Structure and bioactivity of an insecticidal trans-defensin from assassin bug venom.

Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P. plagipennis, which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans-defensins, with the same cystine connectivity as human α-defensin (I-VI, II-IV, and III-V). The structure of Pp19 is unique among reported structures of arthropod peptides.

A fully human connective tissue growth factor blocking monoclonal antibody ameliorates experimental rheumatoid arthritis through inhibiting angiogenesis.

BACKGROUND: Connective tissue growth factor (CTGF) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) by facilitating angiogenesis and is a promising therapeutic target for RA treatment. Herein, we generated a fully human CTGF blocking monoclonal antibody (mAb) through phage display technology. RESULTS: A single-chain fragment variable (scFv) with a high affinity to human CTGF was isolated through screening a fully human phage display library. We carried out affinity maturation to elevate its affinity for CTGF and reconstructed it into a full-length IgG1 format for further optimization. Surface plasmon resonance (SPR) data showed that full-length antibody IgG mut-B2 bound to CTGF with a dissociation constant (KD) as low as 0.782 nM. In the collagen-induced arthritis (CIA) mice, IgG mut-B2 alleviated arthritis and decreased the level of pro-inflammatory cytokines in a dose-dependent manner. Furthermore, we confirmed that the TSP-1 domain of CTGF is essential for the interaction. Additionally, the results of Transwell assays, tube formation experiments, and chorioallantoic membrane (CAM) assays showed that IgG mut-B2 could effectively inhibit angiogenesis. CONCLUSION: The fully human mAb that antagonizes CTGF could effectively alleviate arthritis in CIA mice, and its mechanism is tightly associated with the TSP-1 domain of CTGF.

Effect of different sample treatment methods on Low Endotoxin Recovery Phenomenon.

Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, Doratifera vulnerans.

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Doratifera vulnerans Contrary to dogma that defensive venoms are simple in composition, D. vulnerans produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by D. vulnerans Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.

Weaponisation 'on the fly': Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies.

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the KV11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of KV11.1 channel activation to more depolarised potentials, but has no effect on KV1.3, KV2.1, KV10.1, KCa1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these KV channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.

Cytochrome P450-Mediated Metabolic Characterization of a Mono-Carbonyl Curcumin Analog WZ35.

WZ35 is a monocarbonyl analog of curcumin, which had been proved advantage over curcumin in chemical stability and antitumor activity. However, its pharmacokinetic profile has not been determined. In the present study, an ultraperformance liquid chromatography-tandem mass spectrometry assay was developed to detect concentration of WZ35 in rat plasma. Subsequently, pharmacokinetic study showed that the oral bioavailability of WZ35 is 10.56%. Cytochrome P450 (CYP450) plays a major role in metabolizing exogenous substance. The concentration of WZ35 was sharply decreased while incubating with microsome. It's indicated that WZ35 is a substrate of CYP450s. Molecular docking assay showed that WZ35 can combine with CYP2B6 and CYP2C9 to form much more stable complex. The lowest docking energy was generated in complex with CYP2E1. The inhibition of CYP450s by WZ35 was also evaluated. Pan inhibitions of WZ35 on rat CYP3A2, CYP2B1, CYP2C11, CYP2D1, and -CYP2E1 were observed by detecting probe substrates (midazolam, bupropion, tolbutamide, dextromethorphan, chlorzoxazone) and metabolites accordingly. On an average, 80% activities of enzymes were blocked. Mechanistically, the inhibitions of WZ35 on CYP3A2, CYP2B1, CYP2E1 were in a time-dependent manner according to the results of IC50 shift assay. The collective data demonstrated that the oral bioavailability of monocarbonyl analog of curcumin has significantly improved compared to curcumin. It's both the substrate and inhibitor of CYP450s through in a time-dependent mechanism.

Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug Platymeris rhadamanthus.

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug Platymeris rhadamanthus, a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.

Activation of Toll-like receptor 3 promotes pathological corneal neovascularization by enhancement of SDF-1-mediated endothelial progenitor cell recruitment.

Toll-like receptors (TLRs) play an important role in inflammatory and immunological responses, which are intimately related to neovascularization. However, the precise mode of action of TLR3 in neovascularization still remains ambiguous. In this study, we sought to investigate the role of TLR3 in pathological corneal neovascularization (CNV) using a mouse model of alkali-induced CNV. CNV was attenuated in TLR3-deficient mice, and the absence of TLR3 led to decreased production of stromal cell-derived factor 1 (SDF-1), a well-characterized cytokine that regulates the recruitment of endothelial progenitor cells (EPCs) to the sites of neo-angiogenic niches in the injured tissues. Topical administration of polyinosinic-polycytidylic acid [poly (I:C)], a synthetic ligand for TLR3, to the injured cornea promoted CNV in wild type (WT) mice but not in TLR3-deficient mice. In addition, the effect of poly (I:C) on WT mice was abolished by addition of SDF-1 receptor antagonist AMD 3100. Furthermore, poly (I:C) treatment in vitro enhanced the migration of EPCs, whereas the enhanced migration was abolished by AMD 3100. These results indicate an essential role of TLR3 signalling in CNV that involves upregulating SDF-1 production and recruiting EPCs to the sites of injury for neovascularization. Thus, targeting the TLR3 signalling cascade may constitute a novel therapeutic approach for treating neovascularization-related diseases.

Buzz Kill: Function and Proteomic Composition of Venom from the Giant Assassin Fly Dolopus genitalis (Diptera: Asilidae).

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly Dolopus genitalis using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies (Lucilia cuprina) produced rapid contractile paralysis (PD50 at 1 min = 3.1 µg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/µL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/ß defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.

Influence of super-hydrophobic silicone rubber substrate on the growth and differentiation of human lens epithelial cells.

Materials with low cell adhesion are advantageous for production of replacement intraocular lens (IOL) to prevent posterior capsular opacification (PCO). We evaluated the feasibility of compression molding for manufacture of silicone rubber with super-hydrophobic surface and low cell infiltrative characteristics compared to ordinary hydrophobic silicone rubber. Silicone specimens with complex surface topology (super-hydrophobic) or smooth surfaces (hydrophobic) were manufactured by vacuum deforming and molding. Contact angle, microscopic surface structure, and transparency were evaluated. Super-hydrophobic and smooth samples were compared for effects on proliferation, adhesion, and morphology of human lens epithelial cells (hLECs). Epithelial-mesenchymal transition (EMT) was examined by immunofluorescence expression of fibronectin (Fn), Alpha-smooth muscle actin (α-SMA), and vimentin. The surface contact angle of super-hydrophobic silicone was greater than that of smooth silicone (153.8° vs. 116°). The super-hydrophobic surface exhibited a micron-scale palisade structure under scanning electron microscopy (unit length, width, and height of 80, 25, and 25 µm, respectively). However, cell number per 50 × microscopic field on super-hydrophobic surfaces was markedly reduced 24 and 72 h post-seeding compared to smooth surfaces (p < 0.01). Cells were cuboidal or spherical after 72h on super-hydrophobic surfaces, and exhibited numerous surface microvilli with fluff-base polarity, while cells on smooth surfaces exhibited morphological characteristics of EMT. Expression levels of the α-SMA and vimentin were reduced on super-hydrophobic surfaces compared to smooth surfaces. Super-hydrophobic silicon inhibits proliferation, adhesion, and EMT of hLECs, properties that may prevent fibrosis following cataract surgery.

Interleukin 37 promotes angiogenesis through TGF-ß signaling.

IL-37 is a novel pro-angiogenic cytokine that potently promotes endothelial cell activation and pathological angiogenesis in our previous study, but the mechanisms behind the pro-angiogenic effect of IL-37 are less well understood. Extending our observations, we found that TGF-ß interacts with IL-37, and potently enhances the binding affinity of IL-37 to the ALK1 receptor complex, thus allowing IL-37 to signal through ALK1 to activate pro-angiogenic responses. We further show that TGF-ß and ALK1 are required in IL-37 induced pro-angiogenic response in ECs and in the mouse model of Matrigel plug and oxygen-induced retinopathy. The result suggests that IL-37 induces pro-angiogenic responses through TGF-ß, which may act as the bridging molecule that mediates IL-37 binding to the TGF-ß receptor complex.

The Effect of Interleukin 38 on Angiogenesis in a Model of Oxygen-induced Retinopathy.

Interleukin 38 (IL-38) is a novel identified cytokine of IL-1 family in which some members are important in inflammation and angiogenesis. However, the role of IL-38 in regulating angiogenesis is unknown. The aim of the present study is to explore the effect of IL-38 on angiogenesis. Oxygen-induced retinopathy (OIR) of C57BL/6 J mice was induced by exposure of hyperoxia (75% oxygen) from postnatal day 7 (P7) to P12 and then returned to room air. The mice were injected with IL-38. At P17, neovascular region (tufts) and avascular area of the retinas were analyzed. The data showed that administration of IL-38 in vivo inhibited retinal angiogenesis significantly. Furthermore, the addition of IL-38 to the cell cultures attenuated the proliferation, scratch wound healing and tube formation of vascular endothelial cells induced by VEGF significantly. Our findings suggest that IL-38 is an antiangiogenic cytokine in pathophysiological settings and may have therapeutic potential for angiogenesis related diseases.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae).

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

IL-37 Is a Novel Proangiogenic Factor of Developmental and Pathological Angiogenesis.

OBJECTIVE: Angiogenesis is tightly controlled by growth factors and cytokines in pathophysiological settings. Interleukin 37 (IL-37) is a newly identified cytokine of the IL-1 family, some members of which are important in inflammation and angiogenesis. However, the function of IL-37 in angiogenesis remains unknown. We aimed to explore the regulatory role of IL-37 in pathological and physiological angiogenesis. APPROACH AND RESULTS: We found that IL-37 was expressed and secreted in endothelial cells and upregulated under hypoxic conditions. IL-37 enhanced endothelial cell proliferation, capillary formation, migration, and vessel sprouting from aortic rings with potency comparable with that of vascular endothelial growth factor. IL-37 activates survival signals including extracellular signal-regulated kinase 1/2 and AKT in endothelial cells. IL-37 promoted vessel growth in implanted Matrigel plug in vivo in a dose-dependent manner with potency comparable with that of basic fibroblast growth factor. In the mouse model of retinal vascular development, neonatal mice administrated with IL-37 displayed increased neovascularization. We demonstrated further that IL-37 promoted pathological angiogenesis in the mouse model of oxygen-induced retinopathy. CONCLUSIONS: Our findings suggest that IL-37 is a novel and potent proangiogenic cytokine with essential role in pathophy siological settings.

AA092, an annonaceous acetogenin mimetic, attenuates angiogenesis in a mouse model of inflammation-induced corneal neovascularization.

Previous studies demonstrated that annonaceous acetogenin (AA) was an antitumor drug with anti-angiogenic activity. However, the effect of AA on ocular neovascular disorders remains unclear. The aim of the present study is to explore the effect of AA092, an annonaceous acetogenin mimetic, on corneal neovascularization (CNV). In a mouse model of alkali-induced CNV, topical application of AA092 to the injured corneas attenuated CNV. In addition, in vivo treatment with AA092 down-regulated the expression of the pro-angiogenic factors VEGF, b-FGF, TGFß1, EGF but up-regulated the expression of the anti-angiogenic factors Thrombospondin-1 (Tsp-1), Tsp-2 and ADAMTS-1 in the injured corneas. Furthermore, AA092 inhibited the expression of pro-angiogenic factors, migration, proliferation and tube formation by human microvascular endothelial cells (HEMC-1) in vitro. These data indicate that AA092 has therapeutic potential for angiogenesis-associated diseases such as CNV.

Largazole, an inhibitor of class I histone deacetylases, attenuates inflammatory corneal neovascularization.

Histone deacetylases (HDACs) regulate gene transcription by modifying the acetylation level of histone and nonhistone proteins. In this study, we examined the effect of largazole, an inhibitor of class I HDACs, on inflammatory corneal angiogenesis. In a mouse model of alkali-induced corneal neovascularization (CNV), topical application of largazole to the injured corneas attenuated CNV. In addition, in vivo treatment with largazole down-regulated the expression of the pro-angiogenic factors VEGF, b-FGF, TGFß1 and EGF but up-regulated the expression of the anti-angiogenic factors Thrombospondin-1 (Tsp-1), Tsp-2 and ADAMTS-1 in the injured corneas. Furthermore, largazole inhibited the expression of pro-angiogenic factors, migration, proliferation and tube formation by human microvascular endothelial cells (HEMC-1) in vitro. These data indicate that largazole has therapeutic potential for angiogenesis-associated diseases.