AB toxins derived from different pathogens. As toxigenic pathogens broaden their scope and diversity, it amplifies the complexity of the problems posed by their AB toxins. Here, we discuss the history, structure and characteristics of key AB toxins, and report on historical and ongoing research on these toxins. We also explore research avenues that hold great promise in potentially improving clinical management of toxin-mediated diseases in the future.

Botulinum neurotoxins (BoNTs) are emerging as multipurpose therapeutic compounds for the treatment of several different syndromes involving peripheral and central nervous systems, and muscular and musculoskeletal disorders both in human and veterinary medicine. Therefore, the studies of BoNTs are rapidly developing and identifying newly produced BoNT variants. Efforts should be made to clarify the biological and pharmacological characteristics of these novel BoNTs as well as the natural ones. The high potential of BoNTs as a therapeutic compound for medical syndromes lies on its ability to reach a specific cell type bypassing other cells, thus having mild or no side effects. In this paper the recent developments on BoNTs are reviewed with the aim to analyze the current knowledge on BoNTs biological mechanisms of action, immunogenicity, formulations, and therapeutic applications in the veterinary field, highlighting advantages and drawbacks and identifying the gaps to be filled in order to address research priorities.

(1) Background: This manuscript introduces the "Lines and Dots" (LAD) technique, a novel method for administering Botulinum Toxin Type A (BoNT-A) in treating horizontal forehead lines. This technique emphasizes precision in targeting nerve endplates, considering individual anatomical variations, and optimizing treatment efficacy. (2) Methods: The LAD technique focus on using the frontalis muscle contraction dynamics to identify the specific distribution of the supraorbital and supratrochlear nerves It involves creating a grid of predetermined lines and points on the forehead that align with nerve paths and motor endplate zones. (3) Results: By targeting injections along these precise lines and points, the LAD technique ensures more effective utilization of BoNT-A. This approach not only enhances the toxin's effect but also allows for a reduction in the overall dosage. The strategic placement of injections results in a more natural, symmetrical, and sustained therapeutic effect, ensuring an improved aesthetic outcome. (4) Conclusions: The LAD technique represents a significant advancement in the realm of cosmetic injectables. It offers a tailored, patient-specific approach, maximizing the effectiveness of BoNT-A injections for treating forehead lines while minimizing risks and enhancing patient satisfaction. This method underscores the importance of a deep anatomical understanding and precision in cosmetic procedures.