Rapid eradication of vancomycin and methicillin-resistant Staphylococcus aureus by MDP1 antimicrobial peptide coated on photocrosslinkable chitosan hydrogel: in vitro antibacterial and in silico molecular docking studies.

Introduction: Antibiotic resistance and weak bioavailability of antibiotics in the skin due to systemic administration leads to failure in eradication of vancomycin- and methicillin-resistant Staphylococcus aureus (VRSA and MRSA)-associated wound infections and subsequent septicemia and even death. Accordingly, this study aimed at designing a photocrosslinkable methacrylated chitosan (MECs) hydrogel coated by melittin-derived peptide 1 (MDP1) that integrated the antibacterial activity with the promising skin regenerative capacity of the hydrogel to eradicate bacteria by burst release strategy. Methods: The MECs was coated with MDP1 (MECs-MDP1), characterized, and the hydrogel-peptide interaction was evaluated by molecular docking. Antibacterial activities of MECs-MDP1 were evaluated against VRSA and MRSA bacteria and compared to MECs-vancomycin (MECs-vanco). Antibiofilm activity of MECs-MDP1 was studied by our novel 'in situ biofilm inhibition zone (IBIZ)' assay, and SEM. Biocompatibility with human dermal fibroblast cells (HDFs) was also evaluated. Results and Discussion: Molecular docking showed hydrogen bonds as the most interactions between MDP1 and MECs at a reasonable affinity. MECs-MDP1 eradicated the bacteria rapidly by burst release strategy whereas MECs-vanco failed to eradicate them at the same time intervals. Antibiofilm activity of MECs-MDP1 were also proved successfully. As a novel report, molecular docking analysis has demonstrated that MDP1 covers the structure of MECs and also binds to lysozyme with a reasonable affinity, which may explain the inhibition of lysozyme. MECs-MDP1 was also biocompatible with human dermal fibroblast skin cells, which indicates its safe future application. The antibacterial properties of a photocrosslinkable methacrylated chitosan-based hydrogel coated with MDP1 antimicrobial peptide were successfully proved against the most challenging antibiotic-resistant bacteria causing nosocomial wound infections; VRSA and MRSA. Molecular docking analysis revealed that MDP1 interacts with MECs mainly through hydrogen bonds with reasonable binding affinity. MECs-MDP1 hydrogels eradicated the planktonic state of bacteria by burst release of MDP1 in just a few hours whereas MECs-vanco failed to eradicate them. inhibition zone assay showed the anti-biofilm activity of the MECs-MDP1 hydrogel too. These findings emphasize that MECs-MDP1 hydrogel would be suggested as a biocompatible wound-dressing candidate with considerable and rapid antibacterial activities to prevent/eradicate VRSA/MRSA bacterial wound infections.

Highly in vitro anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer cells.

BACKGROUND: Development of promising medicines from natural sources, specially venom, is of highly necessitated to combat against life-threatening cancers. Non-small cell lung cancer (NSCLC) has a significant percentage of mortalities. Melittin, from bee venom, is a potent anticancer peptide but its toxicity has limited its therapeutic applications. Accordingly, this study aims to synthesize niosomes with suitable stability and capacity for carrying melittin as a drug. Additionally, it seeks to evaluate the anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer. METHODS: The niosome was prepared by thin film hydration method. Cytotoxicity and apoptosis were assessed on A549, Calu-3, and MRC5 cells. Real-time PCR was used to determine expression of apoptotic and pro-apoptotic Bax, Bcl2, and Casp3 genes. Immunocytochemistry (ICC) was also used to confirm expression of the abovementioned genes. Furthermore, wound healing assay was performed to compare inhibition effects of melittin-loaded niosomes with free melittin on migration of cancer cells. RESULTS: IC50 values of melittin-loaded niosomes for A549, Calu-3, and MRC5 cells were respectively 0.69 µg/mL, 1.02 µg/mL, and 2.56 µg/mL after 72 h. Expression level of Bax and Casp3 increased '10 and 8' and '9 and 10.5' fold in A549 and Calu-3, whereas Bcl2 gene expression decreased 0.19 and 0.18 fold in the mentioned cell lines. The cell migration inhibited by melittin-loaded niosomes. CONCLUSIONS: Melittin-loaded niosomes had more anti-cancer effects and less toxicity on normal cells than free melittin. Furthermore, it induced apoptosis and inhibited cancer cells migration. Our results showed that melittin-loaded niosomes may be a drug lead and it has the potential to be future developed for lung cancer treatment.

Development of a Functional Nanobody Targeting Programmed Cell Death Protein-1 as Immune Checkpoint Inhibitor.

BACKGROUND: Programmed cell death protein 1 (PD-1) is a membrane receptor that is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, natural killer T cells, and dendritic cells. In cancer, the interaction between PD-1 and its ligand PD-L1 suppresses the activation and function of T lymphocytes, leading to the impairment and apoptosis of tumor-specific T cells. This mechanism allows cancer cells to evade the immune response and promotes tumor progression. METHODS: Recombinant PD-1 protein was produced and used to immunize a camel. A nanobody library was generated from the camel's peripheral blood lymphocytes and screened for PD-1 binding. A specific nanobody (3PD9) was selected and characterized by affinity measurement, western blotting, and flow cytometry analysis. The ability of the selected nanobody to block the inhibitory signal of PD-1 in peripheral blood mononuclear cells (PBMCs) was evaluated by measuring the level of interleukin-2 (IL-2). RESULTS: The selected nanobody showed high specificity and affinity for human PD-1. Western blot and flow cytometry analysis confirmed that 3PD9 could recognize and bind to human PD-1 on the cell surface. It was demonstrated that the level of IL-2 was significantly increased in PBMCs treated with 3PD9 compared to the control group, indicating that the nanobody could enhance the T cell response by disrupting the PD-1/PD-L1 interaction. CONCLUSION: The results suggested that the anti-PD-1 nanobody could be a promising candidate for cancer immunotherapy.

In silico and In vitro Assessment of Antibacterial Activity, LPS Binding Affinity, and Toxicity of the GKY25 Peptide.

INTRODUCTION: Extensively and multi-drug resistant isolates of bacteria (MDR, XDR) have caused significant health problems and are responsible for high morbidity and mortality as well. In this critical condition, the discovery, design, or development of new antibiotics is of great concern. According to this necessity, antimicrobial peptides (AMPs) suggested as promising agents. Accordingly, this study aims to evaluate the GKY25 peptide to develop its future antibacterial applications as well as confirmation of LPS neutralization. METHODS: Predictions of 3D structure and helical wheel projection analysis of the peptide were performed by ITASSER and Heliquest servers. Binding affinity and antibacterial activity were performed using molecular docking and CAMPR4, respectively, followed by experimental binding assay as well as in vitro antibacterial assay. RESULTS: GKY25 was predicted as an alpha-helical peptide, and its helicity showed probable projection of hydrophobic and positively-charged amino acid residues. Docking studies showed binding affinity of GKY25 peptide to gram-positive and outer and inner gram-negative bacterial membranes as -5.7, -6.8, and -4 kcal/mole, respectively. CAMPR4 analysis predicted the peptide as an AMP. Experimental binding assay showed that the peptide binds LPS immediately and their interaction was observed at 274 nm. CONCLUSION: Gathering all in silico and in vitro data together, GKY25 is a good drug lead that could be examined further using clinical isolates of gram-negative bacteria in vitro.

Design of a new electrochemical aptasensor based on screen printed carbon electrode modified with gold nanoparticles for the detection of fumonisin B1 in maize flour.

A new aptasensor for detecting fumonisin B1 (FB1) in the maize samples was developed based on DNA- aptamer recognition and electrochemical technique. A thiol-modified single-stranded DNA (ss-HSDNA) was immobilized on a screen printed carbon electrode (SPCE) electrodeposited by gold nanoparticles (AuNPs). The morphology and structure of SPCE and AuNPs/SPCE were evaluated via scanning electron microscopy (SEM) equipped with energy dispersive spectroscopy (EDS). The SEM results demonstrated that the SPCE had a flat sheet-like structure, and the AuNPs were homogeneously electrodeposited on the SPCE. Cyclic voltammetry (CV) experiments in the [Fe(CN)6]- 3/- 4 solution were conducted to investigate each step of electrode modification as well as aptasensor performance. Aptamer-FB1 interaction prevented the electron transfer permitting the determination of FB1 in the range of 0.5-500 ng/mL with a low detection limit (0.14 ng/mL). The designed aptasensor was also shown high selectivity, acceptable repeatability and reproducibility, good long-term stability, and excellent recovery. Furthermore, there was a strong correlation between the findings achieved via the designed aptasensor and high performance liquid chromatography (HPLC). Therefore, a simple construction process and satisfactory electrochemical performance of the proposed aptasensor have a great potential for the detection of FB1 in maize samples.

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

Background: The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. Methods: In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). Results: The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 µg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 µg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 µg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 µg/mL, and MBEC values ranging from 40 to 10 µg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. Conclusion: In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Fast killing kinetics, significant therapeutic index, and high stability of melittin-derived antimicrobial peptide.

The emergence of multidrug-resistant (MDR) bacteria is a major challenge for antimicrobial chemotherapy. Concerning this issue, antimicrobial peptides (AMPs) have been presented as novel promising antibiotics. Our previous de novo designed melittin-derived peptides (MDP1 and MDP2) indicated their potential as peptide drug leads. Accordingly, this study was aimed to evaluate the kinetics of activity, toxicity, and stability of MDP1 and MDP2 as well as determination of their structures. The killing kinetics of MDP1 and MDP2 demonstrate that all bacterial strains were rapidly killed. MDP1 and MDP2 were ca. 100- and 26.6-fold less hemolytic than melittin and found to be respectively 72.9- and 41.6-fold less cytotoxic than melittin on the HEK293 cell line. MDP1 and MDP2 showed 252- and 132-fold improvement in their therapeutic index in comparison to melittin. MDP1 and MDP2 sustained their activities in the presence of human plasma and were found to be ca. four to eightfold more stable than melittin. Spectropolarimetry analysis of MDP1 and MDP2 indicates that the peptides adopt an alpha-helical structure predominantly. According to the fast killing kinetics, significant therapeutic index, and high stability of MDP1, it could be considered as a drug lead in a mouse model of septicemia infections.

Discovery of Leptulipin, a New Anticancer Protein from theIranian Scorpion, Hemiscorpius lepturus.

Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.

Green synthesis of PEG-coated MIL-100(Fe) for controlled release of dacarbazine and its anticancer potential against human melanoma cells.

In this study, the potential of using MIL-100(Fe) metal-organic framework (MOF) for loading and controlling the release of dacarbazine (DTIC) was evaluated for in vitro treatment of melanoma. The drug loading was performed during the green synthesis of MIL-100(Fe) in an aqueous media without using any harmful solvents, to obtain MIL-DTIC. The surface of this structure was then coated with polyethylene glycol (PEG) in the same aqueous solution to synthesize MIL-DTIC-PEG. The synthesized samples were characterized using various methods. Their release profile was studied in phosphate-buffered saline (PBS) and simulated cutaneous medium (SCM). The cytotoxicity of DTIC and its nano-MOF formulation were investigated against melanoma A375 cell lines. The results revealed that the PEG coating (PEGylation) changed the surface charge of MOF from -2.8 ± 0.9 mV to -42.8 ± 1.2 mV, which can contribute to the colloidal stability of MOF. The PEGylation showed a significant effect on controlled drug release, especially in SCM, which increases the complete release time from 60 h to 12 days. Moreover, both of the drug-containing MOFs showed more toxicity than DTIC and unloaded MOFs, confirming that the cumulative release of drug and better cellular uptake of NPs lead to increased toxicity.

Oxilipin, a New Anti-cancer Phospholipase A2-like Protein from Iranian Caspian Cobra, Naja Naja Oxiana.

The discovery of novel anti-cancer agents from natural resources is highly necessary. Concerning the above problem, the purpose of this study was to discover an anti-cancer compound from Caspian cobra venom. Fractionation of Caspian cobra venom was performed by gel filtration and IEX chromatography. The results showed an anti-cancer protein nominated as Oxilipin. Activity and toxicity of Oxilipin were studied on the colon SW480 cancer cell line using MTT, LDH release, PI staining, morphological cell analysis, hemolysis, and anti-proliferation assays. Oxilipin, an 11kDa protein purified from the venom of the Caspian cobra. LC/MS/MS analysis of obtained protein showed homology with Phospholipase A2 from Naja naja oxiana. 40 µg/ml of Oxilipin can induce an apoptotic effect in SW480 cell line up to 90%; meanwhile, this amount can induce only one-third of cytotoxicity on a normal cell. In this study, Iranian cobra venom was found to have cytotoxic effects on SW480 colon cancer tumor cells, with the least cytotoxicity on normal cells on HEK-293. Given that Oxilipin has slight toxicity on normal cells, it can be hypothesized that the obtained peptide can be considered as a drug lead in an animal model study of colon cancer.

Cellular Immunity in Mice Vaccinated with Recombinant Phospholipase D Toxoid of Hemiscorpius lepturus Scorpion.

Background: Hemiscorpius lepturus is one of the most dangerous scorpions in Iran and the world. Numerous studies have been conducted on phospholipases, especially phospholipase D, in this scorpion's venom, and the results have shown this protein to be the main cause of death. Therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. The present study was conducted to assess the non-toxicity of this toxoid and the safety of the vaccine candidate in BALB/c mice. Methods: The production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using ELISpot assay 28 days following immunization with rPLD toxoid. Results: The unpaired t-test results showed a significant increase in the concentration of IFN-γ cytokine in the vaccinated mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant difference was observed in the levels of IL-4 (P= 0.16) despite an increase in this cytokine. The in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the LD100 of the venom. Moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin. Conclusion: As a vaccine, the toxoid has the potential to increase the Th1 cytokine response and, subsequently, increase acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the venom of Hemiscorpius lepturus.

Eradication of vancomycin-resistant Staphylococcus aureus on a mouse model of third-degree burn infection by melittin: An antimicrobial peptide from bee venom.

Third-degree burn infections caused by antibiotic-resistant bacteria are of high clinical concern. Chemical antibiotics are not promising in eradication of bacterial infections. In this challenging condition, antimicrobial peptides (AMPs) are recently introduced as novel promising agents to overcome the issue. Accordingly, our study aimed to evaluate the efficiency of 'melittin' as natural peptide in bee venom, in eradicating vancomycin resistant Staphylococcus aureus (VRSA) on a mouse model of third-degree burn infection. In vitro pharmacological value of melittin was determined by examining its inhibitory and killing activities on VRSA isolates at different doses and time periods. The action mechanism of 'melittin' was evaluated by fluorescent release assay and Field Emission Scanning Electron Microscopy (FE-SEM) analyses. In vivo activity and toxicity of melittin were also examined on a mouse model of third-degree burn infection. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of melittin on all isolates ranged from '0.125-2 µg/mL' and '0.125-4 µg/mL', respectively. Rapid antibacterial activity of melittin on VRSA isolates was demonstrated by killing kinetics assays. Fluorometric and FE-SEM analyses indicated the membranolytic effects of melittin on VRSA isolates. The colonized VRSA bacteria were eradicated by melittin at 16 µg, in a single dose. No dermal toxicity and in vivo hemolysis were observed in the examined mice. The lack of in vivo toxicity of melittin along with its potent antibacterial activity indicated its promising therapeutic value as a topical drug against S. aureus associated third-degree burn infections.

Discovery of a New Analgesic Peptide, Leptucin, from the Iranian Scorpion, Hemiscorpius lepturus.

Hemiscorpius lepturus scorpion stings do not induce considerable pain based on epidemiological surveys conducted in the southwest part of Iran. Accordingly, this study was aimed to identify the analgesic molecule in H. lepturus venom by analyzing a cDNA library of the scorpion venom gland looking for sequences having homology with known animal venom analgesic peptides. The analgesic molecule is a cysteine rich peptide of 55 amino acids. the synthetic peptide was deprotected and refolded. RP-HPLC, Ellman's, and DLS assays confirmed the refolding accuracy. Circular dichroism (CD) showed helix and beta sheet contents. This peptide, called leptucin, demonstrated 95% analgesic activity at the dose of 0.48 mg/kg in hot plate assay. Leptucin at the doses of 0.32, 0.48, and 0.64 mg/kg showed 100% activity in thermal tail flick test. No hemolysis or cytotoxicity was observed at 8 and 16 µg. Histopathology evaluations indicated no hepatotoxicity, nephrotoxicity, and cardiotoxicity. We thus report that leptucin is the analgesic agent of H. lepturus venom. Regarding the high in vivo efficacy of leptucin and the fact it shows no observable toxicity, it could be suggested as a drug lead in a preclinical study of acute pain as well as the study of its mechanism of action.

Cloning, high-level gene expression and bioinformatics analysis of SP15 and LeIF from Leishmania major and Iranian Phlebotomus papatasi saliva as single and novel fusion proteins: a potential vaccine candidate against leishmaniasis.

BACKGROUND: Early exacerbation of cutaneous leishmaniasis is mainly affected by both the salivary and Leishmania parasite components. Little is known of the vaccine combination made by immunogenic proteins of sandfly saliva (SP15) with Leishmania parasites (LeIF) as a single prophylactic vaccine, namely SaLeish. Also, there are no data available to determine the species-specific sequence of SP15 isolated from the Iranian Phlebotomus papatasi. METHODS: Integrated bioinformatics and genetic engineering methods were employed to design, optimize and obtain a vector-parasite-based vaccine formulation in a whole-length fusion form of LeIF-SP15 against leishmaniasis. Holistic gene optimization was initially performed to obtain a high yield of pure 'whole-SaLeish' expression using bioinformatics analyses. Genomic and salivary gland RNAs of wild-caught P. papatasi were extracted and their complementary DNA was amplified and cloned into pJET vector. RESULTS: The new chimeric protein of whole-SaLeish and randomly selected transcripts of native PpIRSP15 (GenBank accession nos. MT025054 and MN938854, MN938855 and MN938856) were successfully expressed, purified and validated by immunoblotting assay. Furthermore, despite the single amino acid polymorphisms of PpIRSP15 found at positions Y23 and E73 within the population of wild Iranian sandflies, antigenicity and conservancy of PpIRSP15 epitopes remained constant to activate T cells. CONCLUSIONS: The SaLeish vaccine strategy takes advantage of a plethora of vector-parasite immunogenic proteins with potential protective efficacy to stimulate both the innate and specific cellular immune responses against Leishmania parasites.

Interactions of GF-17 derived from LL-37 antimicrobial peptide with bacterial membranes: a molecular dynamics simulation study.

Human cathelicidin LL-37 has recently attracted interest as a potential therapeutic agent, mostly because of its ability to kill a wide variety of pathogens and cancer cells. In this study, we used molecular dynamics simulation aimed to get insights that help to correlate with the antibacterial activity of previously designed LL-37 anticancer derivative (i.e. GF-17). Two independent molecular dynamics simulation involving four units of GF-17 peptide in the mixture (9:1) of 1,2-dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and the pure DPPG lipids were performed. Various properties of membranes such as mass density distributions, area per lipid, bilayer thickness, and lateral diffusion were examined in both systems. The results showed that the thickness of the bilayer was not affected by the presence of GF-17, while the area per lipid and lateral diffusion of lipids showed an increase. Moreover, the potential of the mean force (PMF) method was used to calculate the free energy profile for transferring GF-17 from the bulk water into both kinds of membranes. It revealed that penetration of GF-17 into the DPPG membrane was more favorable than the DPPE/DPPG membrane, and there was no energy barrier for crossing through the bilayer center. Investigation of the radius of gyration (Rg) and root mean square fluctuation (RMSF) of peptides in two membranes showed that GF-17 had more compactness and rigidity in the pure DPPG system. By examining the secondary structure of GF-17 peptide, it was seen that the α-helix, and coil structures in both DPPE/DPPG and pure DPPG membranes are dominant.

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation.

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

Complete neutralization of the lethality of Hemiscorpius lepturus crude venom by a novel anti-recombinant phospholipase D1 IgGs.

Treatment of scorpion envenomation is a challenging issue since serotherapy is implemented by administration of polyvalent equine antisera. In our previous study we discovered that recombinant phospholipase D1 (Hl-RecPLD1) is responsible for the lethality of Hemiscorpius lepturus (H. lepturus) venom in mice. Accordingly, this study was aimed to investigate the protectivity of purified anti-Hl-RecPLD1 IgG against the lethality or major complications of H. lepturus venom. The neutralization efficiency of purified anti-Hl-RecPLD1 IgGs against sphingomyelinase activities of the crude venom and Hl-RecPLD1 was also assessed. Anti-Hl-RecPLD1 IgGs at optimum amount of 3.7 mg completely neutralized one Lethal Dose 100 (LD100) of crude venom in mice. The anti-Hl-RecPLD1 IgGs remarkably reduced the necrosis area from 6.5 to 1 cm2 in rabbit derma, induced by the crude venom. The anti-Hl-RecPLD1 IgGs remarkably reduced the sphingomyelinase and hemolytic activities of crude venom as well. In conclusion, a novel rabbit monovalent IgG against Hl-RecPLD1 was able to completely protect the mice against the lethality of H. lepturus crude venom and reduced its toxicity as well. Such monovalent anti-Hl-RecPLD1 IgGs may have potential applications in serotherapy of H. lepturus envenomation.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Significant Anticancer Activity of a Venom Fraction Derived from the Persian Gulf Sea Anemone, Stichodactyla haddoni.

Chemotherapy is still one of the main therapeutic regimens in cancer patients but its toxicity is a hard challenge for every patient yet. One of the available solutions is tracing for non-toxic anticancer agents from natural resources. Numerous proteins and peptides in the venom of sea anemones are potentially useful agents with pharmacological properties. Concerning to significance of this issue, the current study was aimed to finding a non-toxic anticancer fraction from the venom of the Persian Gulf sea anemone, Stichodactyla haddoni. Anticancer and hemolytic activity of crude venom was evaluated and followed by fractionation using RP-HPLC. Breast, Brain, and Colon cancer cell lines were selected to assessment of anticancer activity and toxicity. IC50 of crude venom on the abovementioned cancer cell lines was as 4.13, 6.58, and 31.54 µg, respectively. According to the results obtained by paired sample t-test and comparison of toxicity of the fractions in normal cell line, F10, designated as hadonin, was determined as the candidate anti-cancer fraction. The non-toxic dose of F10 was 20 ng in which showed respectively 66, 29, and 7 anticancer activities on breast, brain, and colon cancer cell lines. According to results, anticancer activity of hadonin is of high pharmaceutical value to follow its therapeutic potency in animal model. In conclusion, the venom of the Persian Gulf sea anemone contains a potential anticancer agent with reasonable activity at nanogram level against three kinds of cancer cells with no toxicity on normal cells.

Genotoxicity assessment of antiepileptic drugs (AEDs) in human embryonic stem cells.

Several antiepileptic drugs (AEDs) are administrated during pregnancy according to recent therapeutic protocols. Ten percent of pregnant women with epilepsy give birth to offspring with malformations and teratogenic defects. Since the mechanism of action of AEDs is not yet completely understood, therefore, it could be hypothesized that they may cause cyto- or genotoxicity in embryonic fetus cells. To investigate this hypothesis, the genotoxicity and cell survival of AEDs treated human embryonic stem cells (hESCs) were investigated by single-cell gel electrophoresis (Comet assay) and MTS assay, respectively. HESCs (Royan H6 cell line) were treated in-vitro with high therapeutic doses of Carbamazepine, Gabapentin, Lamotrigine, Levetiracetam or Topiramate as monotherapy or combination therapy of each drug with Folic acid. After hESCs pluripotency confirmation, the effect of AEDs on cellular DNA damage of hESCs was investigated. levetiracetam and topiramate were found to damage the DNA significantly compared to untreated cells. The amount of DNA damage produced by carbamazepine and lamotrigine was similar while for gabapentin, the amount of DNA migration was very low and produced less DNA damage than the others. A considerable reduction in DNA damages occurred in genotoxicity in the presence of Folic acid in comparison to AEDs monotherapies. According to our results, all mentioned AEDs caused DNA damage, while Levetiracetam and topiramate caused more extensive DNA damages than the others. Noticeably, the addition of Folic acid to the treated cells decreased the DNA damages considerably.