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ACS Nano Jun 2024We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite...
We have evolved the nanopore-forming macrolittin peptides from the bee venom peptide melittin using successive generations of synthetic molecular evolution. Despite their sequence similarity to the broadly membrane permeabilizing cytolytic melittin, the macrolittins have potent membrane selectivity. They form nanopores in synthetic bilayers made from 1-palmitoyl, 2-oleoyl-phosphatidylcholine (POPC) at extremely low peptide concentrations and yet have essentially no cytolytic activity against any cell membrane, even at high concentration. Here, we explore the structural determinants of macrolittin nanopore stability in POPC bilayers using atomistic molecular dynamics simulations and experiments on macrolittins and single-site variants. Simulations of macrolittin nanopores in POPC bilayers show that they are stabilized by an extensive, cooperative hydrogen bond network comprised of the many charged and polar side chains interacting with each other via bridges of water molecules and lipid headgroups. Lipid molecules with unusual conformations participate in the H-bond network and are an integral part of the nanopore structure. To explore the role of this H-bond network on membrane selectivity, we swapped three critical polar residues with the nonpolar residues found in melittin. All variants have potency, membrane selectivity, and cytotoxicity that were intermediate between a cytotoxic melittin variant called MelP5 and the macrolittins. Simulations showed that the variants had less organized H-bond networks of waters and lipids with unusual structures. The membrane-spanning, cooperative H-bond network is a critical determinant of macrolittin nanopore stability and membrane selectivity. The results described here will help guide the future design and optimization of peptide nanopore-based applications.
Topics: Nanopores; Melitten; Molecular Dynamics Simulation; Phosphatidylcholines; Lipid Bilayers; Hydrogen Bonding; Peptides; Humans
PubMed: 38844421
DOI: 10.1021/acsnano.4c02824 -
Journal of Cellular and Molecular... Jun 2024Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The...
Excessive load on the temporomandibular joint (TMJ) is a significant factor in the development of TMJ osteoarthritis, contributing to cartilage degeneration. The specific mechanism through which excessive load induces TMJ osteoarthritis is not fully understood; however, mechanically-activated (MA) ion channels play a crucial role. Among these channels, Piezo1 has been identified as a mediator of chondrocyte catabolic responses and is markedly increased in osteoarthritis. Our observations indicate that, under excessive load conditions, endoplasmic reticulum stress in chondrocytes results in apoptosis of the TMJ chondrocytes. Importantly, using the Piezo1 inhibitor GsMTx4 demonstrates its potential to alleviate this condition. Furthermore, Piezo1 mediates endoplasmic reticulum stress in chondrocytes by inducing calcium ion influx. Our research substantiates the role of Piezo1 as a pivotal ion channel in mediating chondrocyte overload. It elucidates the link between excessive load, cell apoptosis, and calcium ion influx through Piezo1. The findings underscore Piezo1 as a key player in the pathogenesis of TMJ osteoarthritis, shedding light on potential therapeutic interventions for this condition.
Topics: Chondrocytes; Endoplasmic Reticulum Stress; Apoptosis; Ion Channels; Animals; Temporomandibular Joint; Calcium; Osteoarthritis; Humans; Mice; Signal Transduction; Spider Venoms; Intercellular Signaling Peptides and Proteins
PubMed: 38842129
DOI: 10.1111/jcmm.18472 -
Animals : An Open Access Journal From... May 2024Few mammals are venomous, including one group of primates-slow ( spp.) and pygmy ( spp.) lorises. Hypotheses for the evolutionary function of venom in these primates...
Few mammals are venomous, including one group of primates-slow ( spp.) and pygmy ( spp.) lorises. Hypotheses for the evolutionary function of venom in these primates include defense from predators or ectoparasites, communication or competition with conspecifics, and the capture of prey. We tested the prey capture hypothesis in 75 trials with 22 wild-caught greater slow lorises () housed in a rescue center in Java, Indonesia. We experimentally offered the slow lorises arthropod prey items varying in size, escape potential, and toxicity and recorded venom-related and predatory behaviors using live and video observations. The slow lorises visually targeted arthropod prey, approached it quickly and efficiently, and captured it with a manual grasping motion. They rarely performed venom-related behaviors and seemed to do so in a defensive context. The slow lorises exhibited little variation in pre-capture behavior as a function of prey size or escape potential. In response to noxious prey, the slow lorises performed tongue-flicking and other investigative behaviors that indicate they are using chemosensory input to assess prey characteristics. These data suggest it is unlikely that slow lorises use chemical weapons to subdue arthropod prey and may support, instead, a defensive function for slow loris venom.
PubMed: 38791656
DOI: 10.3390/ani14101438 -
Toxins May 2024Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly...
Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly Morocco, experiences severe envenomations, mainly attributed to and in Morocco, and and in Algeria and Tunisia, with case numbers often underestimated. Current treatment relies mainly on symptomatic approaches, except in Morocco, where management is limited to symptomatic treatment due to controversies regarding specific treatment. In Morocco, between 30,000 and 50,000 scorpion envenomation cases are reported annually, leading to hundreds of deaths, mainly among children. Controversies among clinicians persist regarding the appropriate course of action, often limiting treatments to symptomatic measures. The absence of a specific antivenom for the venoms of the most lethal scorpions further exacerbates the situation. This study aims to address this gap by developing a monovalent antivenom against the endemic and most dangerous scorpion, . The antivenom was produced by immunizing albino rabbits with a mixture of venom collected from high-risk areas in Morocco. Immunizations were performed by subcutaneous injections at multiple sites near the lymphatic system, following an immunization schedule. Production control of neutralizing antibody titers was conducted through immunodiffusion. Once a sufficient antibody titer was achieved, blood collection was performed, and the recovered plasma underwent affinity chromatography. The efficacy of purified IgG was evaluated by determining the ED in mice, complemented by histological and immunohistochemical studies on its ability to neutralize venom-induced tissue alterations and the neutralization of toxins bound to receptors in the studied organs. The monovalent antivenom demonstrated specificity against venom and effective cross-protection against the venom of the scorpions and , highly implicated in lethal envenomations in the Maghreb. This study shows that the developed monovalent antivenom exhibits notable efficacy against local scorpions and a surprising ability to neutralize the most lethal envenomations in North Africa. These results pave the way for a new, more specific, and promising therapeutic approach to countering severe scorpion envenomations, especially in Morocco, where specific treatment is lacking.
Topics: Animals; Humans; Africa, Northern; Antivenins; Morocco; Scorpion Stings; Scorpion Venoms; Scorpions
PubMed: 38787066
DOI: 10.3390/toxins16050214 -
Biomolecules May 2024Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in...
Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia-Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 , but also against other enveloped viruses, making it a potential new candidate therapeutic molecule.
Topics: Scorpion Venoms; Antiviral Agents; Enterovirus A, Human; Humans; Antimicrobial Peptides; Animals; Virus Replication; Chlorocebus aethiops; Vero Cells
PubMed: 38785952
DOI: 10.3390/biom14050545 -
Scientific Reports May 2024The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This...
The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.
Topics: Immunoglobulin E; Cross Reactions; Animals; Humans; Ant Venoms; Ants; Allergens; Epitopes; Recombinant Proteins; Insect Proteins; Female; Adult; Male; Amino Acid Sequence; Middle Aged; Adolescent; Young Adult
PubMed: 38750087
DOI: 10.1038/s41598-024-61843-4 -
International Journal of Molecular... May 2024This research aimed to explore the healing impacts of Melittin treatment on gastrocnemius muscle wasting caused by immobilization with a cast in rabbits. Twenty-four... (Comparative Study)
Comparative Study
This research aimed to explore the healing impacts of Melittin treatment on gastrocnemius muscle wasting caused by immobilization with a cast in rabbits. Twenty-four rabbits were randomly allocated to four groups. The procedures included different injections: 0.2 mL of normal saline to Group 1 (G1-NS); 4 μg/kg of Melittin to Group 2 (G2-4 μg/kg Melittin); 20 μg/kg of Melittin to Group 3 (G3-20 μg/kg Melittin); and 100 μg/kg of Melittin to Group 4 (G4-100 μg/kg Melittin). Ultrasound was used to guide the injections into the rabbits' atrophied calf muscles following two weeks of immobilization via casting. Clinical measurements, including the length of the calf, the compound muscle action potential (CMAP) of the tibial nerve, and the gastrocnemius muscle thickness, were assessed. Additionally, cross-sectional slices of gastrocnemius muscle fibers were examined, and immunohistochemistry and Western blot analyses were performed following two weeks of therapy. The mean regenerative changes, as indicated by clinical parameters, in Group 4 were significantly more pronounced than in the other groups ( < 0.05). Furthermore, the cross-sectional area of the gastrocnemius muscle fibers and immunohistochemical indicators in Group 4 exceeded those in the remaining groups ( < 0.05). Western blot analysis also showed a more significant presence of anti-inflammatory and angiogenic cytokines in Group 4 compared to the others ( < 0.05). Melittin therapy at a higher dosage can more efficiently activate regeneration in atrophied gastrocnemius muscle compared to lower doses of Melittin or normal saline.
Topics: Animals; Rabbits; Melitten; Muscle, Skeletal; Regeneration; Muscular Atrophy; Male
PubMed: 38732255
DOI: 10.3390/ijms25095035 -
International Journal of Molecular... Apr 2024Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for...
Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type Ca3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type Cas but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH to be a concentration-dependent partial inhibitor of Ca3.2 (IC = 1.18 μM) and Ca3.3 (IC = 0.49 μM) depolarized currents but was ineffective at Ca3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type Ca2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH was determined using NMR spectroscopy and used in docking studies to predict its binding site at Ca3.2 and Ca3.3. As both Ca3.2 and Ca3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH produced antiallodynia in both mechanical and thermal pain.
Topics: Animals; Calcium Channels, T-Type; Mice; Scorpion Venoms; Hyperalgesia; Disease Models, Animal; Pain, Postoperative; Calcium; Male; Humans; Calcium Channel Blockers
PubMed: 38731963
DOI: 10.3390/ijms25094745 -
Scientific Reports May 2024It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the...
It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.
Topics: Scorpion Venoms; Antivenins; Animals; Scorpions; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Chromatography, High Pressure Liquid; Tandem Mass Spectrometry; Peptides; Amino Acid Sequence
PubMed: 38710718
DOI: 10.1038/s41598-024-59150-z -
BMC Immunology Apr 2024Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation... (Comparative Study)
Comparative Study
BACKGROUND
Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy.
METHODS
Seventeen patients with an insect venom allergy were included in the study. The established "BAT 1" utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas "BAT 2" uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed.
RESULTS
Seven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen's kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive.
CONCLUSION
The BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.
Topics: Humans; Basophils; Female; Male; Adult; Middle Aged; Flow Cytometry; Arthropod Venoms; Pilot Projects; Animals; Hypersensitivity; Insect Bites and Stings; Bee Venoms; Young Adult; Aged; Antibodies; Adolescent; Basophil Degranulation Test; Venom Hypersensitivity
PubMed: 38678193
DOI: 10.1186/s12865-024-00616-0