Proteomic analysis of two novel peptides from the Odontobuthus doriae scorpion venom.

The venom of the Odontobuthus doriae scorpion, prevalent in East Asia and Iran, has not been fully characterized. This study provides the first proteomic profile of O. doriae venom to explore its potential as a medical. 2D-PAGE analysis revealed 96 protein spots with isoelectric points from 3 to 9 and molecular weights between 6.6 to 205 kDa. Fourteen toxin fractions were isolated via HPLC, and SDS-PAGE showed seven protein bands ranging from 3.8 to 182 kDa. MALDI-TOF MS identified Peptide 1 and Peptide 2, resembling Hemoglobin beta-2 chain and Chaperonin HSP60 and suggest potential therapeutic applications for P1 and P2.

Anticancer and antibacterial effects of Iranian viper (Vipera latifii) venom; an in-vitro study.

Viper venom contains antibacterial and cytotoxic components. The aim of this study was to identify and evaluate the antimicrobial and cytotoxic properties of the crude venom of Vipera latifii (V. latifii). Lyophilized venom of V. latifii was quantified by Bradford method and its antibacterial activity (6.25-400 µg/ml) was assessed using the MTT, MIC, Disc diffusion, and Well diffusion assays. Also, its cytotoxic activity was investigated using MTT reduction, Neutral uptake, and Comet assay on human liver cancer (HepG2) cell line. Crude venom showed antibacterial effects against Bacillus subtilis and Staphylococcus aureus, but was not effective on Escherichia coli. Also, the crude venom showed apoptotic and necrotic effects on human liver cancer cells. The venom of V. latifii can inhibit the growth of bacteria and cancer cells. These findings suggest that this may be a potential source of molecules with antibacterial and anticancer characteristics.

Simultaneous targeted inhibition of Sox2-Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells.

Transcriptional master regulators like Sox2 and Oct4, which are expressed in various human tumors, have been shown to cause tumor growth promotion as well as epithelial dysplasia by means of interfering with progenitor cell differentiation. In order to investigate the potential of Sox2-Oct4 transcription factor decoy (TFD) strategy for differentiation therapy, mouse embryonic stem cells (mESCs) were used in this study as a model of cancer stem cells (CSCs). Sox2-Oct4 complex decoy ODNs (cd-ODNs) were designed according to their elements in the promoter region of Sox2 gene. DNA-protein interactions between decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Then, decoy and scrambled ODNs were transfected into mESCs with lipofectamine under 2 inhibitors (2i) conditions. Fluorescence and confocal microscopy, cell viability, cell cycle and apoptosis analysis, alkaline phosphatase, embryoid body formation assay, and real-time PCR were used to conduct further investigations. EMSA data showed that Sox2-Oct4 decoy ODNs bound specifically to their recombinant proteins. The results revealed that the synthesized complex decoy can concomitantly target Sox2 and Oct4, which subsequently represses the stemness properties of mESCs compared to controls through decreasing cell viability, arresting cell cycle in G0 /G1 phases, inducing apoptosis, and modulating differentiation in mESCs despite the presence of 2i/LIF in cell culture. While cd-ODN strategy seems to offer great promise for cancer therapy, further studies are still required to put this powerful investigative tool in practice for a wide range of human cancers.

Nano-encapsulation of chicken immunoglobulin (IgY) in sodium alginate nanoparticles: In vitro characterization.

Controlled delivery of therapeutic agents by alginate nanoparticles became an attractive issue in the gastric organ. Some therapeutic agents such as proteins could not tolerate in severe condition in the gastrointestinal tract. In the present study, four concentrations of a specific IgY as a prophylactic agent against E. coli O157: H7 was entrapped in 0.2% w/v sodium alginate nanoparticles by ionic gelation method. Depending on the IgY concentration entrapment efficacy was 28.31-99.84%. The physicochemical and structural characteristics of free and IgY-loaded Alg NPs revealed that the individual particles exhibited a spherical shape with a diameter of 45-85 nm, and a negatively charged surface with a zeta potential value of 26-36 mV. In vitro release study showed a high significant difference of released amounts of IgY at 10% and 99.84% in simulated gastric fluid (pH 1.2) and simulated intestine fluid (pH 6.8), respectively. Also, the quality and activity of released IgY from Alg NPs not changed. The cytotoxicity of different concentrations of Alg NPs on the Vero cells was measured. Our results indicated that Alg NPs prepared from 0.2%w/v stock solution could be appropriate candidates for efficient and safe delivery of IgY through the gastrointestinal tract.

Hesperidin inhibits collagen-induced arthritis possibly through suppression of free radical load and reduction in neutrophil activation and infiltration.

Rheumatoid arthritis is a chronic inflammatory disease characterized by the destruction of articular cartilage and bone in a chronic phase. Pathology of rheumatoid arthritis suggests autoimmunity linked to inflammation. In our study, rheumatoid arthritis was induced in Wistar rats by intradermal injections of 100 µl of emulsion containing bovine type II collagen in complete Freund's adjuvant at the base of the tail. Disease developed about 13 ± 1 days after immunization and treatment with hesperidin (HES) at a dose of 160 mg kg(-1) body weight was given after onset of disease daily until 20th day. The effect of treatment in the rats was monitored by clinical scoring, biochemical parameters and histological evaluations in joints. A steady increase in the articular elastase, nitric oxide and lipid peroxidation was observed in joints of arthritic rats as compared to control, whereas a significant decrease in reduced glutathione, superoxide dismutase activity and catalase was observed in collagen-induced arthritis rats as compared to control group. The results from the present work indicate that the treatment with hesperidin was effective in bringing about significant changes on all the parameters studied in collagen-induced arthritis rats. These data confirm that erosive destruction of the joint cartilage in collagen-induced arthritis is due free radicals released by activated neutrophils and produced by other biochemical pathways. In the present study, an attempt has been made to amelioration of the disease process by a natural product. These results suggest that oral administration of HES could be effective for treating human RA patients.

Investigating the inhibitory and penetrating properties of three novel anticancer and antimicrobial scorpion peptides via molecular docking and molecular dynamic simulation.

The two types of bladder cancer, muscle invasive and non-muscle invasive (NMIBC), are among the most prevalent cancers worldwide. Despite this, even though muscle-invasive bladder cancer is more deadly, NMIBC requires more therapy due to a greater recurrence rate and more extended and expensive care. Immunotherapy, intravesical chemotherapy, cystoscopy, and transurethral resection (TUR) are among the treatments available. Crude scorpion venomand purified proteins and peptides, can suppress cancer metastasis in an in vitro or in vivo context, suppress cancer growth, halt the cell cycle, and cause cell apoptosis, according to an increasing number of experimental and preclinical studies. In this research, three novels discovered peptides (P2, P3 and P4. ProteomeXchange: PXD036231) from Buthotus saulcyi and, Odontobuthus doriae scorpions were used along with a peptide called pantinin (as a control). The phylogenetic tree showed that the peptides belong to Chaperonin HSP60, Chrysophsin2 and Pheromone-binding protein2, respectively. These peptides were docked with four known antigens, BAGE, BLCAP, PRAME and ROR1 related to bladder cancer and three bacterial antigens FliC, FliD and FimH to investigate their antimicrobial and anticancer properties. The results showed that peptides 2 and 3 have the best binding rate. The MD simulation results also confirmed the binding of peptides 2 and 3 to antigens. The penetration power of peptides 2 and 3 in the membrane of cancer cells and bacterial cells was also simulated, and the results of RMSD and PD confirmed it. QSAR suggests that peptides 2 and 3 can act as anti-cancer and anti-microbial peptides.Communicated by Ramaswamy H. Sarma.

Evaluation of Anticancer and Cytotoxic Effects of Genistein on PC3 Prostate Cell Line under Three-Dimensional Culture Medium

Background: Prostate cancer is a major cause of disease and mortality among men. Genistein (GNT) is an isoflavone found naturally in legumes. Isoflavones, a subset of phytoestrogens, are structurally similar to mammalian estrogens. This study aimed to evaluate the anticancer and cytotoxic effects of GNT on PC3 cell line under three dimensional (3D) culture medium. Methods: The 3D culture was created by encapsulating the PC3 cells in alginate hydrogel. MTT assay, neutral red uptake, comet assay, and cytochrome C assay were used to study the anticancer and cytotoxic effects of GNT at 120, 240, and 480 µM concentrations. Also, nitric oxide (NO), catalase, and glutathione assay levels were determined to evaluate the effect of GNT on the cellular stress. The culture medium was used as the negative control. Results: GNT reduced the production of cellular NO and increased the production of catalase and glutathione, confirming the results of the NO test. Evaluation of the toxicity effect of GNT at the concentrations of 120, 240, and 480 µM using comet assay showed that this chemical agent induces apoptosis in PC3 cells in a dose-dependent manner. As the level of cytochrome C in PC3 cells treated with different concentrations of GNT was not significantly different from that of the control, GNT could induce apoptosis in PC3 cells through the non-mitochondrial pathway. Conclusion: The findings of this study disclose that the anticancer effect of GNT on PC3 cells under 3D culture conditions could increase the effectiveness of treatment. Also, the cell survival rate is dependent on GNT concentration.

Combined Effect of Neutron Radiation and Curcumin on Breast Cancer Cells Cytotoxicity in 3D Culture Medium

Introduction: Introduction: Chemotherapy, biotherapy, and radiotherapy play a limited but important role in treating breast cancer. For more efficient treatment, combination therapy could be an appropriate option. In this study, radiotherapy using neutron radiation emitted from a 241Am-Be neutron source, as well as biotherapy using curcumin (80 µM) was combined to investigate the efficiency of treatment towards MCF-7 breast cancer in a three dimensional (3D) culture medium. Methods: Methods: MTT, neutral red uptake assay, nitric oxide, glutathione assay, catalase, cytochrome c, comet assay, and caspase-3 were used to determine the effect of neutron radiation and also neutron and curcumin combination on the viability of cancer cells. Results: Results: The results of cytotoxicity test showed that neutron irradiation with or without curcumin at 5, 10, 15, and 20 h reduced the survival of tumor cells. Moreover, the rate of apoptosis due to the neutron effect at different irradiation times enhanced with the increasing time. Conclusion: Conclusion: Due to the significant anticancer effect of curcumin in 3D culture, using this molecule before or after neutron therapy is recommended.

Increased spontaneous apoptosis of rat primary neurospheres in vitro after experimental autoimmune encephalomyelitis.

Survival of neuronal progenitors (NPCs) is a critical determinant of the regenerative capacity of brain following cellular loss. Herein, we report for the first time, the increased spontaneous apoptosis of the first acute phase of Experimental Autoimmune Encephalomyelitis (EAE) derived neurospheres in vitro. Neuronal as well as oligodendroglial loss occurs during experimental autoimmune encephalomyelitis (EAE). This loss is replenished spontaneously by the concomitant increase in the NPC proliferation evidenced by the presence of thin myelin sheaths in the remodeled lesions. However, remyelination depends upon the survival of NPCs and their lineage specific differentiation. We observed significant increase (P < 0.001) in number of BrdU (+) cells in ependymal subventricular zone (SVZ) in EAE rats. EAE derived NPCs showed remarkable increase in S-phase population which was indeed due to the decrease in G-phase progeny suggesting activation of neuronal progenitor cells (NPCs) from quiescence. However, EAE derived neurospheres showed limited survival in vitro which was mediated by the significantly (P < 0.01) depolarized mitochondria, elevated Caspase-3 (P < 0.001) and fragmentation of nuclear DNA evidenced by single cell gel electrophoresis. Our results suggest EAE induced spontaneous apoptosis of NPCs in vitro which may increase the possibility of early stage cell death in the negative regulation of the proliferative cell number and may explain the failure of regeneration in human multiple sclerosis.

Scorpion (Odontobuthus doriae) venom induces apoptosis and inhibits DNA synthesis in human neuroblastoma cells.

Scorpion and its organs have been used to cure epilepsy, rheumatism, and male impotency since medieval times. Scorpion venom which contains different compounds like enzyme and non-enzyme proteins, ions, free amino acids, and other organic inorganic substances have been reported to posses antiproliferative, cytotoxic, apoptogenic, and immunosuppressive properties. We for the first time report the apoptotic and antiproliferative effects of scorpion venom (Odontobuthus doriae) in human neuroblastoma cells. After exposure of cells to medium containing varying concentrations of venom (10, 25, 50, 100, and 200 µg/ml), cell viability decreased to 90.75, 75.53, 55.52, 37.85, and 14.30%, respectively, after 24 h. Cells expressed morphological changes like swelling, inhibition of neurite outgrowth, irregular shape, aggregation, rupture of membrane, and release of cytosolic contents after treatment with venom. Lactate dehydrogenase (LDH) level increased in 50 and 100 µg/ml as compared to control, but there was no significant increase in LDH level at a dose of 10 and 20 µg/ml. Two concentrations viz. 50 and 100 µ/ml were selected because of the profound effect of these concentrations on the cellular health and population. Treatment with these two concentrations induced reactive nitrogen intermediates and depolarization in mitochondria. While caspase-3 activity increased in a concentration-dependent manner, only 50 µg/ml was able to fragment DNA. It was interesting to note that at higher dose, i.e., 100 µg/ml, the cells were killed, supposedly by acute necrosis. DNA synthesis evidenced by bromodeoxyuridine (BrdU) incorporation was inhibited in a concentration-dependent manner. The cells without treatment incorporated BrdU with high affinity confirming their cancerous nature whereas very less incorporation was noticed in treated cells. Our results show apoptotic and antiproliferative potential of scorpion venom (O. doriae) in human neuroblastoma cells. These properties make scorpion venom a valuable therapeutic agent in cancer research.

Scorpion (Androctonus crassicauda) venom limits growth of transformed cells (SH-SY5Y and MCF-7) by cytotoxicity and cell cycle arrest.

The purpose of study was to examine the cytotoxic and anti-cancer properties along with addressing the plausible pathway followed by scorpion venom to reduce cell viability in SH-SY5Y and MCF-7 cells. Following exposure of cells with scorpion venom, cytotoxicity was estimated using MTT and lactate dehydrogenase assays. Apoptotic effects were measured by assessment of mitochondrial membrane potential, reactive nitrogen species, DNA fragmentation, and caspase-3 activity whereas antiproliferative effect was assayed using BrdU incorporation. Our results indicate that scorpion venom causes suppression of proliferation by arresting S-phase and induction of apoptosis through increased nitric oxide production, caspase-3 activity and depolarization of mitochondrial membrane. Induction of apoptosis and arrest of DNA synthesis are critical determinant factors for development of anti cancer drugs. These properties may lead to isolation of effective molecule(s) with potential anticancer activity from scorpion venom of Androctonus crassicauda.

Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice.

BACKGROUND: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX-1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice. METHODS: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata. RESULTS: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD501 of jellyfish venom. CONCLUSION: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.

Immunological detection of AcAMP antimicrobial peptide secreted by Aspergillus clavatus.

BACKGROUND AND OBJECTIVES: Aspergillus clavatus antimicrobial peptide (AcAMP) is a fungi-derived peptide with a broad spectrum of activity against pathogenic bacteria and fungi. Natural antimicrobial peptides, including AcAMP, have attracted many attentions in the development of new natural antibiotics against pathogenic bacteria, especially multidrug resistant ones. MATERIALS AND METHODS: In the present study, acamp gene was codon-optimized and chemically synthesized in pUC57 cloning vector, subcloned into pET28a (+) expression vector and transferred into competent Escherichia coli BL21 (DE3) cells. The expression of AcAMP was induced by addition of Isopropyl ß- d-1-thiogalactopyranoside (IPTG) and the expressed peptide was purified by Ni-NTA. BALB/c mice were immunized with the purified peptide and the ability of the immunized mice sera for the detection of the native AcAMP secreted by A. clavatus IRAN 142C was examined through ELISA and Western blotting techniques. RESULTS: Both ELISA and Western blotting demonstrated the ability of the sera of the immunized mice to detect the native AcAMP. CONCLUSION: The results of the present work show that the raised antibody against recombinant AcAMP can be used to detect AcAMP peptide, an issue which paves the way to develop detection kits for the detection of AcAMP-producing organisms, purification of this valuable peptide for further investigations.

In-vitro Study of Hottentotta Schach Crude Venom Anticancer Effects on MCF-7 and Vero Cell Lines.

Scorpion venoms contain potentially useful pharmacological agents. Several studies demonstrate that the venoms of some scorpions induce apoptosis and inhibit the growth of cancer cells; therefore, they have been investigated for isolating anticancer components. In this study, antitumor effects of Hottentotta schach crude venom on MCF-7 (breast cancer cell line) as test group and Vero (African green monkey kidney normal cell line) as control group were analyzed. Cell toxicity was analyzed using MTT and neutral red (NR) uptake assays and apoptosis induction was analyzed using comet assay and caspase-3 activity. Oxidative stress following Hottentotta schach crude venom treatment was analyzed using nitrite oxide (NO) determination assay, reduced glutathione (GSH) and catalase enzyme activity assays. Results showed that crude venom (25-200 µg/mL) induced apoptosis and inhibited the growth of MCF-7 and to a lesser extent in Vero cell lines. Nitrite oxide concentration increased while glutathione concentration and catalase enzyme activity were decreased in MCF-7 cells; however, results in Vero cells were reversed completely. It can be concluded that Hottentotta schach crude venom disturbs the oxidation and reduction potential in cancer cells and ultimately induce apoptosis. So this venom can be used as a good source for isolation and designing new anticancer drugs.

A new species of Eryx (Serpentes: Erycidae) from Iran.

We describe a new species of the genus Eryx Daudin, 1803 from southern Iran that is morphologically closely related to the Indian sand boa, E. johnii. The new species, Eryx sistanensis sp. nov. has a distribution range from Zabol in the Sistan Region to the southern parts of Sistan Baluchistan, as well as Hormozgan Province of Iran. Morphologically, E. sistanensis sp. nov. differs from E. johnii by having fewer dorsal scale rows at midbody and the tail tip is not as blunt as E. johnii. The genetic distance (p-distance) between the new species and the Indian sand boa is considerable (9.1% for cytb and 11.8% for COI).

Hippocampal neurodegeneration in experimental autoimmune encephalomyelitis (EAE): potential role of inflammation activated myeloperoxidase.

Experimental Autoimmune Encephalomyelitis (EAE) is a well-established animal model of human multiple sclerosis (MS). The effect of this inflammatory disease on hippocampus has not been addressed. Keeping in view the above consideration an attempt was made to delineate the effect of EAE on the hippocampus of Wistar rats. The assessment of the damage to the hippocampus was done 16 days post induction by the immunolocalization of ChAT (choline acetyl transferase). ChAT decreased remarkably after induction that revealed cholinergic neuronal degeneration in the hippocampus. Subsequently, many biochemical parameters were assessed to ascertain inflammatory activation of nitric oxide and associated oxidative damage as a putative mechanism of the cholinergic degeneration. Nitric oxide metabolites increased significantly (P < 0.05) with enhancement of MPO (Myeloperoxidase activity) (P < 0.001) in the MOG (myelin oligodendrocyte protein) group as compared to the controls. Peroxidation of biomembranes increased (P < 0.001), while reduced glutathione depleted (P < 0.001) with parallel decrease in catalase (P < 0.01) and superoxide dismutase enzyme activity (P < 0.001) in the MOG group. Our results show a strong role of peroxidase dependent oxidation of nitrite and oxidative stress in cholinergic degeneration in EAE.

Osteoblast-seeded bioglass/gelatin nanocomposite: a promising bone substitute in critical-size calvarial defect repair in rat.

INTRODUCTION: Amid the plethora of methods to repair critical bone defects, there is no one perfect approach. In this study, we sought to evaluate a potent 3-dimensional (3D) bioactive SiO2-CaO-P2O5 glasses (bioglass)/gelatin (gel) scaffold for its biocompatibility by seeding cells as well as for its regenerative properties by animal implantation. METHODS: Osteoblast cells were seeded onto nanocomposite scaffolds to investigate the process of critical-size calvarial defect via new bone formation. Scanning electron microscopy (SEM) was used to validate topography of the scaffolds, its homogeneity and ideal cellular attachment. Proliferation assay and confocal microscopy were used to evaluate its biocompatibility. To validate osteogenesis of the bioactive nanocomposite scaffolds, they were first implanted into rats and later removed and analyzed at different time points post mortem using histological, immunohistochemical and histomorphometric methods. RESULTS: Based on in vitro results, we showed that our nanocomposite is highly cell-compatible material and allows for osteoblasts to adhere, spread and proliferate. In vivo results indicate that our nanocomposite provides a significant contribution to bone regeneration and is highly biodegradable and biocompatible. So, seeded scaffolds with osteoblasts enhanced repair of critical bone defects via osteogenesis. CONCLUSIONS: We demonstrate the feasibility of engineering a nanocomposite scaffold with an architecture resembling the human bone, and provide proof-of-concept validation for our scaffold using a rat animal model.

Quercetin prevents protein nitration and glycolytic block of proliferation in hydrogen peroxide insulted cultured neuronal precursor cells (NPCs): Implications on CNS regeneration.

Survival along with optimal proliferation of neuronal precursors determines the outcomes of the endogenous cellular repair in CNS. Cellular-oxidation based cell death has been described in several neurodegenerative disorders. Therefore, this study was aimed at the identification of the potent targets of oxidative damage to the neuronal precursors and its effective prevention by a natural flavonoid, Quercetin. Neuronal precursor cells (NPCs), Nestin+ and GFAP (Glial fibrillary acidic protein)+ were isolated and cultured from adult rat SVZ (subventricular zone). These cells were challenged with a single dose of H2O2 (50µM) and/or pre-treated with different concentrations of Quercetin. H2O2 severely limited the cellular viability and expansion of the neurospheres. Cellular-oxidation studies revealed reduction in glutathione dependent redox buffering along with depletion of enzymatic cellular antioxidants that might potentiate the nitrite (NO2(-)) and superoxide anion (O2(-)) mediated peroxynitrite (ONOO(-)) formation and irreversible protein nitration. We identified depleted PK-M2 (M2 isoform of pyruvate kinase) activity and apoptosis of NPCs revealed by the genomic DNA fragmentation and elevated PARP (poly ADP ribose polymerase) activity along with increased Caspase activity initiated by severely depolarised mitochondrial membranes. However, the pre-treatment of Quercetin in a dose-response manner prevented these changes and restored the expansion of neurospheres preferably by neutralizing the oxidative conditions and thereby reducing peroxynitrite formation, protein nitration and PK-M2 depletion. Our results unravel the potential interactions of oxidative environment and respiration in the survival and activation of precursors and offer a promise shown by a natural flavonoid in the protective strategy for neuronal precursors of adult brain.