Isolation, characterization and antimicrobial properties of hepatopancreas lectin of the freshwater crab Oziotelphusanaga.

Lectins are versatile proteins that specifically recognize and interact with sugar moieties expressed on the cell surface. The potential of lectin in drug targeting and delivery has instigated interest to identify natural lectins. Crabs have been identified as a rich source of lectin because the innate immune system is activated on encounter of pathogens and helps in the production of lectin. Although the presence of lectins in crab's hemolymph is well documented, little information about lectin in hepatopancreas, a vital organ for immunity and digestion in crustaceans, is currently available. A calcium dependent lectin (75 kDa) was purified from the hepatopancreas of the freshwater crab Oziotelphusa naga by bioadsorption and fetuin linked Sepharose 4B affinity chromatography technique. The isolated hepatopancreas lectin is calcium dependent and maximum agglutination was observed with rabbit erythrocytes. The hemagglutinating activity of the hepatopancreas lectin was effectively inhibited by sugars, such as α-lactose, GlcNAc, trehalose and NeuAc. Compared to sialylated N-glycosylated proteins including transferrin and apo transferrin, sialylated O-glycosylated proteins like fetuin exhibited stronger inhibitory effect. The ability of erythrocytes to bind hepatopancreas lectin has been diminished by desialylation of the potent inhibitor, indicating the significance of sialic acid in lectin-ligand interactions. The purified hepatopancreas lectin showed a broad spectrum of antimicrobial activity against bacteria Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, E. coli and fungi Candida albicans and Aspergillus niger. The findings of this study demonstrate the significance of hepatopancreas lectin as a multifunctional defense protein that inhibits the growth of bacteria and fungi.

Identification of a Double-ß-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

ß-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian ß-defensin 11 (AvBD11) is unique, with two ß-defensin motifs with a broad range of antimicrobial activities. However, a double-sized ß-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-ß-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to ß-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD in vivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-ß-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

A New Gene SCY3 Homologous to Scygonadin Showing Antibacterial Activity and a Potential Role in the Sperm Acrosome Reaction of Scylla paramamosain.

In the study, a new gene homologous to the known antimicrobial peptide Scygonadin was identified in mud crab Scylla paramamosain and named SCY3. The full-length sequences of cDNA and genomic DNA were determined. Similar to Scygonadin, SCY3 was dominantly expressed in the ejaculatory ducts of male crab and the spermatheca of post-mating females at mating. The mRNA expression was significantly up-regulated after stimulation by Vibrio alginolyticus, but not by Staphylococcus aureus. The recombinant protein rSCY3 had a killing effect on Micrococcus luteus and could improve the survival rate of mud crabs infected with V. alginolyticus. Further analysis showed that rSCY3 interacted with rSCY1 or rSCY2 using Surface Plasmon Resonance (SPR, a technology for detecting interactions between biomolecules using biosensor chips) and Mammalian Two-Hybrid (M2H, a way of detecting interactions between proteins in vivo). Moreover, the rSCY3 could significantly improve the sperm acrosome reaction (AR) of S. paramamosain and the results demonstrated that the binding of rSCY3, rSCY4, and rSCY5 to progesterone was a potential factor affecting the sperm AR by SCYs on. This study lays the foundation for further investigation on the molecular mechanism of SCYs involved in both immunity and physiological effects of S. paramamosain.

The dynamic changes of genes revealed that persistently overexpressed genes drive the evolution of cyflumetofen resistance in Tetranychus cinnabarinus.

Changes in gene expression are associated with the evolution of pesticide resistance in arthropods. In this study, transcriptome sequencing was performed in 3 different resistance levels (low, L; medium, M; and high, H) of cyflumetofen-resistant strain (YN-CyR). A total of 1 685 genes, including 97 detoxification enzyme genes, were upregulated in all 3 stages, of which 192 genes, including 11 detoxification enzyme genes, showed a continuous increase in expression level with resistance development (L to H). RNA interference experiments showed that overexpression of 7 genes (CYP392A1, TcGSTd05, CCE06, CYP389A1, TcGSTz01, CCE59, and CYP389C2) is involved in the development of cyflumetofen resistance in Tetranychus cinnabarinus. The recombinant CYP392A1 can effectively metabolize cyflumetofen, while CCE06 can bind and sequester cyflumetofen in vitro. We compared 2 methods for rapid screening of resistance molecular markers, including short-term induction and 1-time high-dose selection. Two detoxification enzyme genes were upregulated in the field susceptible strain (YN-S) by induction with 20% lethal concentration (LC20 ) of cyflumetofen. However, 16 detoxification enzyme genes were upregulated by 1-time selection with LC80 of cyflumetofen. Interestingly, the 16 genes were overexpressed in all 3 resistance stages. These results indicated that 1 685 genes that were upregulated at the L stage constituted the basis of cyflumetofen resistance, of which 192 genes in which upregulation continued to increase were the main driving force for the development of resistance. Moreover, the 1-time high-dose selection is an efficient way to rapidly obtain the resistance-related genes that can aid in the development of resistance markers and resistance management in mites.

Effects of copper on non-specific immunity and antioxidant in the oriental river prawn (Macrobrachium nipponense).

The copper, as heavy metal has important impacts on the antioxidant and immune defense systems in aquatic organisms, and the toxic effects of copper can be accumulated and magnified with the food chain, thus posing a threat to food safety as well as ecosystems. This study explored the response of the antioxidant system and non-specific immunity in M. nipponense to copper stress. Low concentration of copper (0.05, 0.1 mg L-1) had positive effects on the non-specific immunity in M. nipponense, while the non-specific immunity in M. nipponense could be affect negatively or even be inhibited by high copper concentration (0.15 mg L-1). Even low concentrations of copper could cause oxidative stress, and high copper concentration (0.15 mg L-1) could induce oxidative damage and even apoptosis, and thus causing damage to the antioxidant defense system in M. nipponense. Low concentration of copper could affect the gill and hepatopancreas structure in M. nipponense, but high level oxidative stress caused by high copper concentration could cause oxidative damage to these tissue, resulting in the destruction of gill and hepatopancreas. This study provides the safety concentration for using copper-containing fish drugs in the actual culture of M. nipponense and provides basic data for the toxicity mechanism of copper to M. nipponense.

Characterization of the Dual Functions of LvCrustinVII from Litopenaeus vannamei as Antimicrobial Peptide and Opsonin.

Crustin are a family of antimicrobial peptides that play an important role in protecting against pathogens infection in the innate immune system of crustaceans. Previously, we identified several novel types of crustins, including type VI and type VII crustins. However, their immune functions were still unclear. In the present study, the immune function of type VII crustin LvCrustinVII were investigated in Litopenaeus vannamei. LvCrustinVII was wildly expressed in all tested tissues, with relatively high expression levels in hepatopancreas, epidermis and lymphoid organ. Upon Vibrio parahaemolyticus infection, LvCrustinVII was significantly upregulated in hepatopancreas. Recombinant LvCrustinVII (rLvCrustinVII) showed strong inhibitory activities against Gram-negative bacteria Vibrio harveyi and V. parahaemolyticus, while weak activities against the Gram-positive bacteria Staphylococcus aureus. Binding assay showed that rLvCrustinVII could bind strongly to V. harveyi and V. parahaemolyticus, as well as the cell wall components Glu, LPS and PGN. In the presence of Ca2+, rLvCrustinVII could agglutinate V. parahaemolyticus and enhance hemocyte phagocytosis. The present data partially illustrate the immune function of LvCrustinVII, which enrich our understanding on the functional mechanisms of crustins and provide useful information for application of this kind of antimicrobial peptides.

Antimicrobial, Insecticidal and Cytotoxic Activity of Linear Venom Peptides from the Pseudoscorpion Chelifer cancroides.

Linear cationic venom peptides are antimicrobial peptides (AMPs) that exert their effects by damaging cell membranes. These peptides can be highly specific, and for some, a significant therapeutic value was proposed, in particular for treatment of bacterial infections. A prolific source of novel AMPs are arthropod venoms, especially those of hitherto neglected groups such as pseudoscorpions. In this study, we describe for the first time pharmacological effects of AMPs discovered in pseudoscorpion venom. We examined the antimicrobial, cytotoxic, and insecticidal activity of full-length Checacin1, a major component of the Chelifer cancroides venom, and three truncated forms of this peptide. The antimicrobial tests revealed a potent inhibitory activity of Checacin1 against several bacteria and fungi, including methicillin resistant Staphylococcus aureus (MRSA) and even Gram-negative pathogens. All peptides reduced survival rates of aphids, with Checacin1 and the C-terminally truncated Checacin11-21 exhibiting effects comparable to Spinosad, a commercially used pesticide. Cytotoxic effects on mammalian cells were observed mainly for the full-length Checacin1. All tested peptides might be potential candidates for developing lead structures for aphid pest treatment. However, as these peptides were not yet tested on other insects, aphid specificity has not been proven. The N- and C-terminal fragments of Checacin1 are less potent against aphids but exhibit no cytotoxicity on mammalian cells at the tested concentration of 100 µM.

A g-Type Lysozyme from Deep-Sea Hydrothermal Vent Shrimp Kills Selectively Gram-Negative Bacteria.

Lysozyme is a key effector molecule of the innate immune system in both vertebrate and invertebrate. It is classified into six types, one of which is the goose-type (g-type). To date, no study on g-type lysozyme in crustacean has been documented. Here, we report the identification and characterization of a g-type lysozyme (named LysG1) from the shrimp inhabiting a deep-sea hydrothermal vent in Manus Basin. LysG1 possesses conserved structural features of g-type lysozymes. The recombinant LysG1 (rLysG1) exhibited no muramidase activity and killed selectively Gram-negative bacteria in a manner that depended on temperature, pH, and metal ions. rLysG1 bound target bacteria via interaction with bacterial cell wall components, notably lipopolysaccharide (LPS), and induced cellular membrane permeabilization, which eventually caused cell lysis. The endotoxin-binding capacity enabled rLysG1 to alleviate the inflammatory response induced by LPS. Mutation analysis showed that the bacterial binding and killing activities of rLysG1 required the integrity of the conserved α3 and 4 helixes of the protein. Together, these results provide the first insight into the activity and working mechanism of g-type lysozyme in crustacean and deep-sea organisms.

SpCrus2 Glycine-Rich Region Contributes Largely to the Antiviral Activity of the Whole-Protein Molecule by Interacting with VP26, a WSSV Structural Protein.

Crustins are cysteine-rich cationic antimicrobial peptides with diverse biological functions including antimicrobial and proteinase inhibitory activities in crustaceans. Although a few crustins reportedly respond to white spot syndrome virus (WSSV) infection, the detailed antiviral mechanisms of crustins remain largely unknown. Our previous research has shown that SpCrus2, from mud crab Scylla paramamosain, is a type II crustin containing a glycine-rich region (GRR) and a cysteine-rich region (CRR). In the present study, we found that SpCrus2 was upregulated in gills after WSSV challenge. Knockdown of SpCrus2 by injecting double-stranded RNA (dsSpCrus2) resulted in remarkably increased virus copies in mud crabs after infection with WSSV. These results suggested that SpCrus2 played a critical role in the antiviral immunity of mud crab. A GST pull-down assay showed that recombinant SpCrus2 interacted specifically with WSSV structural protein VP26, and this result was further confirmed by a co-immunoprecipitation assay with Drosophila S2 cells. As the signature sequence of type II crustin, SpCrus2 GRR is a glycine-rich cationic polypeptide with amphipathic properties. Our study demonstrated that the GRR and CRR of SpCrus2 exhibited binding activities to VP26, with the former displaying more potent binding ability than the latter. Interestingly, pre-incubating WSSV particles with recombinant SpCrus2 (rSpCrus2), rGRR, or rCRR inhibited virus proliferation in vivo; moreover, rSpCrus2 and rGRR possessed similar antiviral abilities, which were much stronger than those of rCRR. These findings indicated that SpCrus2 GRR contributed largely to the antiviral ability of SpCrus2, and that the stronger antiviral ability of GRR might result from its stronger binding activity to the viral structural protein. Overall, this study provided new insights into the antiviral mechanism of SpCrus2 and the development of new antiviral drugs.

Antarctic krill-derived peptides with consecutive Glu residues enhanced iron binding, solubility, and absorption.

Three peptides containing three glutamic acid (Glu) residues at different positions derived from Antarctic krill were obtained to investigate their iron-binding properties, digestive stability, and effectiveness on enhancing iron solubility and absorption. Results indicated that Fe2+ bound to the carbonyl, carboxyl, or hydroxyl groups of DELEDSLER, EEEFDATR, and DTDSEEEIR at stoichiometric ratios of 0.453, 0.466, and 0.490, respectively. DTDSEEEIR with three consecutive Glu in the middle of the sequence possessed higher iron-binding ability and iron release potential than EEEFDATR with three consecutive Glu in the N-terminal, and DELEDSLER with three discontinuous Glu showed the lowest values. Although EEEFDATR showed remarkably lower digestion stability than DTDSEEEIR, the effect of EEEFDATR-iron on iron solubility and absorption was comparable to that of DTDSEEEIR-iron, but better than that of DELEDSLER-iron and FeSO4. Thus, peptides with consecutive Glu have the potential as an effective iron carrier to improve iron absorption.

Angiotensin I-Converting Enzyme (ACE-I) Inhibition and Antioxidant Peptide from a Squilla Species.

BACKGROUND: Oratosquilla woodmasoni is one of the marine squilla species, which is found in the entire Asia-Pacific region. This current study assesses the species as the main basis of both ACEi and antioxidant peptide. OBJECTIVE: To isolate the ACEi peptide derived from O. woodmasoni and examine its ACE inhibition along with antioxidant potential. MATERIALS AND METHODS: The squilla muscle protein was hydrolysed using alcalase and trypsin enzymes for 12 hours and tested for DH. The hydrolysates were examined for their ACEi activity and then the best hydrolysate was sequentially purified in various chromatographical methods. The purified peptide was studied for anti-oxidant and functional properties, followed by amino acid sequencing. The purified peptide was also evaluated for its toxicity by in vitro cell viability assay. RESULTS: The DH% was found to be 47.13 ± 0.72% and 89.43 ± 2.06% for alcalase and trypsin, respectively. The alcalase 5th-hour hydrolysate was detected with potent activity (65.97 ± 0.56%) using ACEi assay and was primarily fractionated using ultrafiltration; the maximum inhibitory activity was found with 77.04 ± 0.52% in 3-10 kDa fraction. Subsequently, the fraction was purified using IEC and GFC, in which the AC1-A2 fraction had higher antihypertensive activity (70.85 ± 0.78%). The non-toxic fraction showed hexapeptide HVGGCG with molecular weight 529 Da with great potential of antioxidant activity along with functional property. CONCLUSION: This peptide could be developed as a potential ACE-inhibitory and antioxidant agent.

Biochemical and cytotoxic evaluation of latroeggtoxin-VI against PC12 cells.

Latroeggtoxin-VI (LETX-VI) is a peptide neurotoxin discovered from Latrodectus tredecimguttatus eggs. In the current study, the action features of the neurotoxin on PC12 cells were systematically investigated. LETX-VI could promote dopamine release from PC12 cells in the absence and presence of Ca2+, involving an even more complex action mechanism in the presence of Ca2+ and when the treatment time was longer. Although LETX-VI enchanced the autophagy and secretion activity in PC 12 cells, it showed no remarkable influence on the proliferation, cell cycle, apoptosis and ultrastructure of the cells. Pulldown combined with CapLC-MS/MS analysis suggested that LETX-VI affected PC12 cells by interacting with multiple proteins involved in the metabolism, transport, and release of neurotransmitters, particularly dopamine. The low cytotoxicity and effective regulatory action of LETX-VI on PC12 cells suggest the potential of the active peptide in the development of drugs for the treatment of some dopamine-related psychotic diseases and cancers.

Structural modelling and thermostability of a serine protease inhibitor belonging to the Kunitz-BPTI family from the Rhipicephalus microplus tick.

rBmTI-A is a recombinant serine protease inhibitor that belongs to the Kunitz-BPTI family and that was cloned from Rhipicephalus microplus tick. rBmTI-A has inhibitory activities on bovine trypsin, human plasma kallikrein, human neutrophil elastase and plasmin with dissociation constants in nM range. It is characterized by two inhibitory domains and each domain presents six cysteines that form three disulfide bonds, which contribute to the high stability of its structure. Previous studies suggest that serine protease inhibitor rBmTI-A has a protective potential against pulmonary emphysema in mice and anti-inflammatory potential. Besides that, rBmTI-A presented a potent inhibitory activity against in vitro vessel formation. In this study, the tertiary structure of rBmTI-A was modeled. The structure stabilization was evaluated by molecular dynamics analysis. Circular dichroism spectroscopy data corroborated the secondary structure found by the homology modelling. Also, in circular dichroism data it was shown a thermostability of rBmTI-A until approximately 70 °C, corroborated by inhibitory assays toward trypsin.

Production of an invertebrate lysozyme of Scylla paramamosain in E.coli and evaluation of its antibacterial, antioxidant and anti-inflammatory effects.

Lysozymes, which are secreted in many organisms, including invertebrates, mammals, plants, bacteria and fungus, exhibit antimicrobial, antiviral, antioxidant, and anti-inflammatory activities. Splys-i is an invertebrate-type (i-type) lysozyme isolated from Scylla paramamosain in 2017 and is involved in immune defense against bacteria. However, the antibacterial, antioxidant, and anti-inflammatory activities of Splys-i remain to be elucidated. In the current study, the expression parameters (including IPTG concentration, induction temperature, and induction duration) of Splys-i in Escherichia coli were optimized to achieve high-level yield through shake-flask cultivation with approximately 120 mg of Splys-i obtained from 1 L of LB medium. The purified Splys-i displayed low cytotoxicity to RAW264.7 macrophage cells and low hemolytic activity against erythrocytes of mouse, rat, and rabbit, respectively, and exhibited potent antibacterial activity against both Gram-positive and -negative bacteria with minimum concentrations ranging from 15 to 90 µg/mL. The antibacterial property of Splys-i was also unaffected when treated with various temperature, pHs, and salinity, respectively, and Splys-i showed resistance to proteinase digestion. Radical-scavenging rate assay (including ABTS+, DPPH, hydroyl free radical, and superoxide anion) indicated that Splys-i was an efficient antioxidant. Splys-i also exerted anti-inflammatory effect through the inhibition of IκBα and NF-κB(P65) phosphorylation, thereby reducing the secretion of pro-inflammatory cytokines. All these results suggested that Splys-i can be prepared from E. coli with potent biological property.

A Novel Antimicrobial Peptide Sparanegtin Identified in Scylla paramamosain Showing Antimicrobial Activity and Immunoprotective Role In Vitro and Vivo.

The abuse of antibiotics in aquaculture and livestock no doubt has exacerbated the increase in antibiotic-resistant bacteria, which imposes serious threats to animal and human health. The exploration of substitutes for antibiotics from marine animals has become a promising area of research, and antimicrobial peptides (AMPs) are worth investigating and considering as potential alternatives to antibiotics. In the study, we identified a novel AMP gene from the mud crab Scylla paramamosain and named it Sparanegtin. Sparanegtin transcripts were most abundant in the testis of male crabs and significantly expressed with the challenge of lipopolysaccharide (LPS) or Vibrio alginolyticus. The recombinant Sparanegtin (rSparanegtin) was expressed in Escherichia coli and purified. rSparanegtin exhibited activity against Gram-positive and Gram-negative bacteria and had potent binding affinity with several polysaccharides. In addition, rSparanegtin exerted damaging activity on the cell walls and surfaces of P. aeruginosa with rougher and fragmented appearance. Interestingly, although rSparanegtin did not show activity against V. alginolyticus in vitro, it played an immunoprotective role in S. paramamosain and exerted an immunomodulatory effect by modulating several immune-related genes against V. alginolyticus infection through significantly reducing the bacterial load in the gills and hepatopancreas and increasing the survival rate of crabs.

LP2, the first lanthipeptide GPCR agonist in a human pharmacokinetics and safety study.

Introduction of a lanthionine into a peptide may enhance target affinity, target specificity and proteolytic resistance. This manuscript reports preclinical safety studies and the first-in-human study with the lanthipeptide AT2R agonist LP2, a structural analog of cAng-(1-7), whose N-terminus was protected against aminopeptidases by the presence of a d-lysine. None of the preclinical studies, including an in vitro multitarget panel, behavioral, respiratory and cardiovascular measurements, genotoxicity and toxicity studies in rat and dog, posed any safety concern. Due to lack of toxicity the maximum tolerated dose was not reached neither in rat nor in dog. In the human dose escalation study, healthy male volunteers received a single 1 mL subcutaneous injection (0.001 mg, 0.01 mg or 0.1 mg) of LP2 or matching placebo. In contrast to angiotensin II which has a T1/2 in plasma of < 1 min, LP2 has a T1/2 of approximately 2.1-2.6 hours. The fraction of the dose excreted unchanged in urine ranged from 84.73 ± 10.4 % at a dose of 0.001 mg to 66.4 ± 3.9 % at 0.1 mg. There were no deaths, serious adverse events or subject withdrawals as a result of an adverse event. The incidence of adverse events was 16.7 %; each was mild in severity. One adverse event, peripheral coldness, was considered to be possibly related to LP2 at 0.001 mg LP2. None of the results was considered to pose a clinically relevant safety concern. This study supports the potential for the therapeutic use of lanthipeptides.

Cyclic gomesin, a stable redesigned spider peptide able to enter cancer cells.

Anticancer chemo- and targeted therapies are limited in some cases due to strong side effects and/or drug resistance. Peptides have received renascent interest as anticancer therapeutics and are currently being considered as alternatives and/or as complementary to biologics and small-molecule drugs. Gomesin, a disulfide-rich host defense peptide expressed in the Brazilian spider Acanthoscurria gomesiana selectively targets and disrupts cancer cell membranes. In the current study, we employed a range of biophysical methodologies with model membranes and bioassays to investigate the use of a cyclic analogue of gomesin as a drug scaffold to internalize cancer cells. We found that cyclic gomesin can internalize cancer cells via endocytosis and direct membrane permeation. In addition, we designed an improved non-disruptive and non-toxic cyclic gomesin analogue by incorporating D-amino acids within the scaffold. This improved analogue retained the ability to enter cancer cells and can be used as a scaffold to deliver drugs. Efforts to investigate the internalization mechanism used by host defense peptides, and to improve their stability, potency, selectivity and ability to permeate cancer cell membranes will increase the opportunities to repurpose peptides as templates for designing alternative anticancer therapeutic leads.

Biological activities of a recombinant fortilin from Fenneropenaeus merguiensis.

Human Fortilin, an antiapoptotic protein, has also been implicated in several diseases; however, several potential uses of fortilin have also been proposed. Bearing the implications of fortilin in mind, fortilin analog, which has no complication with diseases, is required. Since a recombinant full-length fortilin from Fenneropenaeus merguiensis (rFm-Fortilin (FL)) reported only 44% (3e-27) homologous to human fortilin, therefore the biological activities of the Fm-Fortilin (FL) and its fragments (F2, F12, and F23) were investigated for potential use against HEMA toxicity from filling cement to pulp cell. The rFm-Fortilin FL, F2, 12, and F23 were expressed and assayed for proliferation activity. The rFm-Fortilin (FL) showed proliferation activity on human dental pulp cells (HDPCs) and protected the cells from 2-hydroxy-ethyl methacrylate (HEMA) at 1-20 ng/ml. In contrast, none of the rFm-Fortilin fragments promoted HDPC growth that may be due to a lack of three conserved amino acid residues together for binding with the surface of Rab GTPase for proliferative activity. In addition, rFm-Fortilin (FL) activated mineralization and trend to suppressed production of proinflammatory cytokines, including histamine (at 10 ng/ml) and TNF-α (at 100 ng/ml). Besides, the rFm-Fortilin (FL) did not mutate the Chinese hamster ovary (CHO) cell. Therefore, the rFm-Fortilin (FL) has the potential use as a supplementary medical material to promote cell proliferation in patients suffering severe tooth decay and other conditions.

Arg-substituted VmCT1 analogs reveals promising candidate for the development of new antichagasic agent.

VmCT1 is an antimicrobial peptide (AMP) isolated from the venom of the scorpion Vaejovis mexicanus with antimicrobial, anticancer and antimalarial activities, which the rational design with Arg-substitution has yielded AMPs with higher antimicrobial activity than VmCT1. Chagas is a neglected tropical disease, becoming the development of new antichagasic agents is urgent. Thus, we aimed to evaluate the antichagasic effect of VmCT1 and three Arg-substituted analogues, as well their action mechanism. Peptides were tested against the epimastigote, trypomastigote, amastigote forms of Trypanossoma cruzi Y strain and against LLC-MK2 mammalian cells. The mechanism of action of these peptides was evaluated by means of flow cytometry and scanning electron microscopy. VmCT1 presented activity against all three forms of T. cruzi, with EC50 against trypomastigote forms of 1.37 µmol L-1 and selectivity index (SI) of 58. [Arg]3-VmCT1, [Arg]7-VmCT1 and [Arg]11-VmCT1 also showed trypanocidal effect, but [Arg]11-VmCT1 had the best effect, being able to decrease the EC50 against trypomastigote forms to 0.8 µmol L-1 and increase SI to 175. Necrosis was cell death pathway of VmCT1, as well [Arg]7-VmCT1 and [Arg]11-VmCT1, such as observed by membrane damage in flow cytometry analyses and scanning-electron-microscopy. In conclusion, [Arg]11-VmCT1 revealed promising as a candidate for new antichagasic therapeutics.

Proteotranscriptomic Insights into the Venom Composition of the Wolf Spider Lycosa tarantula.

Spider venoms represent an original source of novel compounds with therapeutic and agrochemical potential. Whereas most of the research efforts have focused on large mygalomorph spiders, araneomorph spiders are equally promising but require more sensitive and sophisticated approaches given their limited size and reduced venom yield. Belonging to the latter group, the genus Lycosa ("wolf spiders") contains many species widely distributed throughout the world. These spiders are ambush predators that do not build webs but instead rely strongly on their venom for prey capture. Lycosa tarantula is one of the largest species of wolf spider, but its venom composition is unknown. Using a combination of RNA sequencing of the venom glands and venom proteomics, we provide the first overview of the peptides and proteins produced by this iconic Mediterranean spider. Beside the typical small disulfide rich neurotoxins, several families of proteins were also identified, including cysteine-rich secretory proteins (CRISP) and Hyaluronidases. Proteomic analysis of the electrically stimulated venom validated 30 of these transcriptomic sequences, including nine putative neurotoxins and eight venom proteins. Interestingly, LC-MS venom profiles of manual versus electric stimulation, as well as female versus male, showed some marked differences in mass distribution. Finally, we also present some preliminary data on the biological activity of L. tarantula crude venom.