|
1
|
Zhang Y, Yang L, Wang H, Zhang H, Feng Y, Tan T. High-efficiency heterologous expression of functional RVG29-BoNT/A light chain fusion protein in Bacillus subtilis based on a combinatorial strategy. Int J Biol Macromol 2025; 310:143226. [PMID: 40246127 DOI: 10.1016/j.ijbiomac.2025.143226] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2025] [Revised: 04/05/2025] [Accepted: 04/14/2025] [Indexed: 04/19/2025]
Abstract
Botulinum neurotoxin type A (BoNT/A) has been used as an injectable therapeutic agent in medical aesthetics, neuroscience research, and other medical fields. In this study, the non-endotoxin-producing and food-safe bacterium Bacillus subtilis WB800N was selected as the host strain to express functional RVG29-BoNT/A light chain (Lc) fusion proteins through a combinatorial strategy involving promoter engineering, signal peptide optimization, and overexpression of transport-related proteins and molecular chaperones. The RVG29 peptide, known for its neuronal targeting ability, was fused to the Lc of BoNT/A to enhance its transmembrane delivery and substrate cleavage activity. Furthermore, a series of promoter screening, tandem promoter construction, and site-specific mutations were conducted to maximize transcriptional activity of the target protein. Moreover, a library of 244 native signal peptides was constructed, and high-throughput fluorescence screening identified Sec pathway-related signal peptides that significantly improved secretion efficiency. Meanwhile, transport-related proteins, particularly FtsY, and molecular chaperones like GroEL/DnaJ, were overexpressed to further enhance the secretion and soluble expression of the RVG29-Lc. Additionally, knockdown of the hrcA gene, a repressor of heat-shock genes, improved the expression of molecular chaperones, as well as the RVG29-Lc. The combinatorial strategy achieved a final intracellular expression level of 500.33 ± 5.00 mg/L and the secretion level of 75.15 ± 4.33 mg/L under the same experimental conditions. This work presents a robust framework for the high-efficiency production of functional RVG29-Lc fusion proteins in B. subtilis, offering valuable insights into the industrial-scale production of therapeutic BoNT-derived proteins.
Collapse
Affiliation(s)
- Yuhan Zhang
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Key Laboratory of Green Chemicals Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Synthetic Bio-manufacturing Technology Innovation Center, Beijing 102209, China
| | - Lingguang Yang
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; College of Chemical and Biological Engineering, Yichun University, Yichun 336000, China
| | - Hao Wang
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Key Laboratory of Green Chemicals Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Synthetic Bio-manufacturing Technology Innovation Center, Beijing 102209, China
| | - Haobo Zhang
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Key Laboratory of Green Chemicals Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Synthetic Bio-manufacturing Technology Innovation Center, Beijing 102209, China
| | - Yue Feng
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Key Laboratory of Green Chemicals Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Synthetic Bio-manufacturing Technology Innovation Center, Beijing 102209, China.
| | - Tianwei Tan
- National Energy R&D Center for Biorefinery, Beijing University of Chemical Technology, Beijing 100029, China; State Key Laboratory of Green Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Key Laboratory of Green Chemicals Biomanufacturing, Beijing University of Chemical Technology, Beijing 100029, China; Beijing Synthetic Bio-manufacturing Technology Innovation Center, Beijing 102209, China.
| |
Collapse
|
|
2
|
Elgendy YS, Elzoghby S, AbuBakr N. Effect of zinc or copper supplementation on the efficacy and sustainability of botulinum toxin A "Botox" injection in masseter muscle of albino rats. JOURNAL OF STOMATOLOGY, ORAL AND MAXILLOFACIAL SURGERY 2024; 126:102156. [PMID: 39550001 DOI: 10.1016/j.jormas.2024.102156] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/14/2024] [Accepted: 11/14/2024] [Indexed: 11/18/2024]
Abstract
OBJECTIVES This study aimed to evaluate whether oral zinc or copper supplementation affected the efficacy and sustainability of botulinum toxin-A (BTX-A) injection in masseter muscle of albino rats. MATERIALS AND METHODS 32 adult male albino rats were allocated equally into four groups: group I (control), group II received 10U BTX-A injection, group III received 10U BTX-A injection + zinc (1 mg, 4 days pre-injection), and group IV received 10U BTX-A injection + copper (0.04 mg, 7 days post-injection). Rats were euthanized at 2 and 12 weeks (4 rats per subgroup) after injection. The masseter muscle was examined via histological, histochemical, histomorphometrical and real-time polymerase chain reaction (qRT-PCR) analyses. RESULTS The histopathological results of the BTX-A group showed atrophied muscle fibers with increased atrophy with time compared to the control group. The BTX-A + zinc group displayed more atrophy compared to BTX-A group. Conversely, the BTX-A + copper group demonstrated improved histology of muscle fibers compared to BTX-A and BTX-A + zinc groups. Histomorphometric analysis of Masson trichrome staining at 2 and 12 weeks revealed that collagen area percentage was the highest in the BTX-A + copper and control groups, followed by BTX-A and BTX-A + zinc groups. At 12 weeks, the nuclear factor kappa beta (NF-κB) mRNA expression was significantly higher in BTX-A + zinc and BTX-A groups compared to BTX-A + copper group and relative to the control group. CONCLUSION Zinc supplementation significantly improved the effectiveness and durability of BTX-A, whereas copper supplementation reduced its efficacy.
Collapse
Affiliation(s)
- Yasmin Saad Elgendy
- Department of Oral Biology, Faculty of Dentistry, Cairo University, Cairo, Egypt
| | - Sanaa Elzoghby
- Department of Oral Biology, Faculty of Dentistry, Cairo University, Cairo, Egypt
| | - Nermeen AbuBakr
- Department of Oral Biology, Faculty of Dentistry, Cairo University, Cairo, Egypt.
| |
Collapse
|
|
3
|
Moron H, Gagnard-Landra C, Guiraud D, Dupeyron A. Contribution of Single-Fiber Evaluation on Monitoring Outcomes Following Injection of Botulinum Toxin-A: A Narrative Review of the Literature. Toxins (Basel) 2021; 13:toxins13050356. [PMID: 34067540 PMCID: PMC8156529 DOI: 10.3390/toxins13050356] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2021] [Revised: 05/12/2021] [Accepted: 05/13/2021] [Indexed: 11/24/2022] Open
Abstract
Botulinum toxin-A (BoNT-A) blocks acetylcholine release at the neuromuscular junction (NMJ) and is widely used for neuromuscular disorders (involuntary spasms, dystonic disorders and spasticity). However, its therapeutic effects are usually measured by clinical scales of questionable validity. Single-fiber electromyography (SFEMG) is a sensitive, validated diagnostic technique for NMJ impairment such as myasthenia. The jitter parameter (µs) represents the variability of interpotential intervals of two muscle fibers from the same motor unit. This narrative review reports SFEMG use in BoNT-A treatment. Twenty-four articles were selected from 175 eligible articles searched in Medline/Pubmed and Cochrane Library from their creation until May 2020. The results showed that jitter is sensitive to early NMJ modifications following BoNT-A injection, with an increase in the early days’ post-injection and a peak between Day 15 and 30, when symptoms diminish or disappear. The reappearance of symptoms accompanies a tendency for a decrease in jitter, but always precedes its normalization, either delayed or nonexistent. Increased jitter is observed in distant muscles from the injection site. No dose effect relationship was demonstrated. SFEMG could help physicians in their therapeutic evaluation according to the pathology considered. More data are needed to consider jitter as a predictor of BoNT-A clinical efficacy.
Collapse
Affiliation(s)
- Hélène Moron
- Department of Functional Exploration of the Nervous System and Acupuncture, CHU Nîmes, Univ Montpellier, 30029 Nîmes, France;
- EuroMov DHM, IMT Ales, Univ Montpellier, 34090 Montpellier, France;
- CAMIN, INRIA, Univ Montpellier, 34090 Montpellier, France;
- Correspondence:
| | - Corine Gagnard-Landra
- Department of Functional Exploration of the Nervous System and Acupuncture, CHU Nîmes, Univ Montpellier, 30029 Nîmes, France;
| | - David Guiraud
- CAMIN, INRIA, Univ Montpellier, 34090 Montpellier, France;
| | - Arnaud Dupeyron
- EuroMov DHM, IMT Ales, Univ Montpellier, 34090 Montpellier, France;
- Department of Physical and Rehabilitation Medicine, CHU Nîmes, Univ Montpellier, 30029 Nîmes, France
| |
Collapse
|
|
4
|
Nawrocki S, Cha J. Botulinum toxin: Pharmacology and injectable administration for the treatment of primary hyperhidrosis. J Am Acad Dermatol 2019; 82:969-979. [PMID: 31811879 DOI: 10.1016/j.jaad.2019.11.042] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2019] [Revised: 11/15/2019] [Accepted: 11/22/2019] [Indexed: 01/17/2023]
Abstract
Hyperhidrosis is a dermatological condition defined by excessive sweating beyond thermoregulatory needs with significant effects on patients' quality of life. Hyperhidrosis is categorized as primary or secondary: primary hyperhidrosis is mostly focal and idiopathic, whereas secondary hyperhidrosis is commonly generalized and caused by an underlying medical condition or use of medications. Various surgical and nonsurgical therapies exist for primary hyperhidrosis. Although botulinum toxin is one of the deadliest toxins known, when used in small doses, it is one of the most effective therapies for primary hyperhidrosis. Botulinum toxin injections are widely used as a second-line primary hyperhidrosis treatment option once topical treatment strategies have failed. This article provides an overview of the commercially available botulinum toxin formulations and their applications in the treatment of primary hyperhidrosis.
Collapse
Affiliation(s)
- Shiri Nawrocki
- Rutgers-Robert Wood Johnson Medical School, Department of Dermatology, Piscataway, New Jersey.
| | - Jisun Cha
- Thomas Jefferson University, Department of Dermatology, Philadelphia, Pennsylvania
| |
Collapse
|
|
5
|
Balanta-Melo J, Torres-Quintana MA, Bemmann M, Vega C, González C, Kupczik K, Toro-Ibacache V, Buvinic S. Masseter muscle atrophy impairs bone quality of the mandibular condyle but not the alveolar process early after induction. J Oral Rehabil 2018; 46:233-241. [PMID: 30468522 DOI: 10.1111/joor.12747] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2018] [Revised: 11/15/2018] [Accepted: 11/17/2018] [Indexed: 12/27/2022]
Abstract
BACKGROUND Masseter muscle function influences mandibular bone homeostasis. As previously reported, bone resorption markers increased in the mouse mandibular condyle two days after masseter paralysis induced with botulinum toxin type A (BoNTA), followed by local bone loss. OBJECTIVE This study aimed to evaluate the bone quality of both the mandibular condyle and alveolar process in the mandible of adult mice during the early stage of a BoNTA-induced masseter muscle atrophy, using a combined 3D histomorphometrics and shape analysis approach. METHODS Adult BALB/c mice were divided into an untreated control group and an experimental group; the latter received one single BoNTA injection in the right masseter (BoNTA-right) and saline in the left masseter (Saline-left). 3D bone microstructural changes in the mandibular condyle and alveolar process were determined with high-resolution microtomography. Additionally, landmark-based geometric morphometrics was implemented to assess external shape changes. RESULTS After 2 weeks, masseter mass was significantly reduced (P-value <0.001). When compared to Saline-left and untreated condyles, BoNTA-right condyles showed significant bone loss (P-value <0.001) and shape changes. No significant bone loss was observed in the alveolar processes of any of the groups (P-value >0.05). CONCLUSION Condyle bone quality deteriorates at an early stage of BoNTA-induced masseter muscle atrophy, and before the alveolar process is affected. Since the observed bone microstructural changes resemble those in human temporomandibular joint degenerative disorders, the clinical safety of BoNTA intervention in the masticatory apparatus remains to be clarified.
Collapse
Affiliation(s)
- Julián Balanta-Melo
- Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.,School of Dentistry, Universidad del Valle, Cali, Colombia.,Max Planck Weizmann Center for Integrative Archaeology and Anthropology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | | | - Maximilian Bemmann
- Max Planck Weizmann Center for Integrative Archaeology and Anthropology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Carolina Vega
- Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile
| | | | - Kornelius Kupczik
- Max Planck Weizmann Center for Integrative Archaeology and Anthropology, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.,Center for Quantitative Analysis in Dental Anthropology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile
| | - Viviana Toro-Ibacache
- Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.,Center for Quantitative Analysis in Dental Anthropology, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.,Department of Human Evolution, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
| | - Sonja Buvinic
- Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile.,Center for Exercise, Metabolism and Cancer Studies CEMC2016, Faculty of Medicine, Universidad de Chile, Santiago, Chile
| |
Collapse
|
|
6
|
Botulinum Toxin Induced Atrophy: An Uncharted Territory. Toxins (Basel) 2018; 10:toxins10080313. [PMID: 30072597 PMCID: PMC6115806 DOI: 10.3390/toxins10080313] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2018] [Revised: 07/30/2018] [Accepted: 07/31/2018] [Indexed: 11/29/2022] Open
Abstract
Botulinum neurotoxins (BoNTs) produce local chemo-denervation by cleaving soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins. Botulinum neurotoxins are therapeutically indicated in several neurological disorders and have been in use for three decades. The long-term efficacy, safety, and side effects of BoNTs have been well documented in the literature. However, the development of muscle atrophy following chronic exposure to BoNTs has not received sufficient attention. Muscle atrophy is not only cosmetically distressing, but also has an impact on future injections. An extensive literature search was conducted on atrophy and mechanisms of atrophy. Five hundred and four relevant articles in the English language were reviewed. This review revealed the surprising lack of documentation of atrophy within the literature. In addition, as demonstrated in this review, the mechanisms and the clinical factors that may lead to atrophy have also been poorly studied. However, even with this limited information it is possible to indicate factors that could modify the clinical approach to botulinum toxin injections. This review highlights the need for further study of atrophy following BoNT injections.
Collapse
|
|
7
|
Nassri A, Ramzan Z. Pharmacotherapy for the management of achalasia: Current status, challenges and future directions. World J Gastrointest Pharmacol Ther 2015; 6:145-55. [PMID: 26558149 PMCID: PMC4635155 DOI: 10.4292/wjgpt.v6.i4.145] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Revised: 09/06/2015] [Accepted: 10/16/2015] [Indexed: 02/06/2023] Open
Abstract
This article reviews currently available pharmacological options available for the treatment of achalasia, with a special focus on the role of botulinum toxin (BT) injection due to its superior therapeutic effect and side effect profile. The discussion on BT includes the role of different BT serotypes, better pharmacological formulations, improved BT injection techniques, the use of sprouting inhibitors, designer recombinant BT formulations and alternative substances used in endoscopic injections. The large body of ongoing research into achalasia and BT may provide a stronger role for BT injection as a form of minimally invasive, cost effective and efficacious form of therapy for patients with achalasia. The article also explores current issues and future research avenues that may prove beneficial in improving the efficacy of pharmacological treatment approaches in patients with achalasia.
Collapse
|
|
8
|
Matak I, Lacković Z. Botulinum neurotoxin type A: Actions beyond SNAP-25? Toxicology 2015; 335:79-84. [PMID: 26169827 DOI: 10.1016/j.tox.2015.07.003] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2015] [Revised: 07/01/2015] [Accepted: 07/04/2015] [Indexed: 01/29/2023]
Abstract
Botulinum neurotoxin type A (BoNT/A), the most potent toxin known in nature which causes botulism, is a commonly used therapeutic protein. It prevents synaptic vesicle neuroexocytosis by proteolytic cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25). It is widely believed that BoNT/A therapeutic or toxic actions are exclusively mediated by SNAP-25 cleavage. On the other hand, in vitro and in vivo findings suggest that several BoNT/A actions related to neuroexocytosis, cell cycle and apoptosis, neuritogenesis and gene expression are not necessarily mediated by this widely accepted mechanism of action. In present review we summarize the literature evidence which point to the existence of unknown BoNT/A molecular target(s) and modulation of unknown signaling pathways. The effects of BoNT/A apparently independent of SNAP-25 occur at similar doses/concentrations known to induce SNAP-25 cleavage and prevention of neurotransmitter release. Accordingly, these effects might be pharmacologically significant. Potentially the most interesting are observations of antimitotic and antitumor activity of BoNT/A. However, the exact mechanisms require further studies.
Collapse
Affiliation(s)
- Ivica Matak
- Laboratory of Molecular Neuropharmacology, Department of Pharmacology and Croatian Brain Research Institute, University of Zagreb School of Medicine, Šalata 11, 10000 Zagreb, Croatia
| | - Zdravko Lacković
- Laboratory of Molecular Neuropharmacology, Department of Pharmacology and Croatian Brain Research Institute, University of Zagreb School of Medicine, Šalata 11, 10000 Zagreb, Croatia.
| |
Collapse
|