A Novel Peptidomimetic Insecticide: Dippu-AstR-Based Rational Design and Biological Activity of Allatostatin Analogs.

Insect neuropeptides play an essential role in regulating growth, development, reproduction, nerve conduction, metabolism, and behavior in insects; therefore, G protein-coupled receptors of neuropeptides are considered important targets for designing green insecticides. Cockroach-type allatostatins (ASTs) (FGLamides allatostatins) are important insect neuropeptides in Diploptera punctata that inhibit juvenile hormone (JH) synthesis in the corpora allata and affect growth, development, and reproduction of insects. Therefore, the pursuit of novel insecticides targeting the allatostatin receptor (AstR) holds significant importance. Previously, we identified an AST analogue, H17, as a promising candidate for pest control. Herein, we first modeled the 3D structure of AstR in D. punctata (Dippu-AstR) and predicted the binding mode of H17 with Dippu-AstR to study the critical interactions and residues favorable to its bioactivity. Based on this binding mode, we designed and synthesized a series of H17 derivatives and assessed their insecticidal activity against D. punctata. Among them, compound Q6 showed higher insecticidal activity than H17 against D. punctata by inhibiting JH biosynthesis, indicating that Q6 is a potential candidate for a novel insect growth regulator (IGR)-based insecticide. Moreover, Q6 exhibited insecticidal activity against Plutella xylostella, indicating that these AST analogs may have a wider insecticidal spectrum. The underlying mechanisms and molecular conformations mediating the interactions of Q6 with Dippu-AstR were explored to understand its effects on the bioactivity. The present work clarifies how a target-based strategy facilitates the discovery of new peptide mimics with better bioactivity, enabling improved IGR-based insecticide potency in sustainable agriculture.

[Comparison of internal quality and nutrients between male and female Eupolyphaga sinensis].

According to the Chinese Pharmacopoeia(2020 edition), Eupolyphaga Steleophaga is prepared from female Eupolyphaga sinensis. To explore the quality difference between male and female E. sinensis, the present study determined indexes in the Chinese Pharmacopoeia, anticoagulant activity, and content of primary metabolites, amino acids, and trace elements in male and female E. sinensis before and after sexual maturity. The results showed that the content of ash, water, extract, and aflatoxin of the larvae and female adults of E. sinensis met the Chinese Pharmacopoeia standards, while the content of ash and extract of male adults failed to meet the standards. After sexual maturity, the content of water-soluble proteins, crude fat, and extract of female E. sinensis was higher than that of the other three groups(P<0.05). Although the content of total protein, total amino acid(TAA), essential amino acid(EAA), non-essential amino acid(NEAA), branched-chain amino acid(BCAA), functional amino acid(FAA), and delicious amino acid(DAA) of male adults was higher than that of the other groups(P<0.05), the anticoagulant activity was the weakest(P<0.05). Before sexual maturity, the content of Fe in E. sinensis larvae was significantly higher than that in adults(P<0.05). The content of K and Mg in female larvae was the highest, while the content of Ca was the lowest(P<0.05). The content of Cu in male adults was higher than that in the other three groups(P<0.05). In conclusion, except for male adults, the larvae and female adults of E. sinensis meet the Chinese Pharmacopoeia standards. There is no difference in the internal quality of male and female E. sinensis larvae. It is recommended to collect female adults as much as possible. Male adults are rich in proteins and amino acids with high fibrinolytic activities, which can be developed in the future.

Functionally modified chitotriosidase catalytic domain for chitin detection based on split-luciferase complementation.

In this study, we applied a luciferase-fragment complementation assay for chitin detection. When luciferase-fragment fused chitin-binding proteins were mixed with chitin, the reconstituted luciferase became active. The recombinant chitin-binding domain (CBD) and a functionally modified catalytic domain (CatD) of human chitotriosidase were employed for this method. We designed the CatD mutant as a chitin-binding protein with diminished chitinolytic activity. The non-wash assay using the CatD mutant had higher sensitivity than CBD for chitin detection and proved to be a structure-specific biosensor for chitin, including crude biomolecules (from fungi, mites, and cockroaches). The CatD mutant recognized a chitin-tetramer as the minimal binding unit and bound chitin at KD 99 nM. Furthermore, a sandwich ELISA using modified CatD showed a low limit of quantification for soluble chitin (13.6 pg/mL). Altogether, our work shows a reliable method for chitin detection using the potential capabilities of CatD.

Characterization of a polysaccharide from Eupolyphaga sinensis walker and its effective antitumor activity via lymphocyte activation.

A polysaccharide (ESPS) purified from Eupolyphaga sinensis Walker by ion exchange chromatography and gel chromatography was investigated, including its structure characterization and antitumor activity. The results showed that ESPS was composed of rhamnose, fucose, arabinose, xylose, glucose, and galactose in a molar ratio of 7.4: 3.1: 13.9: 9.3: 39.7: 26.5, with the mean weight (Mw) of 2.14 × 104Da; the main chain of ESPS was mainly composed of → 4) - α - D - Glcp - (1 â†’ and → 3) - ß - D - Galp - (1 →, and the side chains were connected to the main chain through the O-6 atom of glucose and O-4 and O-6 atom of galactose. In addition, ESPS promoted the lymphocyte proliferation and inhibited liver cancer cells growth through enhancing lymphocyte activity in vitro, mainly NK cells. Moreover, ESPS markedly stimulated immunity in H22-bearing mice by increasing the spleen and thymus indices and effectively inhibited H22 cell growth in vivo. These data indicated that ESPS was a polysaccharide component possessing high anti-hepatocellular carcinoma activity, representing a potential immunotherapy candidate for the treatment of liver cancer.

Optimal conditions for the storage of German cockroach extract.

Allergen extracts are commonly utilized for diagnosis and immunotherapy; however, the stability of protease­rich extracts is important for a precise diagnosis and treatment efficacy. The present study determines the optimal conditions for the storage of German cockroach allergen extract. Cockroach extracts were reconstituted in four buffers: normal saline (NS), 50% glycerol in NS, 0.3% phenol in NS, or 0.3% phenol and 50% glycerol in NS. The extracts in different buffers were stored either at room temperature (18­26˚C, RT) or refrigerated (2­8˚C). Subsequently, the protein concentration and allergen content (Bla g 1 and Bla g 2) in the extracts were examined for the course of one year. Extract potency was estimated by inhibition ELISA. At least 90.5% protein, 94.4% Bla g 1, 65.2% Bla g 2, and 91.4% potency remained after one year when 50% glycerol NS was added to the extract with refrigeration. However, less than 13.7% protein, 17.1% Bla g 1, 0% Bla g 2 and 32.5% potency were maintained after one year when 50% glycerol NS was not added to the extract and was maintained at RT. The addition of 0.3% phenol NS did not show significant effects on extract stability. The addition of 50% glycerol NS and refrigerated storage temperature were found to be important factors for increasing the shelf life of protease­rich cockroach extract.

High level of methylmercury exposure causes persisted toxicity in Nauphoeta cinerea.

Methylmercury (MeHg+) is a neurotoxicant abundantly present in the environment. The long-term effects of MeHg+ have been investigated in rodents, yet data on the long-term or persisted toxicity of MeHg+ in invertebrates is scanty. Here, we examined the acute, intermediate, and chronic effects upon dietary administration of MeHg+ in nymphs of Nauphoeta cinerea. Besides, the potential reversibility of the toxic effects of MeHg+ after a detoxification period was evaluated. Nymphs were exposed to diets containing 0 (control), 2.5, 25, and 100 µg MeHg+/g of diet for 10, 30, and 90 days. Additional groups of nymphs were fed with the same dose of MeHg+ for 30 days and then were subjected to a detoxification period for 60 days. The nymphs exposed to 100 µg MeHg+/g succumbed to a high mortality rate, along with multiple biochemical (increase of reactive oxygen species production and glutathione S-transferase activity, as well as decrease in the acetylcholinesterase activity) and behavioral alterations. We observed delayed mortality rate and behavioral alterations in nymphs exposed to 100 µg MeHg+/g for 30 days and subsequently subjected to 60 days of detoxification. However, the biochemical alterations did not persist throughout the detoxification period. In conclusion, our results established the persistent toxic effect of MeHg+ even after a prolonged detoxification period and evidenced the use of N. cinerea as an alternative model to study the toxicity of MeHg+.

Polypeptides extracted from Eupolyphaga sinensis walker via enzymic digestion alleviate UV radiation-induced skin photoaging.

The traditional medicine-Eupolyphaga sinensis walker is a rich source of functional proteins and peptides. In this study, Eupolyphaga sinensis walker were identified to include fifteen amino acids, of which hydrophobic amino acids accounted for 46.7%. Eupolyphaga sinensis walker polypeptides (EPs) were extracted by sequentially hydrolyzing with pepsin and trypsin. EPs could effectively scavenge 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·), superoxide anion radical (O2-), and hydroxyl radical (OH-), and reduce ferric solution in vitro. EPs also enhanced the activities of Ssuperoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPH-Px), increased the contents of hydroxyproline (HYP), and reduced the content of Malondialdehyde (MDA) in photoaged mice skin. Histological study confirmed that EPs improved UV irradiation-induced damage of skin texture and morphology. Therefore, the extracted EPs are effective antioxidants and can serve as powerful treatment for skin photoaging.

3D-QSAR based optimization of insect neuropeptide allatostatin analogs.

Allatostatins (AST) are neuropeptides originally described as inhibitors of juvenile hormone (JH) synthesis in insects. Consequently, they have been considered as potential lead compounds for the discovery of new insect growth regulators (IGRs). In the present work, receptor-based three-dimensional quantitative structure-activity relationship (3D-QSAR) was studied with 48 AST analogs, and a general approach for novel potent bioactive AST analogs is proposed. Hence, six novel AST analogs were designed and synthesized. Bioassays indicated that the majority novel analogs exhibited potent JH inhibitory activity, especially analog A6 (IC50: 3.79 nmol/L), which can be used as lead compound to develop new IGRs.

Determination of vitamin B12 in four edible insect species by immunoaffinity and ultra-high performance liquid chromatography.

Insects are rich in major nutrients, such as protein and fat. Recently, minor nutrients like vitamins have become the subjects of interest in insects. Hence, this study reports on the development and validation of a method for the determination of vitamin B12 in mealworm (Tenebrio molitor larvae), cricket (Gryllus assimilis), grasshopper (Locusta migratoria) and cockroach (Shelfordella lateralis), using an ultra-high performance liquid chromatography approach with preliminary immunoaffinity chromatography sample preparation. The method was validated regarding linearity, specificity, accuracy and precision, as well as limits of detection/quantification, and was found to be satisfactory for the desired application. Found levels of vitamin B12 were 1.08 µg/100 g for mealworm, 2.88 µg/100 g for cricket, 0.84 µg/100 g for grasshopper, and 13.2 µg/100 g dry weight for cockroach, representing the first validated report on the content of vitamin B12 in edible insects. Observed interferences are likely caused by the presence of pseudovitamin B12.

[Simultaneous determination of aflatoxin B_1,B_2,G_1,G_2,M_1,M_2 in Eupolyphaga Steleophaga by immunoaffinity column clean-up and HPLC-FLD with post-column photochemical derivatization].

The contamination of aflatoxin B_1,B_2,G_1,G_2,M_1 and M_2 in Eupolyphaga Steleophaga was determined by immunoaffinity column clean-up and HPLC-FLD with post-column photochemical derivatization. Chromatographic separations were carried out using a Cloversil C_(18) column( 4. 6 mm×250 mm,5 µm) that were eluted in isocratic with methanol-acetonitrile-water( 20 ∶ 20 ∶ 60) as the mobile phase. The excitation wavelength and the emission wavelength of fluorescence detector were maintained at 360 nm and 450 nm,respectively. The flow rate was 0. 8 m L·min~(-1),and the column temperature was 30 ℃ ． The sample was prepared using the immunoaffinity column,then the recovery was measured with 75. 47%-101. 8% with RSD values lower than 6. 7%. A total of 20 batches of Eupolyphaga Steleophaga samples were assayed. According to the Chinese Pharmacopoeia( 2015 edition,part 1),the aflatoxin B_1 limit should be less than 5 µg·kg~(-1),and the sum of aflatoxins( AFB_1,AFB2,AFG_1,AFG_2) should be less than 10 µg·kg-1. Therefore,the positive rate of the 20 samples was 50. 0%,and 7 batches of samples exceeded the standard,and the over-standard rate was as high as 70. 0%. Among them,aflatoxins B_1,B_2,G_1,G_2,M_1,and M_2 were detected in three batches( SD-1,AH-1,AH-3),and aflatoxins B_1,B2,G1,G2,and M1 were detected in one batch( AH-7). The results showed that the newly developed method in this work is suitable for the simultaneous determination of six aflatoxins in Eupolyphaga Steleophaga,and also suggested that it should be of high values to take the contamination with aflatoxins into concerns.

Plasticizer, di(2-ethylhexyl)phthalate (DEHP) enhances cockroach allergen extract-driven airway inflammation by enhancing pulmonary Th2 as well as Th17 immune responses in mice.

In recent decades, there has been a gradual increase in the prevalence of asthma. Various factors including environmental pollutants have contributed to this phenomenon. Plasticizer, di(2-ethylhexyl)phthalate (DEHP) is one of the commonest environmental pollutants due to its association with plastic products. DEHP gets released from plastic products easily leading to respiratory exposure in humans. As a consequence, DEHP is associated with allergic asthma in humans and animals. DEHP is reported to act as an adjuvant in ovalbumin-induced mouse models of asthma at high doses. However, these studies mostly looked into the role of DEHP on Th2 cytokines/eosinophilic inflammation without investigating the role of airway epithelial cells (AECs)/dendritic cells (DCs)/Th17 cells. Its adjuvant activity with natural allergens such as cockroach allergens at tolerable daily intake needs to be explored. Cockroach allergens and DEHP may be inhaled together due to their coexistence in work place as well as household environments. Therefore, effect of DEHP was assessed in cockroach allergens extract (CE)-induced mouse model of asthma. Airway inflammation, histopathology, mucus secretion, and immune responses related to Th2/Th17/DCs and AECs were assessed in mice with DEHP exposure alone and in combination with CE. Our study shows that DEHP converts CE-induced eosinophilic inflammation into mixed granulocytic inflammation by promoting Th2 as well as Th17 immune responses. This was probably due to downregulation of E-cadherin in AECs, and enhancement of costimulatory molecules (MHCII/CD86/CD40)/pro-inflammatory cytokines (IL-6/MCP-1) in DCs by DEHP. This suggests that DEHP facilitates development of mixed granulocytic airway inflammation in the presence of a natural allergen.

School exposure and asthma.

OBJECTIVE: To provide a comprehensive overview of common school exposures and the association between school exposures and pediatric asthma morbidity. DATA SOURCES: A comprehensive literature review was performed using PubMed. STUDY SELECTIONS: Full-length, peer-reviewed studies published in English were considered for review. In vivo, in vitro, and animal studies were excluded. Studies of school exposure to cockroach, mouse, dust mite, dog, cat, molds, pollution, and endotoxin associated with asthma and asthma morbidity were considered. RESULTS: The current literature establishes an association between school exposure and pediatric asthma morbidity. There is a need for ongoing research to evaluate the effects of school-based environmental interventions on asthma morbidity. CONCLUSION: It is evident that the indoor school environment is a significant reservoir of allergens, molds, pollutants, and endotoxin and that there is an association between school exposure and pediatric asthma morbidity. School-based interventions have the potential for substantial individual, community, and public health benefit. It is important that researchers continue to study the health effects associated with school exposures and assess cost-effectiveness of multifaceted school-based interventions.

A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry.

A method for rapid quantitation of insect juvenile hormones (JH) and intermediates in the biosynthetic pathway, both in vitro and in vivo (hemolymph and whole body), has been developed using GC-MS/MS. This method is as simple as the radiochemical assay (RCA), the most commonly used method for measurement of JH biosynthesis in vitro, without need for further purification and derivatization, or radioactive precursors or ligands. It shows high sensitivity, accuracy and reproducibility. Linear responses were obtained the range of 1-800 ng/mL (approximately 4-3000 nM). Recovery efficiencies for farnesol, farnesal, methyl farnesoate and JH III were approximately 100% in vitro and over 90% in vivo, with excellent reproducibility at three different spike levels. Titer of JH III in the hemolymph was relatively low at day 0 (adult female emergence) (79.68 ±â€¯5.03 ng/mL) but increased to a maximum of 1717 ng/mL five days later. In whole body, JH III quantity reached a maximum on day 4 (845.5 ±â€¯87.9 ng/g) and day 5 (679.7 ±â€¯164.6 ng/g) and declined rapidly thereafter. It is in agreement with the hemolymph titer changes and biosynthetic rate of JH in vitro. Comparison with the results of inhibition of JH biosynthesis by two known inhibitors (allatostatin (AST) mimic H17 and pitavastatin) using RCA and GC-MS/MS, showed that there was little difference between the two methods In contrast to other methods, the present method with GC-MS/MS can be used to elucidate the mechanism of inhibition by inhibitors of JH biosynthesis without any derivatization and purification. This method is applicable to screening of JH inhibitors and the study of inhibitory mechanisms with high sensitivity and accurate quantification. It may also be useful for the determination of JH titer in other Arthropods.

MECHANISMS UNDERLYING ACTION OF XINMAILONG INJECTION, A TRADITIONAL CHINESE MEDICINE IN CARDIAC FUNCTION IMPROVEMENT.

BACKGROUND: As a bioactive composite extracted from American cockroach, Xinmailong injection (XML) is used for the treatment of congestive heart failure (CHF) in China. Clinical data has provided evidence that XML has positive inotropic properties. The objective of this study was to assess the mechanisms involved in the therapeutical effect of XML on CHF. MATERIALS AND METHODS: The effects of XML on the cardiac function in isolated rat heart were measured. A Ca2+ imaging technology was used in rat cardiomyocytes (H9c2 cells) to reveal the role of XML on Ca2+ channels. Meanwhile, the effects of XML on the activities of Na+/K+ ATPase and sodium/calcium exchanger were measured. In addition, the level of reactive oxygen species and the protein expressions for the superoxide dismutase and hemeoxygenase were determined in the cardiomyocytes. RESULTS: The results showed that XML increased the electrical impulse-induced [Ca2+]i in H9c2 cells, which was dependant on extracellular Ca2+ and was abolished by ML218-HCl (a T-type Ca2+channels antagonist) but not nimodipine (a L-type Ca2+channels antagonist). Ouabain, a Na+/K+-ATPase inhibitor, increased the electrical impulse-induced [Ca2+]i, which was significantly inhibited by XML. Moreover, XML markedly inhibited the Na+/K+ ATPase activity in H9c2 cells. In addition, XML notably reduced the production of reactive oxygen species and enhanced the protein expressions of antioxidant enzymes including superoxide dismutase 1, superoxide dismutase 2 and hemeoxygenase 1 in H9c2 cell. CONCLUSION: Our findings pave the ways to the better understandings of the therapeutic effects of XML on cardiovascular system.

Biological, mechanical, optical and physicochemical properties of natural chitin films obtained from the dorsal pronotum and the wing of cockroach.

In previous studies, chitin-based films were produced from chitin nanofibers in dust form and fully characterized. However, chitin films naturally present in many organisms have not been isolated and characterized. Herein, structurally intact chitin films were successfully extracted from the dorsal pronotum and the wing of cockroach. Despite using the same extraction procedure, important differences were observed. Especially, hydrophobicity, transparency, antifungal and antibacterial biofilm activities of wing chitin film were recorded notably higher than those of chitin film from the dorsal pronotum. However, better mechanical properties were observed for chitin film from the dorsal pronotum. Notably, among the tested bacteria, two common pathogens could not form biofilms on the surface of the films. This study clearly demonstrated natural chitin films obtained from an insect can provide a new perspective to chitin-based applications where chitin films with high thermal stability, transparency, resistance to bacterial biofilm formation and antifungal activity are needed.

Discovery of Rab1 binding sites using an ensemble of clustering methods.

Targeting non-native-ligand binding sites for potential investigative and therapeutic applications is an attractive strategy in proteins that share common native ligands, as in Rab1 protein. Rab1 is a subfamily member of Rab proteins, which are members of Ras GTPase superfamily. All Ras GTPase superfamily members bind to native ligands GTP and GDP, that switch on and off the proteins, respectively. Rab1 is physiologically essential for autophagy and transport between endoplasmic reticulum and Golgi apparatus. Pathologically, Rab1 is implicated in human cancers, a neurodegenerative disease, cardiomyopathy, and bacteria-caused infectious diseases. We have performed structural analyses on Rab1 protein using a unique ensemble of clustering methods, including multi-step principal component analysis, non-negative matrix factorization, and independent component analysis, to better identify representative Rab1 proteins than the application of a single clustering method alone does. We then used the identified representative Rab1 structures, resolved in multiple ligand states, to map their known and novel binding sites. We report here at least a novel binding site on Rab1, involving Rab1-specific residues that could be further explored for the rational design and development of investigative probes and/or therapeutic small molecules against the Rab1 protein. Proteins 2017; 85:859-871. © 2016 Wiley Periodicals, Inc.

Identification and characterization of antibacterial compound(s) of cockroaches (Periplaneta americana).

Infectious diseases remain a significant threat to human health, contributing to more than 17 million deaths, annually. With the worsening trends of drug resistance, there is a need for newer and more powerful antimicrobial agents. We hypothesized that animals living in polluted environments are potential sources of antimicrobials. Under polluted milieus, organisms such as cockroaches encounter different types of microbes, including superbugs. Such creatures survive the onslaught of superbugs and are able to ward off disease by producing antimicrobial substances. Here, we characterized antibacterial properties in extracts of various body organs of cockroaches (Periplaneta americana) and showed potent antibacterial activity in crude brain extract against methicillin-resistant Staphylococcus aureus and neuropathogenic Escherichia coli K1. The size-exclusion spin columns revealed that the active compound(s) are less than 10 kDa in molecular mass. Using cytotoxicity assays, it was observed that pre-treatment of bacteria with lysates inhibited bacteria-mediated host cell cytotoxicity. Using spectra obtained with LC-MS on Agilent 1290 infinity liquid chromatograph, coupled with an Agilent 6460 triple quadruple mass spectrometer, tissues lysates were analysed. Among hundreds of compounds, only a few homologous compounds were identified that contained the isoquinoline group, chromene derivatives, thiazine groups, imidazoles, pyrrole-containing analogs, sulfonamides, furanones, and flavanones and known to possess broad-spectrum antimicrobial properties and anti-inflammatory, anti-tumour, and analgesic properties. Further identification, characterization, and functional studies using individual compounds can act as a breakthrough in developing novel therapeutics against various pathogens including superbugs.

Analysis of 10 Environmental Allergen Components of the American Cockroach in Taiwan.

BACKGROUND: Cockroaches are important sources of indoor airborne allergens. The American cockroach (Periplaneta americana) is the second leading inhalant allergen causing allergic airway diseases in Taiwan. We previously reported a difference in pathogenicity of different allergen components from American cockroaches. OBJECTIVE: To analyze the environmental profile of American cockroach allergen components. METHODS: Polyclonal antibodies were generated to recombinant American cockroach allergens, Per a 1 through Per a 10. The levels of each allergen in (1) whole-body extracts and feces from American cockroaches and in (2) fresh-frozen 6-month-old and 12-month-old dead American cockroaches were evaluated by immunoblotting and quantified. Levels of allergen components from patients' household dust samples were determined by competition enzyme-linked immunosorbent assay. RESULTS: Per a 1, 2, and 10 proteins were present predominantly in roach feces, whereas other allergen components were found predominantly in roach bodies. There was a time-dependent decrease in total levels of some allergen proteins. Although levels of Per a 4, 5, 6, and 9 significantly decreased to less 20% of the basal level, there was no significant change in levels of Per a 2, 7, and 10 after 1-year decomposition. The most abundant allergen components in 20 dust samples from patients' houses were Per a 9, Per a 10, and Per a 2. CONCLUSION: The concentration of 10 American cockroach allergen components differed in the environment. Per a 2 and Per a 10 can be used as markers of long-term environmental cockroach control and Per a 9 as current status of control in patients' houses.

First protein and peptide characterization of the tarsal adhesive secretions in the desert locust, Schistocerca gregaria, and the Madagascar hissing cockroach, Gromphadorhina portentosa.

Peptides and proteins have been largely neglected in the analysis of insect tarsal adhesives. After extraction of the protein fraction of the tarsal secretion of the desert locust, Schistocerca gregaria, and Madagascar hissing cockroach, Gromphadorhina portentosa, we combined Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) analyses for protein mass detection. In both these insects, SDS-PAGE analysis revealed several protein bands ranging from 8-190 kDa in both the tarsal secretion and the tibia control sample. Two (S. gregaria) and one (G. portentosa) protein bands exclusively occurred in the tarsal secretion and can be considered to belong to peptides and proteins specific to this secretion. MALDI-TOF analyses revealed 83 different proteins/peptides of 1-7 kDa in S. gregaria, and 48 of 1-11 kDa in G. portentosa. 59 (S. gregaria) and 27 (G. portentosa) proteins exclusively occurred in the tarsal secretion. In G. portentosa, a characteristic series of signal peaks occurred in the range of c. 10-12 kDa, each peak being approximately 160 Da apart. Such a pattern is indicative of proteins modified by glycosylation. Our approach demonstrates that extensive sampling involving considerable time and manpower to sample the adhesive fluid directly from the tarsi opens up a perspective for extracting peptides and proteins in sufficient quantities. This makes them accessible to the field of proteomics and thus to elucidate their possible function in the adhesive process.

Testing small molecule analogues of the Acanthocheilonema viteae immunomodulator ES-62 against clinically relevant allergens.

ES-62 is a glycoprotein secreted by the filarial nematode Acanthocheilonema viteae that protects against ovalbumin (OVA)-induced airway hyper-responsiveness in mice by virtue of covalently attached anti-inflammatory phosphorylcholine (PC) residues. We have recently generated a library of small molecule analogues (SMAs) of ES-62 based around its active PC moiety as a starting point in novel drug development for asthma and identified two compounds - termed 11a and 12b - that mirror ES-62's protective effects. In this study, we have moved away from OVA, a model allergen, to test the SMAs against two clinically relevant allergens - house dust mite (HDM) and cockroach allergen (CR) extract. We show that both SMAs offer some protection against development of lung allergic responses to CR, in particular reducing eosinophil infiltration, whereas only SMA 12b is effective in protecting against eosinophil-dependent HDM-induced allergy. These data therefore suggest that helminth molecule-induced protection against model allergens may not necessarily translate to clinically relevant allergens. Nevertheless, in this study, we have managed to demonstrate that it is possible to produce synthetic drug-like molecules based on a parasitic worm product that show therapeutic potential with respect to asthma resulting from known triggers in humans.