A pathological joint-liver axis mediated by matrikine-activated CD4+ T cells.

The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4+ T cells in peripheral blood and spleen. Deletion of CD4+ T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4+ T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint-liver axis mediated by matrikine-activated CD4+ T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4+ T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences.

Exploration of antimicrobial and anticancer activities of L-amino acid oxidase from Egyptian Naja haje venom.

Hepatocellular carcinoma and bacterial resistance are major health burdens nowadays. Thus, providing new therapies that overcome that resistance is of great interest, particularly those derived from nature rather than chemotherapeutics to avoid cytotoxicity on normal cells. Venomous animals are among the natural sources that assisted in the discovery of novel therapeutic regimens. L-amino acid oxidase Nh-LAAO (140 kDa), purified from Egyptian Naja haje venom by a successive two-step chromatography protocol, has an optimal pH and temperature of 8 and 37 °C. Under standard assay conditions, Nh-LAAO exhibited the highest specificity toward L-Arg, L-Met and L-Leu, with Km and Vmax values of 3.5 mM and 10.4 µmol/min/ml, respectively. Among the metal ions, Ca+2, Na+, and K+ ions are activators, whereas Fe+2 inhibited LAAO activity. PMSF and EDTA slightly inhibited the Nh-LAAO activity. In addition, Nh-LAAO showed antibacterial and antifungal activities, particularly against Gentamicin-resistant P. aeruginosa and E. coli strains with MIC of 18 ± 2 µg/ml, as well as F. proliferatum and A. parasiticus among the selected human pathogenic strains. Furthermore, Nh-LAAO exhibited anti-proliferative activity against cancer HepG2 and Huh7 cells with IC50 of 79.37 and 60.11 µg/ml, respectively, with no detectable effect on normal WI-38 cells. Consequently, the apoptosis % of the HepG2 and Huh7 cells were 12 ± 1 and 34.5 ± 2.5 %, respectively, upon Nh-LAAO treatment. Further, the Nh-LAAO arrested the HepG2 and Huh7 cell cycles in the G0/G1 phase. Thus, the powerful selective cytotoxicity of L-amino acid oxidase opens up the possibility as a good candidate for clinical cancer therapy.

Cobra (Naja naja) venom L-amino acid oxidase (NNLAAO70) induces apoptosis and secondary necrosis in human lung epithelial cancer cells.

Snake venom L-amino acid oxidases (LAAOs) are flavoenzymes with diverse physiological and pharmacological effects. These enzymes are found to showcase anticoagulant, antiplatelet, cytotoxicity and other biological effects in bite victims. However, the exact mechanism through which they exhibit several biological properties is not yet fully understood. The current study focussed on the purification of cobra venom LAAO and the functional characterization of purified LAAO. A novel L-amino acid oxidase NNLAAO70 with a molecular weight ~70 kDa was purified from the venom of an Indian spectacled cobra (Naja naja). NNLAAO70 showed high substrate specificity for L-His, L-Leu, and L-Arg during its LAAO activity. It inhibited adenosine di-phosphate (ADP) and collagen-induced platelet aggregation process in a dosedependent manner. About 60% inhibition of collagen-induced and 40% inhibition of ADP-induced platelet aggregation was observed with a 40 µg/ml dose of NNLAAO70. NNLAAO70 exhibited bactericidal activity on Bacillus subtilis, Escherichia coli, Bacillus megaterium, and Pseudomonas fluorescens. NNLAAO70 also showed cytotoxicity on A549 cells in vitro. It showed severe bactericidal activity on P. fluorescens and lysed 55% of cells. NNLAAO70 also exhibited drastic cytotoxicity on A549 cells. At 1 lg/ml dosage, it demonstrated a 60% reduction in A549 viability and induced apoptosis upon 24-h incubation. H2O2 released during oxidative deamination reactions played a major role in NNLAAO70-induced cytotoxicity. NNLAAO70 significantly increased intracellular reactive oxygen species (ROS) levels in A549 cells by six fold when compared to untreated cells. Oxidative stress-mediated cell injury is the primary cause of NNLAAO70-induced apoptosis in A549 cells and prolonged oxidative stress caused DNA fragmentation and activated cellular secondary necrosis.

Differential effects of the venoms of Russell's viper and Indian cobra on human myoblasts.

Local tissue damage following snakebite envenoming remains a poorly researched area. To develop better strategies to treat snakebites, it is critical to understand the mechanisms through which venom toxins induce envenomation effects including local tissue damage. Here, we demonstrate how the venoms of two medically important Indian snakes (Russell's viper and cobra) affect human skeletal muscle using a cultured human myoblast cell line. The data suggest that both venoms affect the viability of myoblasts. Russell's viper venom reduced the total number of cells, their migration, and the area of focal adhesions. It also suppressed myogenic differentiation and induced muscle atrophy. While cobra venom decreased the viability, it did not largely affect cell migration and focal adhesions. Cobra venom affected the formation of myotubes and induced atrophy. Cobra venom-induced atrophy could not be reversed by small molecule inhibitors such as varespladib (a phospholipase A2 inhibitor) and prinomastat (a metalloprotease inhibitor), and soluble activin type IIb receptor (a molecule used to promote regeneration of skeletal muscle), although the antivenom (raised against the Indian 'Big Four' snakes) has attenuated the effects. However, all these molecules rescued the myotubes from Russell's viper venom-induced atrophy. This study demonstrates key steps in the muscle regeneration process that are affected by both Indian Russell's viper and cobra venoms and offers insights into the potential causes of clinical features displayed in envenomed victims. Further research is required to investigate the molecular mechanisms of venom-induced myotoxicity under in vivo settings and develop better therapies for snakebite-induced muscle damage.

Intramuscular Bleeding and Formation of Microthrombi during Skeletal Muscle Damage Caused by a Snake Venom Metalloprotease and a Cardiotoxin.

The interactions between specific snake venom toxins and muscle constituents are the major cause of severe muscle damage that often result in amputations and subsequent socioeconomic ramifications for snakebite victims and/or their families. Therefore, improving our understanding of venom-induced muscle damage and determining the underlying mechanisms of muscle degeneration/regeneration following snakebites is critical to developing better strategies to tackle this issue. Here, we analysed intramuscular bleeding and thrombosis in muscle injuries induced by two different snake venom toxins (CAMP-Crotalus atrox metalloprotease (a PIII metalloprotease from the venom of this snake) and a three-finger toxin (CTX, a cardiotoxin from the venom of Naja pallida)). Classically, these toxins represent diverse scenarios characterised by persistent muscle damage (CAMP) and successful regeneration (CTX) following acute damage, as normally observed in envenomation by most vipers and some elapid snakes of Asian, Australasian, and African origin, respectively. Our immunohistochemical analysis confirmed that both CAMP and CTX induced extensive muscle destruction on day 5, although the effects of CTX were reversed over time. We identified the presence of fibrinogen and P-selectin exposure inside the damaged muscle sections, suggesting signs of bleeding and the formation of platelet aggregates/microthrombi in tissues, respectively. Intriguingly, CAMP causes integrin shedding but does not affect any blood clotting parameters, whereas CTX significantly extends the clotting time and has no impact on integrin shedding. The rates of fibrinogen clearance and reduction in microthrombi were greater in CTX-treated muscle compared to CAMP-treated muscle. Together, these findings reveal novel aspects of venom-induced muscle damage and highlight the relevance of haemostatic events such as bleeding and thrombosis for muscle regeneration and provide useful mechanistic insights for developing better therapeutic interventions.

Synthetic Peptide Fragments of the Wtx Toxin Reduce Blood Pressure in Rats under General Anesthesia.

Previously, it was shown that the non-conventional toxin WTX from the venom of the cobra Naja kaouthia, when administered intravenously, caused a decrease in blood pressure (BP) and an increase in heart rate (HR) in rats [13]. To identify the site of the toxin molecule responsible for these effects, we studied the influence of synthetic peptide fragments of the WTX on BP and HR in normotensive male Sprague-Dawley rats under general anesthesia induced by Telazol and Xylazine. It was found that peptides corresponding to the WTX central polypeptide loop, stabilized by a disulfide bond, at intravenous injection at concentrations from 0.1 to 1.0 mg/mL caused a dose-dependent decrease in BP, with the HR increasing only in the first 5-10 min after administration. Thus, WTX fragments corresponding to the central polypeptide loop reproduce the decrease in blood pressure caused by the toxin.

Oxineur, a novel peptide from Caspian cobra Naja naja oxiana against HT-29 colon cancer.

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 µg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Pharmacological Screening of Venoms from Five Brazilian Micrurus Species on Different Ion Channels.

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1ß1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1ß2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

Egyptian cobra (Naja haje haje) venom phospholipase A2: a promising antiviral agent with potent virucidal activity against simian rotavirus and bovine coronavirus.

Viral infections are linked to a variety of human diseases. Despite the achievements made in drug and vaccine development, several viruses still lack preventive vaccines and efficient antiviral compounds. Thus, developing novel antiviral agents is of great concern, particularly the natural products that are promising candidates for such discoveries. In this study, we have purified an approximately 15 kDa basic phospholipase A2 (PLA2) enzyme from the Egyptian cobra Naja haje haje venom. The purified N. haje PLA2 showed a specific activity of 22 units/mg protein against 6 units/mg protein for the whole crude venom with 3.67-fold purification. The antiviral activity of purified N. haje PLA2 has been investigated in vitro against bovine coronavirus (BCoV) and simian rotavirus (RV SA-11). Our results showed that the CC50 of PLA2 were 33.6 and 29 µg/ml against MDBK and MA104 cell lines, respectively. Antiviral analysis of N. haje PLA2 showed an inhibition of BCoV and RV SA-11 infections with a therapeutic index equal to 33.6 and 16, respectively. Moreover, N. haje PLA2 decreased the BCoV and RV SA-11 titers by 4.25 log10 TCID50 and 2.5 log10 TCID50, respectively. Thus, this research suggests the potential antiviral activity of purified N. haje PLA2 against BCoV and RV SA-11 infections in vitro.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Kv1 channels regulate variations in spike patterning and temporal reliability in the avian cochlear nucleus angularis.

Diverse physiological phenotypes in a neuronal population can broaden the range of computational capabilities within a brain region. The avian cochlear nucleus angularis (NA) contains a heterogeneous population of neurons whose variation in intrinsic properties results in electrophysiological phenotypes with a range of sensitivities to temporally modulated input. The low-threshold potassium conductance (GKLT) is a key feature of neurons involved in fine temporal structure coding for sound localization, but a role for these channels in intensity or spectrotemporal coding has not been established. To determine whether GKLT affects the phenotypical variation and temporal properties of NA neurons, we applied dendrotoxin-I (DTX), a potent antagonist of Kv1-type potassium channels, to chick brain stem slices in vitro during whole cell patch-clamp recordings. We found a cell-type specific subset of NA neurons that was sensitive to DTX: single-spiking NA neurons were most profoundly affected, as well as a subset of tonic-firing neurons. Both tonic I (phasic onset bursting) and tonic II (delayed firing) neurons showed DTX sensitivity in their firing rate and phenotypical firing pattern. Tonic III neurons were unaffected. Spike time reliability and fluctuation sensitivity measured in DTX-sensitive NA neurons was also reduced with DTX. Finally, DTX reduced spike threshold adaptation in these neurons, suggesting that GKLT contributes to the temporal properties that allow coding of rapid changes in the inputs to NA neurons. These results suggest that variation in Kv1 channel expression may be a key factor in functional diversity in the avian cochlear nucleus.NEW & NOTEWORTHY The dendrotoxin-sensitive voltage-gated potassium conductance typically associated with neuronal coincidence detection in the timing pathway for sound localization is demonstrated to affect spiking patterns and temporal input sensitivity in the intensity pathway in the avian auditory brain stem. The Kv1-family channels appear to be present in a subset of cochlear nucleus angularis neurons, regulate spike threshold dynamics underlying high-pass membrane filtering, and contribute to intrinsic firing diversity.

Thrombolysis, clotting, and genotoxic activities modulated by essential oils extracted from Lippia alba

Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action

Anti-Cancer Effect of Moroccan Cobra Naja haje Venom and Its Fractions against Hepatocellular Carcinoma in 3D Cell Culture.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer in adults, the fifth most common malignancy worldwide and the third leading cause of cancer related death. An alternative to the surgical treatments and drugs, such as sorafenib, commonly used in medicine is necessary to overcome this public health problem. In this study, we determine the anticancer effect on HCC of Moroccan cobra Naja haje venom and its fraction obtained by gel filtration chromatography against Huh7.5 cancer cell line. Cells were grown together with WI38 human fibroblast cells, LX2 human hepatic stellate cell line, and human endothelial cells (HUVEC) in MCTS (multi-cellular tumor spheroids) models. The hepatotoxicity of venom and its fractions were also evaluated using the normal hepatocytes cell line (Fa2N-4 cells). Our results showed that an anti HCC activity of Moroccan cobra Naja haje venom and, more specifically, the F7 fraction of gel filtration chromatography exhibited the greatest anti-hepatocellular carcinoma effect by decreasing the size of MCTS. This effect is associated with a low toxicity against normal hepatocytes. These results strongly suggest that the F7 fraction of Moroccan cobra Naja haje venom obtained by gel filtration chromatography possesses the ability to inhibit cancer cells proliferation. More research is needed to identify the specific molecule(s) responsible for the anticancer effect and investigate their mechanism of action.

The effects of Naja sumatrana venom cytotoxin, sumaCTX on alteration of the secretome in MCF-7 breast cancer cells following membrane permeabilization.

Naja sumatrana venom cytotoxin (sumaCTX) is a basic protein which belongs to three-finger toxin family. It has been shown to induce caspase-dependent, mitochondrial-mediated apoptosis in MCF-7 cells at lower concentrations. This study aimed to investigate the alteration of secretome in MCF-7 cells following membrane permeabilization by high concentrations of sumaCTX, using label-free quantitative (LFQ) approach. The degree of membrane permeabilization of sumaCTX was determined by lactate dehydrogenase (LDH) assay and calcein-propidium iodide (PI) assays. LDH and calcein-PI assays revealed time-dependent membrane permeabilization within a narrow concentration range. However, as toxin concentrations increased, prolonged exposure of MCF-7 cells to sumaCTX did not promote the progression of membrane permeabilization. The secretome analyses showed that membrane permeabilization was an event preceding the release of intracellular proteins. Bioinformatics analyses of the LFQ secretome revealed the presence of 105 significantly distinguished proteins involved in metabolism, structural supports, inflammatory responses, and necroptosis in MCF-7 cells treated with 29.8 µg/mL of sumaCTX. Necroptosis was presumably an initial stress response in MCF-7 cells when exposed to high sumaCTX concentration. Collectively, sumaCTX-induced the loss of membrane integrity in a concentration-dependent manner, whereby the cell death pattern of MCF-7 cells transformed from apoptosis to necroptosis with increasing toxin concentrations.

What Therapeutic Regimen Will Be Optimal for Initial Clinical Trials of Pig Organ Transplantation?

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen.

Mambalgin-1 pain-relieving peptide locks the hinge between α4 and α5 helices to inhibit rat acid-sensing ion channel 1a.

Acid-sensing ion channels (ASICs) are proton-gated cationic channels involved in pain and other processes, underscoring the potential therapeutic value of specific inhibitors such as the three-finger toxin mambalgin-1 (Mamb-1) from snake venom. A low-resolution structure of the human-ASIC1a/Mamb-1 complex obtained by cryo-electron microscopy has been recently reported, implementing the structure of the chicken-ASIC1/Mamb-1 complex previously published. Here we combine structure-activity relationship of both the rat ASIC1a channel and the Mamb-1 toxin with a molecular dynamics simulation to obtain a detailed picture at the level of side-chain interactions of the binding of Mamb-1 on rat ASIC1a channels and of its inhibition mechanism. Fingers I and II of Mamb-1 but not the core of the toxin are required for interaction with the thumb domain of ASIC1a, and Lys-8 of finger I potentially interacts with Tyr-358 in the thumb domain. Mamb-1 does not interfere directly with the pH sensor as previously suggested, but locks by several contacts a key hinge between α4 and α5 helices in the thumb domain of ASIC1a to prevent channel opening. Our results provide an improved model of inhibition of mammalian ASIC1a channels by Mamb-1 and clues for further development of optimized ASIC blockers.

Anti-inflammatory Activity of Caspian Cobra (Naja naja oxiana) Snake Venom on the Serum Level of Interleukin-27 and Histopathological Changes in Myelin Oligodendrocyte Glycoprotein-experimental Autoimmune Encephalomyelitisinduced Mice.

Multiple sclerosis (MS) is considered a chronic disease of the central nervous system, with a strong neurodegenerative component. The exact mechanism of MS is not clear. However, the therapeutic strategies for controlling MS are based on immune modulation and inflammation control. Regarding this, the present study was conducted to investigate the influence of snake venom on the suppression of the immune system after the induction of experimental autoimmune encephalomyelitis (EAE) in mice. For this purpose, C57BL/6 female mice, divided into three groups, were selected to be induced by EAE. Groups 2 and 3 received flank injection with the emulsion of myelin oligodendrocyte glycoprotein (MOG 35-55), as well as complete Freund adjuvant, followed by the administration of pertussis toxin. Furthermore, the treatment group, as an immune-modulator, received cobra venom (CV) after EAE induction. The mice were then evaluated daily based on clinical symptoms, weight changes (within 26 days), histopathological analysis, and serum levels of interleukin 27 (IL-27) for neurological motor deficits. The clinical signs of MOG-EAE in C57BL/6 mice began 9-14 days post-immunization. Histopathological results also revealed that CV-treated EAE mice, compared to the untreated EAE group, witnessed a significant reduction in the intensity of inflammatory cells in parenchymal sections. Furthermore, the increase of IL-27 levels was significant in the CV-treated group (P=0.001), compared with those in the EAE and control groups. Based on results obtained in the present study, it may be concluded that Naja naja oxiana snake venom is a potential immunomodulatory agent that can be effective in the treatment of MS.

The Fraction of the Snake Venom, Its Leishmanicidal Effect, and the Stimulation of an Anti-Leishmania Response in Infected Macrophages.

BACKGROUND AND AIMS: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOVF11) on Leishmania infantum and its broad mode of action. METHODS: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue), macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme- linked immunosorbent assay (ELISA) on L. infantum promastigote and amastigote stages were used. IC50 values of L. infantum stages, CC50 value, and apoptosis were also analyzed. RESULTS: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent manner compared to the untreated control group. Interleukin (IL)-12, TNF-α, and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity, which displayed a significant reduction (p < 0.001). CONCLUSION: NNOV-F11 possessed a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions, which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic-like mechanisms, and inhibited L-ARG activity, which collectively led to the parasite death. Further studies are crucial to assess the effect of the NNOV-F11 on animal models or clinical settings.

The potassium channel subunit Kvß1 serves as a major control point for synaptic facilitation.

Analysis of the presynaptic action potential's (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvß1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvß1 blocked all broadening of the APsyn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.