Determination of cyclic adenosine phosphate and protein content in dormant chlamydospore and nondormant chlamydospore of Arthrobotrys flagrans.

Arthrobotrys flagrans, a nematode-eating fungus, is an effective component of animal parasitic nematode biocontrol agents. In the dried formulation, the majority of spores are in an endogenous dormant state. This study focuses on dormant chlamydospore and nondormant chlamydospore of A. flagrans to investigate the differences in cyclic adenosine monophosphate (cAMP) and protein content between the two types of spores. cAMP and soluble proteins were extracted from the nondormant chlamydospore and dormant chlamydospore of two isolates of A. flagrans. The cAMP Direct Immunoassay Kit and Bradford protein concentration assay kit (Coomassie brilliant blue method) were used to detect the cAMP and protein content in two types of spores. Results showed that the content of cAMP in dormant spores of both isolates was significantly higher than that in nondormant spores (p < 0.05). The protein content of dormant spores in DH055 bacteria was significantly higher than that of nondormant spores (p < 0.05). In addition, the protein content of dormant spores of the SDH035 strain was slightly higher than that of nondormant spores, but the difference was not significant (p > 0.05). The results obtained in this study provide evidence for the biochemical mechanism of chlamydospore dormancy or the germination of the nematophagous fungus A. flagrans.

Chlamydospore dormancy and predatory activity of nematophagous fungus Duddingtonia flagrans.

The chlamydospores of Duddingtonia flagrans are an essential survival and reproductive structure and also an effective ingredient for the biocontrol of parasitic nematodes in livestock. In this study, entering and exiting dormancy conditions and predatory activity of the fungal chlamydospores were conducted. During this fungal growth process, the cultivation time is negatively correlated with spore germination rates. After the spores were processed by vacuum drying for 168 h, their germination rate dropped to 0.94%. In contrast, the percentage of living spores remained 54.82%, suggesting that the spores entered structural dormancy in the arid environment. Meanwhile, the efficacies of the spore against Haemonchus contortus larvae were 93.05% (0 h), 92.19% (16 h), 92.77% (96 h), and 86.45% (168 h), respectively. After dormant spores were stored at 4°C, -20°C, and 28°C (RH90 ~ 95%) for 7 days, their germination rate began to increase significantly (p < 0.05). For in vitro predation assay under the condition of 28°C (RH90 ~ 95%), the predation rate was significantly higher on the 7th day after incubation than that on the 3rd day (p < 0.05). During the period when spores were stored at room temperature for 8 months, their germination rate decreased in the first 5 months and then increased slowly to reach a peak in the 7th month. However, the reduction rate of H. contortus L3 in feces captured by spores remained above 71% for the first 7 months. These results will help us increase the end products yield and the quality of biological control of parasitic nematodes in livestock.

Microstructure characterization of different types of chlamydospores in Arthrobotrys flagrans.

The morphological and structural differences of different types of chlamydospore of Arthrobotrys flagrans, a nematophagous fungus, were studied under light microscope and electron microscope to provide a reference for the biological control of parasitic nematodiasis. In this study, A. flagrans isolate F088 dormant chlamydospore and nondormant chlamydospore were selected as the research objects. The structural differences of these spores were observed by optical microscopy through lactol cotton blue, Trypan blue, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) staining. FunXite -1, 4',6-diamidino-2-phenylindole, and calcofluor white staining were used to observe the metabolic activity, cell wall, and nucleus differences of the two types of spores under fluorescence microscope. Ultrastructure of the two kinds of spores was observed using scanning electron microscope (SEM) and transmission electron microscope (TEM). Since lacto phenol cotton blue, trypan blue staining cannot distinguish dormant spores from dead spores, MTT assay was performed. Fluorescence microscopy observation showed that the cytoplasmic metabolic activity of nondormant spores was stronger than that of dormant spores. The nucleus of dormant spores was bright blue, and their fluorescence was stronger than that of nondormant spores. The cell wall of nondormant spores produced stronger yellow-green fluorescence than that of dormant spores. Ultrastructural observation showed that there were globular protuberances on the surface of the two types of spores but with no significant difference between them. The inner wall of dormant spore possesses a thick zona pellucida with high electron density which was significantly thicker than that of nondormant spores, and their cytoplasm is also changed. In this study, the microstructure characteristics of dormant and nondormant chlamydospores of A. flagrans fungi were preliminarily clarified, suggesting that the state of cell wall and intracellular materials were changed after spores entered to dormancy.

Anti-osteosarcoma effect of antiserum against cross antigen TPD52 between osteosarcoma and Trichinella spiralis.

BACKGROUND: Trichinella spiralis (T. spiralis) is a parasite occurring worldwide that has been proven to have antitumour ability. However, studies on the antitumour effects of cross antigens between the tumour and T. spiralis or antibodies against cross antigens between tumours and T. spiralis are rare. METHODS: To study the role of cross antigens between osteosarcoma and T. spiralis, we first screened the cDNA expression library of T. spiralis muscle larvae to obtain the cross antigen gene tumour protein D52 (TPD52), and prepared fusion protein TPD52 and its antiserum. The anti-osteosarcoma effect of the anti-TPD52 antiserum was studied using cell proliferation and cytotoxicity assays as well as in vivo animal models; preliminary data on the mechanism were obtained using western blot and immunohistochemistry analyses. RESULTS: Our results indicated that TPD52 was mainly localized in the cytoplasm of MG-63 cells. Anti-TPD52 antiserum inhibited the proliferation of MG-63 cells and the growth of osteosarcoma in a dose-dependent manner. The tumour inhibition rate in the 100 µg treatment group was 61.95%. Enzyme-linked immunosorbent assay showed that injection of anti-TPD52 antiserum increased the serum levels of IFN-γ, TNF-α, and IL-12 in nude mice. Haematoxylin and eosin staining showed that anti-TPD52 antiserum did not cause significant pathological damage. Apoptosis of osteosarcoma cells was induced by anti-TPD52 antiserum in vivo and in vitro. CONCLUSIONS: Anti-TPD52 antiserum exerts an anti-osteosarcoma effect by inducing apoptosis without causing histopathological damage.

New Constituents from the Low Polar Fraction of the Fruits of Crataegus dahurica and Their Anti-Inflammatory Activity in RAW264.7 Cells.

The fruit of Crataegus dahurica Koehne was used to treat the disease of infantile indigestion and dyspepsia as an ethnic medicine and food. As a continuous work on finding the active constituents from the edible herbs, four new biphenyl derivatives (1-4), together with two known compounds (5 and 6), were obtained from the petroleum ether fraction of the fruits of C. dahurica. Their structures were determined by the extensive 1D and 2D NMR spectra and HR-MS spectrometry. Furthermore, the anti-inflammatory activities of all the isolated compounds were investigated, in which compound 4 showed moderately inhibitory effects on NO production in RAW264.7 cells without inducing cytotoxicity.

Morphological variability, molecular phylogeny, and biological characteristics of the nematophagous fungus Duddingtonia flagrans.

This study aims to investigate the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Duddingtonia flagrans isolates. We isolated 13 isolates of D. flagrans and found features that have never been reported before, such as two to three septa incluing club-shaped conidia. Meanwhile, we conducted molecular phylogenetic analysis of the seven isolates and tested the radical growth of the isolates under different pH values, temperatures, and media. The capturing ability against infective larvae (L3) of Cooperia spp. in yak was detected in vitro. Finally, one isolate was selected for scanning electron microscopy (SEM) to investigate the trap formation process. The fungal sequence was obtained and submitted to GenBank (Accession no. KY288614.1, KU881774.1, KP257593.1, KY419119.1, MF488979.1, MF488980.1, and MF488981.1), and the tested isolates were identified as D. flagrans. Except for three isolates, the radial growth of the other isolates on 2% corn meal agar and 2% water agar exhibited faster growth than on other media. The fungus could not grow at 10 and 40°C but grew within 11 to 30°C. Moreover, it did not grow at pH 1-3 and 13-14, but instead at pH 4-12. In the in vitro experimental, L3s were reduced by 94.36%, 88.15%, and 91.04% for SDH035, DH055, and F088, respectively. SEM results showed that at 8 hr post addition of nematodes, some of the latter were captured. In the later stages of the interaction of the fungus with nematodes, a large number of chlamydospores were produced, especially on the predation trap. Results of the present study provided information about the molecular phylogenetic analysis, morphological variability, nematode-capturing ability, and other biological properties of Chinese Arthrobotrys flagrans isolates before administering them for biocontrol.

Induction and Suppression of Innate Antiviral Responses by Hepatitis A Virus.

Hepatitis A virus (HAV) belongs to the family Picornaviridae. It is the pathogen of acute viral hepatitis caused by fecal-oral transmission. RNA viruses are sensed by pathogen-associated pattern recognition receptors (PRRs) such as Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA5). PRR activation leads to production of type 1 interferon (IFN-α/ß), serving as the first line of defense against viruses. However, HAV has developed various strategies to compromise the innate immune system and promote viral propagation within the host cells. The long coevolution of HAV in hosts has prompted the development of effective immune antagonism strategies that actively fight against host antiviral responses. Proteases encoded by HAV can cleave the mitochondrial antiviral signaling protein (MAVS, also known as IPS-1, VISA, or Cardif), TIR domain- containing adaptor inducing IFN-ß (TRIF, also known as TICAM-1) and nuclear factor-κB (NF-κB) essential modulator (NEMO), which are key adaptor proteins in RIG-I-like receptor (RLR), TLR3 and NF-κB signaling, respectively. In this mini-review, we summarize all the recent progress on the interaction between HAV and the host, especially focusing on how HAV abrogates the antiviral effects of the innate immune system.

Decrease in phosphorylated ERK indicates the therapeutic efficacy of a clinical PI3Kα-selective inhibitor CYH33 in breast cancer.

PI3Ks are frequently hyper-activated in breast cancer and targeting PI3Kα has exhibited promising but variable response in preclinical and clinical settings. CYH33 is a novel PI3Kα-selective inhibitor in phase I clinical trial. We investigated the efficacy of CYH33 against breast cancer and explored potential predictive biomarkers. CYH33 potently restrained tumor growth in mice bearing human breast cancer cell xenografts and in R26-Pik3caH1047R;MMTV-Cre transgenic mice. CYH33 significantly inhibited proliferation of a panel of human breast cancer cells, while diversity in sensitivity has been observed. Cells harboring activating PIK3CA mutation, amplified HER2 were more responsive to CYH33 than their counterparts. Besides, cells in HER2-enriched or luminal subtype were more sensitive to CYH33 than basal-like breast cancer. Sensitivity to CYH33 has been further revealed to be associated with induction of G1 phase arrest and simultaneous inhibition of Akt and ERK. Sensitivity of patient-derived xenograft to CYH33 was also positively correlated with decrease in phosphorylated ERK. Taken together, CYH33 is a promising PI3Kα inhibitor for breast cancer treatment and decrease in ERK phosphorylation may indicate its efficacy, which provides useful clues for rational design of the ongoing clinical trials.

In Vitro, in Vivo, and Interaction Studies of Nematophagous Fungus Arthrobotrys thaumasia ( Monacrosporium thaumasium) with the Larvae of Trichostrongylides of Sheep.

It is important to isolate potential candidates from the local isolates of nematophagous fungi and to investigate interaction between the fungal strains and gastrointestinal nematodes for the biological control of parasitic nematodes in livestock. In the present study, we assessed the in vitro predatory activity and the viability of isolates of Arthrobotrys thaumasia ( Monacrosporium thaumasium) after passage through the gastrointestinal tract of sheep. The predatory process of a representative isolate selected against the larvae of trichostrongylids was prepared with a scanning electron microscope (SEM). In vitro experiments tested the ability of 9 native isolates of A. thaumasia to prey on larvae of feces of sheep infected with natural mixed nematodes ( Haemonchus contortus, Trichostongylus colubriformis, Marshallagia mongolica). These isolates of A. thaumasia decreased infectivity of third stage infective larvae (L3) by 75.54-99.97%; 7 isolates decreased infectivity by more than 90%. In vivo experiments also demonstrated significant reductions of L3 numbers in the feces treated with the 9 isolates after passing through the gastrointestinal tract of sheep, and these decreases ranged from 51.68 to 88.16%. The isolates tested were re-isolated in 5-g sub-samples of feces from sheep in each treatment group, indicating that these isolates had the capacity to prey upon larvae of trichostrongylids after the passage through gastrointestinal tract. SEM shows that at 6 hr after the larvae were added, including the second stage larvae (L2) and L3 of trichostrongylids, the isolate NBS 005 caught them; at 8 hr after being caught L2 was penetrated by the fungus while penetration of L3 occurred at 12 hr; at 78 hr post-capture L2 was completely destroyed by the fungus while complete digestion of L3 occurred at 84 hr.

In vitro and in vivo studies of nematophagous fungi Arthrobotrys musiformis and Arthrobotrys robusta against the larvae of the trichostrongylides.

Six isolates of Arthrobotrys musiformis and five isolates of Arthrobotrys robusta were assessed in in vitro test regarding the capacity of prey larvae of the natural mixed trichostrongylides. In 5 isolates of A. robusta, the decrease percentage of infective larvae (L3) of trichostrongylides ranged from 97.71%-99.98% and for the isolates of A. musiformis, 5 isolates ranged from 97.99%-99.95% and only NF015 isolate 60.72%. In the following, the isolate (NPS045) of A. musiformis was selected to assess its excretion time in feces after oral administration of goats. Regarding L3 reduction rate, results demonstrated by NPS045 at each time point after fungal administration were 31.65% (12 h), 51.25% (24 h), 41.07% (48 h), 6.44% (72 h), 0% (96 h) and (120 h) (p<0.05) respectively, when compared to the control group. In the plates of the treated groups, the presence of the isolate (NPS045) was detected in samples at 12, 24 and 48 h after the fungus dose and 72 h later was not done. All native isolates of nematophagous fungi, including 6 isolates of A. musiformis and 5 isolates of A. robusta were assessed in vivo regarding the capacity of supporting the passage through goat gastrointestinal tract. The 3 isolates of A. musiformis could be able to pass through the digestive tract of goats without complete loss of ability of preying larvae of trichostrongylides in feces and their efficacies ranged from 47.60% to 55.93%. The two isolates of A. robusta survived the passage and the percentage reduction of L3 in feces were 41.96% and 66.97%, respectively. The remaining isolates were negative for both the efficacy of L3 reduction and the fungal examination in feces. In this study, the native isolates whose efficacies are good in vivo test have preliminarily demonstrated to be potential for the biological control of small ruminant parasite.

In vitro predatory activity of Arthrobotrys oligospora and after passing through gastrointestinal tract of small ruminants on infective larvae of trichostrongylides.

In vitro predatory activity of 157 native isolates of Arthrobotrys oligospora from China on larvae of trichostrongylides (Trichostrongylus colubriformis and Haemonchus contortus) in feces of sheep were assessed. The results showed that 135 of tested isolates of A. oligospora reduced the development of trichostrongylide larvae in feces by 90-99.99%, 11 isolates by 80-89.46% and 11 isolates by 14.58-78.82%. To understand their capacity of passing through gastrointestinal tract of sheep, 50 native isolates of A. oligospora were selected and assessed in sheep. Among these isolates, 16 isolates significantly reduced the number of larvae developing in the feces (P < 0.05); their percentage reduction of L3 ranged from 42.87% to 99.51% and the isolates tested were harvested in 5 g sub-samples of from sheep in each treatment group, indicating that these isolates had the capacity of preying larvae of trichostrongylides after the passage through gastrointestinal tract of sheep. The remaining isolates of A. oligospora were not able to survive after passage through gastrointestinal tract of sheep. In the following, the 16 isolates that presented more or less viability after sheep gastrointestinal passage were selected and assessed in goats. The results showed that the 11 isolates out of them could be able to pass through the digestive tract of goats without loss of ability of preying larvae of trichostrongylides in feces and their efficacies ranged from 53.88% to 94.28%, and that the isolates tested were harvested in 5 g sub-samples of feces from goats in each treatment group. In the current study, these isolates which demonstrated outstanding properties in vitro and could survive in the passage through the alimentary tract of sheep and goat should be potential candidates as a possible feed additive.

In vitro and in vivo studies of the native isolates of nematophagous fungi from China against the larvae of trichostrongylides.

To screen potential nematophagous fungi candidates for the biological control of parasitic nematodes in livestock, in vitro and in vivo studies of the native isolates of nematophagous fungi against the larvae of trichostrongylides were conducted. The in vitro predatory activity of 16 native nematophagous fungal isolates on the larvae of trichostrongylides in sheep feces was assessed. In the ten isolates of Duddingtonia flagrans, the reduction percentage for the infective larvae (L3) of Trichostrongylus colubriformis ranged from 57.21 to 99.83%, and that of Haemonchus contortus ranged from 62.12 to 99.88%. The analysis of the same assay on five isolates of Arthrobotrys superba and one isolate of A. cookedickinson (Monacrosporium cystosporum) showed comparable results with those for D. flagrans. To determine the excretion time of fungal isolates in feces after oral administration, D. flagrans (SDH035) were studied in vivo in sheep and rabbits. Results showed that the tested fungal isolates existed in sheep feces from 12 to 72 h after fungal treatment, and the fungal excretion in rabbit feces occurred at 4 h, reached a peak at 10 h, and declined gradually 18 h after oral administration. All the native fungal isolates were assessed after passing through the gastrointestinal tract of sheep. Treatment with isolates of D. flagrans significantly reduced the number of developing larvae in the feces, and the efficacies ranged from 55.15 to 98.82%. One out of the five isolates of A. superba and A. cookedickinson (BS002) survived after passing through the gastrointestinal tract, and the L3 reduction rates were 83.79 and 81.33%, respectively. Results of the present study provide information about the in vitro predatory activity of nematophagous fungi from China on the L3 of trichostrongylides and their ability to pass through the gastrointestinal tract before administering them for biocontrol.

Screening of different sample types associated with sheep and cattle for the presence of nematophagous fungi in China.

A total of 1502 samples, including feces of sheep (793) and cattle (348), pasture soil (118), dung compost (147) and barn soil (96), were examined between October 2012 and August 2014 to discover potential strains of nematophagous fungi for the biological control of livestock-parasitic nematodes. These samples were collected from 87 sites located in 48 counties of 20 provinces (autonomous regions/municipalities) of China. Fungi were identified down to a species level. Four hundred and seventy-seven isolates, which were distributed in 8 genera and 28 taxa, were identified as nematophagous fungi. Nematode-trapping fungi included 17 species and one unidentified species of Arthrobotrys, two of Dactylella, Drechslerella dactyloides, and Duddingtonia flagrans. Five identified species and two unidentified species of endoparasitic fungi were isolated. The predominant species from all regions were Arthrobotrys oligospora, followed by Arthrobotrys musiformis, Arthrobotrys (Monacrosporium) thaumasiun, and Arthrobotrys (Monacrosporium) microscaphoides. Species with adhesive networks were the most frequently isolated. Among the endoparasitic fungi, Podocrella harposporifera (Harposporium anguillulae) was the most common species, followed by Harposporium lilliputanum and Harposporium arcuatum. Based on Shannon diversity index, the diversity levels of nematophagous fungi were relatively higher in samples associated with cattle, barn soil, and subtropical monsoon climate zone. Three species isolated from this study, namely, Duddingtonia flagrans, Arthrobotrys salina (Monacrosporium salinum), and Arthrobotrys oligospora var. sarmatica, are newly recorded in China, and 20 species (including one unidentified species) are newly recorded in sheep and cattle barn soils worldwide.

Isolation and Characterization of China Isolates of Duddingtonia flagrans, a Candidate of the Nematophagous Fungi for Biocontrol of Animal Parasitic Nematodes.

The nematophagous fungus Duddingtonia flagrans has been investigated as a biological agent for the control of gastrointestinal nematodes infecting domestic animals in other countries. However, D. flagrans has not been detected in China. In this study 1,135 samples were examined from 2012 to 2014; 4 D. flagrans isolates (SDH 035, SDH 091, SFH 089, SFG 170) were obtained from the feces of domestic animals and dung compost. The 4 isolates were then characterized morphologically. The SDH 035 strain was characterized by sequencing the ITS1-5.8S rDNA-ITS2 region. A BLAST search showed that the SDH 035 strain (GenBank KP257593) was 100% identical to Arthrobotrys flagrans (AF106520) and was identified as D. flagrans. The morphological plasticity of the isolated strain and the interaction of this strain with the nematode targets were observed by subjecting the infected trichostrongylide L3 to scanning electron microscopy. At 6 and 8 hr after trichostrongylide L(3) was added, hyphal ramifications were observed and L(3) were captured, respectively. Scanning electron micrographs were obtained at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hr, where 0 is the time when trichostrongylide L(3) were first captured by the fungus. The details of the capture process by the fungus are also described. Chlamydospores were observed in the body of L(3) in the late stage of digestion. A sticky substance and bacteria could be observed in contact areas between predation structures and nematode cuticle.

Isolation, identification, and characterization of the nematophagous fungus Monacrosporium salinum from China.

Nematophagous fungi are considered to have the best potential as biological agents for the control of gastrointestinal nematodes in domestic animals. However, relatively few studies have been conducted with the genus Monacrosporium, especially with strains native to China. In the present study, we isolated and identified nematophagous fungi from fresh sheep feces. A pure fungal strain was molecularly characterized, and its nematophagous activity was evaluated. The morphological plasticity of the isolated strain, as well as its interaction with the nematode targets, was observed by scanning electron microscopy of the infected Trichostrongylus colubriformis L3 and the free-living nematode Caenorhabditis elegans. Three isolated fungal strains from the 30 fresh fecal samples of sheep from Inner Mongolia, China exhibited predatory activity; however, only a single strain was successfully purified (SF 0459). The SF 0459 strain was characterized by morphological analysis of its conidia and sequencing of its ITS1-5.8S rDNA-ITS2 region. This strain was identified to be Monacrosporium salinum (GenBank ID: KP036623). Nematophagous fungus helper bacteria were found at the interaction points between fungi and nematodes. The percentage of live T. colubriformis L3 was reduced by 83.79-88.69% based on the in vitro assay.