Proteomic analysis of excretory-secretory products from young adults of Angiostrongylus cantonensis

BACKGROUND Angiostrongyliasis is caused by the nematode Angiostrongylus cantonensis and can lead to eosinophilic meningitis and meningoencephalitis in humans. The young adult worms play central pathogenic roles in the central nervous system (CNS); however, the underlying mechanism is unclear. Excretory-secretory products (ESPs) are good investigation targets for studying the relationship between a host and its parasite. OBJECTIVES We aimed to profile, identify, and characterise the proteins in the ESPs of A. cantonensis young adults. METHODS The ESPs of young adult worms were collected from culture medium after incubation ranging from 24 to 96 h. Proteomic and bioinformatics analyses were performed to characterise the ESPs. FINDINGS A total of 51 spots were identified, and the highly expressed proteins included two protein disulphide isomerases, one calreticulin, and three uncharacterised proteins. Subsequently, approximately 254 proteins were identified in the ESPs of A. cantonensis young adults via liquid chromatography-mass spectrometry (LC-MS/MS) analysis, and these were further classified according to their characteristics and biological functions. Finally, we identified the immunoreactive proteins from a reference map of ESPs from A. cantonensis young adults. Approximately eight proteins were identified, including a protein disulphide isomerase, a putative aspartic protease, annexin, and five uncharacterised proteins. The study established and identified protein reference maps for the ESPs of A. cantonensis young adults. MAIN CONCLUSIONS The identified proteins may be potential targets for the development of diagnostic or therapeutic agents for human angiostrongyliasis.

Stimulation of IL-1ß and IL-6 through NF-κB and sonic hedgehog-dependent pathways in mouse astrocytes by excretory/secretory products of fifth-stage larval Angiostrongylus cantonensis.

BACKGROUND: Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. Previous studies have shown that the Sonic hedgehog (Shh) signaling pathway may reduce cell apoptosis by inhibiting oxidative stress in A. cantonensis infection. In this study, we investigated the relationship between cytokine secretion and Shh pathway activation after treatment with excretory/secretory products (ESP) of fifth-stage larval A. cantonensis (L5). RESULTS: The results showed that IL-1ß and IL-6 levels in mouse astrocytes were increased. Moreover, ESP stimulated the protein expression of Shh pathway molecules, including Shh, Ptch, Smo and Gli-1, and induced IL-1ß and IL-6 secretion. The transcription factor nuclear factor-κB (NF-κB) plays an important role in inflammation, and it regulates the expression of proinflammatory genes, including cytokines and chemokines, such as IL-1ß and TNF-α. After ESP treatment, NF-κB induced IL-1ß and IL-6 secretion in astrocytes by activating the Shh signaling pathway. CONCLUSIONS: Overall, the data presented in this study showed that ESP of fifth-stage larval A. cantonensis stimulates astrocyte activation and cytokine generation through NF-κB and the Shh signaling pathway.

Molecular Identification of Diphyllobothrium latum from a Pediatric Case in Taiwan.

Human diphyllobothriasis is a parasitic disease caused by ingestion of larvae (plerocercoids) in raw or undercooked fish and commonly found in temperate areas. Rare cases were reported in tropical or subtropical areas especially in children. The first documented case of pediatric diphyllobothriasis in Taiwan had been reported 11 years ago. Here, we report another 8-year-old girl case who presented with a live noodle-like worm hanging down from her anus, with no other detectable symptoms. We pulled the worm out and found the strobila being 260 cm in length. Examination of gravid proglottids showed that they were wider than their lengths, containing an ovoid cirrus sac in the anterior side and the rosette-shaped uterus. Eggs extracted from the uterus were ovoid and operculated. Diphyllobothrium latum was confirmed by molecular analysis of the mitochondrial DNA cytochrome c oxidase subunit 1 (cox1) gene. The girl was treated with a single oral dose of praziquantel, and no eggs or proglottids were observed from her stool in the subsequent 3 months. The reemergence of human diphyllobothriasis in non-endemic countries is probably due to prevalent habit of eating imported raw fish from endemic areas. This pediatric case raised our concern that human diphyllobothriasis is likely underestimated because of unremarkable symptoms.

Activation of Sonic Hedgehog Leads to Survival Enhancement of Astrocytes via the GRP78-Dependent Pathway in Mice Infected with Angiostrongylus cantonensis.

Angiostrongylus cantonensis infection may cause elevation of ROS and antioxidants in the CSF of infected mice. Astrocytes may protect the surrounding neurons from oxidative stress-induced cell death by secreting Sonic hedgehog (Shh) via the PI3-K/AKT/Bcl-2 pathway. This study was conducted to determine the role of the Shh signaling pathway in A. cantonensis-infected BABL/c mice by coculturing astrocytes with living fifth-stage larvae or soluble antigens. The Shh pathway was activated with corresponding increases in the level of the Shh. Glial fibrillary acidic protein (GFAP) and Shh were increased in astrocyte cocultured with living fifth-stage larvae or soluble antigens. The survival of astrocytes pretreated with Shh was significantly elevated in cocultures with the antigens but reduced by its inhibitor cyclopamine. The expression of GRP78 and Bcl-2 was significantly higher in astrocytes pretreated with recombinant Shh. These findings suggest that the expression of Shh may inhibit cell death by activating Bcl-2 through a GRP78-dependent pathway.

Identification and characterization of an asparaginyl endopeptidase from Angiostrongylus cantonensis.

Asparaginyl endopeptidase, also known as legumain, is a family of cysteine proteases in many organisms. In this study, an asparaginyl endopeptidase (Ac-AEP) was identified from the cDNA library of Angiostrongylus cantonensis. The full-length of Ac-AEP was determined to be 1,472 bp with an open reading frame of 1,341 bp encoding a putative protein with 446 amino acids. This putative protein was determined to have 37-65% identity in the amino acid sequences of the asparaginyl endopeptidases of other parasitic helminths. By real-time quantitative PCR analysis, Ac-AEP was revealed to be more abundantly expressed in the female adult worms than in other development stages. A recombinant asparaginyl endopeptidase (rAc-AEP) was then produced by a Pichia pastoris expression system. Posttranslational modification was shown to occur via N-linked glycosylation in this recombinant enzyme. The proteolytic activity of rAc-AEP was inhibited by iodoacetamide but not affected by E64, pepatain A, AEBSF, and EDTA. Moreover, the purified rAc-AEP was recognized by IgG in serum samples from BALB/c or ICR mice with A. cantonensis infection and patients with eosinophilic meningitis. These findings indicate that the rAc-AEP may have the potential for detecting A. cantonensis infection.

Magnetotransport in an aluminum thin film on a GaAs substrate grown by molecular beam epitaxy.

Magnetotransport measurements are performed on an aluminum thin film grown on a GaAs substrate. A crossover from electron- to hole-dominant transport can be inferred from both longitudinal resistivity and Hall resistivity with increasing the perpendicular magnetic field B. Also, phenomena of localization effects can be seen at low B. By analyzing the zero-field resistivity as a function of temperature T, we show the importance of surface scattering in such a nanoscale film.