Immune modulation of goat monocytes by Fasciola gigantica Legumain-1 protein (Fg-LGMN-1).

Legumains belonging to C_13 peptidase family of proteins, and are ubiquitously disseminated among all vertebrate and invertebrate organisms, and have been implicated in innumerable biological and cellular functionality. Herein, we characterized and evaluated immunoregulatory characteristics of Legumain-1 from Fasciola gigantica (Fg-LGMN-1) during its interaction with host immune cells. The isopropyl-ß-d-thiogalactopyranoside (IPTG) stimulated RFg-LGMN-1 protein was positively detected by rat serum containing anti-RFg-LGMN-1 polyclonal antibodies. Furthermore, the uptake of RFg-LGMN-1 by goat monocytes was successfully confirmed using Immunofluorescence Assay (IFA). The immunohistochemical analysis revealed the native localization of LGMN-1 protein on the periphery and internal structures such as suckers, pharynx, and genital pore of the adult parasite, thereby validating its presence in excretory-secretory (ES) products of F. gigantica. The RFg-LGMN-1 co-incubated with concanavalin-A (Con-A) stimulated the increase of interleukin 2 (IL-2), IL-10, and IL-17 in monocytes derived from peripheral blood mononuclear cells (PBMCs) in the concentration-dependent manner. However, the IL-4 cytokine in response to the RFg-LGMN-1 protein declined. These results illuminated the role of LGMN-1 during the parasite-host interface. Our findings elaborated additional evidence that Legumain protein play a role in the manipulating host immune responses during parasite infections. However, further evaluation of RFg-LGMN-1 protein in context of its immunomodulatory roles should be conducted to enhance our understandings of the mechanisms employed by F. gigantica to evade host immune responses.

[Isolation and identification of chemical constituents from aerial parts of Glycyrrhiza uralensis].

The present study investigated the chemical constituents from the aerial parts of Glycyrrhiza uralensis. The ethanol extract of the aerial parts of G. uralensis was separated and purified by different column chromatographies such as macroporous resin, silica gel, and Sephadex LH-20, and through preparative HPLC and recrystallization. Thirteen compounds were isolated and identified as(2S)-6-[(Z)-3-hydroxymethyl-2-butenyl]-5,7,3'-trihydroxy-4'-methoxy-dihydroflavanone(1),(2S)-8-[(E)-3-hydroxymethyl-2-butenyl]-5,7,3',5'-tetrahydroxy-dihydroflavanone(2), α,α'-dihydro-5,4'-dihydroxy-3-acetoxy-2-isopentenylstilbene(3), 6-prenylquercetin(4), 6-prenylquercetin-3-methyl ether(5), formononetin(6), 3,3'-dimethylquercetin(7), chrysoeriol(8), diosmetin(9),(10E,12Z,14E)-9,16-dioxooctadec-10,12,14-trienoic acid(10), 5,7,3',4'-tetrahydroxy-6-prenyl-dihydroflavanone(11), naringenin(12), dibutylphthalate(13). Compounds 1-3 are new compounds, and compounds 10 and 13 are isolated from aerial parts of this plant for the first time.

[Effects of pH value on stachydrine biosynthesis of hydroponic Leonurus japonicus and its physiological mechanism].

The present study explored the physiological mechanism of the effects of different pH treatments on the growth, physiological characteristics, and stachydrine biosynthesis of Leonurus japonicus to provide references for the cultivation and quality control of L. japonicus. Under hydroponic conditions, different pH treatments(pH 5,6,7,8) were set up. The growth, physiology, and the content of stachydrine and total alkaloids of L. japonicus, as well as the content of key intermediate products in stachydrine biosynthesis pathway(i.e., pyruvic acid, α-ketoglutaric acid, glutamic acid, and ornithine) were monitored to explore the physiological mechanism of the effects of pH on the growth and active components of L. japonicus. The results showed that L. japonicus. could grow normally in the pH 5-8 solution. The pH treatment of neutral acidity was more conducive to the accumulation of photosynthetic pigments and the increase in soluble protein in leaves of L. japonicus. to promote its growth and yield. However, since stachydrine is a nitrogen-containing pyrrolidine alkaloid, its synthesis involves the two key rate-limiting steps of nitrogen addition: reductive ammoniation reaction and Schiff base formation reaction. High pH treatments promote the synthesis and accumulation of substrates and products of the above two reactions, indicating that the alkaline environment can promote the nitrogen addition reaction, thereby promoting the biosynthesis and accumulation of stachydrine.

Molecular detection and subtype distribution of Blastocystis in farmed pigs in southern China.

Blastocystis is one of the most common causative agents of intestinal diseases, which can cause enteric diseases in animals and humans. However, limited data is available on the prevalence or subtypes of Blastocystis infections in farmed pigs in southern China. In this study, a total of 396 fecal samples were collected from farmed pigs in three provinces in southern China in 2016, and screened for Blastocystis by PCR amplification of the small subunit rRNA (SSU rRNA) gene fragment. One hundred and seventy (42.93%) of the examined fecal samples were detected Blastocystis-positive, and two known zoonotic subtypes ST1 and ST5 were identified, with ST5 being the predominate subtype. Moreover, gender, age and region were considered as risk factors that associated with Blastocystis infection in farmed pigs. The present study revealed the prevalence and subtypes of Blastocystis infections in farmed pigs in southern China, which provided essential data for the control of Blastocystis infections in pigs, other animals and humans in China.

Toxoplasma gondii induces metabolic disturbances in the hippocampus of BALB/c mice.

Toxoplasma gondii can cross the blood-brain barrier and infect different regions of the brain including the hippocampus. In the present study, we examined the impact of Toxoplasma gondii infection on the metabolism of the hippocampus of female BALB/c mice compared to control mice using ultra-high-performance liquid chromatography-tandem mass spectrometry. Multivariate analysis revealed significant differences between infected and control hippocampi and identified 25, 82, and 105 differential metabolites (DMs) in the infected hippocampi at 7, 14, and 21 days post-infection (dpi), respectively. One DM (sphingosyl-phosphocholine in the sphingolipid metabolism pathway) and 11 dysregulated pathways were detected at all time points post-infection, suggesting their important roles in the neuropathogenesis of T. gondii infection. These pathways were related to neural activity, such as inflammatory mediator regulation of TRP channels, retrograde endocannabinoid signaling, and arachidonic acid metabolism. Weighted correlation network analysis and receiver operating characteristic analysis identified 33 metabolites significantly associated with T. gondii infection in the hippocampus, and 30 of these were deemed as potential biomarkers for T. gondii infection. This study provides, for the first time, a global view of the metabolic perturbations that occur in the mouse hippocampus during T. gondii infection. The potential relevance of the identified metabolites and pathways to the pathogenesis of cognitive impairment and psychiatric disorders are discussed.

[Main chemical constituents in aerial parts of Glycyrrhiza uralensis by UPLC-Q-Exactive Orbitrap-MS].

Chemical constituents from aerial parts of Glycyrrhiza uralensis were analyzed and identified using ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometry(UPLC-Q-Exactive Orbitrap-MS). The chromatographic column of Waters Acquity UPLC BEH-C_(18)(2.1 mm×100 mm, 1.7 µm) was adopted, with acetonitrile-water(0.5% formic acid) as mobile phase at a flow rate of 0.2 mL·min~(-1). Data was collected in positive and negative modes of electrospray ionization(ESI). A total of 55 compounds, including 42 flavonoids, 9 stilbenes, 2 coumarins, 1 lignin and 1 phenolic acid, which were characterized in the aerial parts of G. uralensis based on accurate molecular mass information of molecular and product ions provided by UPLC-Q-Exactive Orbitrap-MS based on comparison with standard substances and references. It is an effective and accurate method to provide chemical information of constituents in aerial parts of G. uralensis, and can provide a reference for further study on pharmacodynamic material basis and resources development and utilization.

First report of Cryptosporidium spp. infection and risk factors in black-boned goats and black-boned sheep in China.

Cryptosporidium is an opportunistic protozoan parasite that can inhabit in the gastrointestinal tract of various hosts. Cryptosporidium infection in black-boned goats and black-boned sheep may pose a threat to the survival and productivity, causing considerable economic losses to the livestock industry. However, it is yet to know whether black-boned goats and black-boned sheep in China are infected with Cryptosporidium. Thus, the objective of the present study was to investigate the prevalence and associated risk factors of Cryptosporidium infection in black-boned goats and black-boned sheep in Yunnan province, China. A total of 590 fecal samples were obtained from black-boned goats and black-boned sheep from five counties in Yunnan province, and the prevalence and species distribution of Cryptosporidium were determined by amplification of the 18S rDNA fragment using the nested PCR. The overall Cryptosporidium prevalence was 13.2% (78/590), with 18.0% (55/305) in black-boned goats and 8.1% (23/285) in black-boned sheep. The age and sampling site were identified as main factors that result in significant differences in Cryptosporidium prevalence. Three species, namely C. muris, C. xiaoi, and C. ubiquitum, were identified in black-boned goats and black-boned sheep in the present study, with C. muris (46/78) as the predominant species. This is the first report of Cryptosporidium infection in black-boned goats and black-boned sheep in China, and the findings will facilitate better understanding, prevention, and control of Cryptosporidium infection in black-boned goats and black-boned sheep in China.

Proteomic analysis of Fasciola gigantica excretory and secretory products (FgESPs) interacting with buffalo serum of different infection periods by shotgun LC-MS/MS.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is an important zoonotic disease in the world. It affects livestock, especially for sheep and cattle, causing major economic loss due to morbidity and mortality. Although the excretory and secretory products (ESPs) of F. hepatica have been relatively well studied, little is known about the interaction between the ESP and host, and the mechanism of the key proteins involved in interaction. In this study, buffaloes were infected by Fasciola gigantica, and infection serum was collected at three different periods (42dpi, 70dpi, and 98dpi). The interaction proteins were pulled down with three different period serum by Co-IP assay, respectively, and then identified by LC-MS/MS analysis. A number of proteins were identified; some of them related to the biological function of the parasite, while most of them the functions were unknown. For the annotated proteins, 13, 5, and 7 proteins were pulled down by the infected serum in 42dpi, 70dpi, and 98dpi, respectively, and 18 proteins could be detected in all three periods. Among them, 13 belong to the cathepsin family, 4 proteins related to glutathione S-transferase, and 3 proteins are calcium-binding protein; other proteins related to catalytic activity and cellular process. This study could provide new insights into the central role played by ESPs in the protection of F. gigantica from the host immune response. At the same time, our research provided material for further studies about the interaction between F. gigantica and host.

[Investigation and analysis on difference of cultivation technique situation of Salvia miltiorrhiza].

Salvia miltiorrhiza is one of the commonly used bulk medicinal materials, which has significant effect on cardiovascular disease, and are heavy demanded in Asia, Europe, North America, Russia and Africa. Consequently, increasing the yield and quality of S. miltiorrhiza has become a major concern worldwide. With the current wild resources of S. miltiorrhiza gradually decreasing, cultivated products occupy most of the markets. However, the cultivation area is widely distributed and the cultivation techniques is different, which lead to the quality and yield of S. miltiorrhiza in consistent. This paper combined visiting survey with document analysis to carry out the cultivation situation of S. miltiorrhiza in main cultivation areas of Shandong, Henan, Sichuan, Shanxi and Hebei provinces. There exist big differences of the ecological environment, mode of cultivation, fertilization, pest control, harvesting processing among the producing areas. We should carry on the ecological suitability zoning analysis and suitable cultivation of each area study to form a pattern of high quality and high yield for the sustainable development of S. miltiorrhiza cultivation.

Identification of two novel host proteins interacting with Toxoplasma gondii 14-3-3 protein by yeast two-hybrid system.

Toxoplasma gondii deploys many effector proteins in order to hijack and manipulate host cell signaling pathways, allowing parasite colonization, subversion of immune responses, and disease progression. T. gondii effector protein 14-3-3 (Tg14-3-3) promotes parasite dissemination inside the body, by enhancing the migratory ability of infected microglia and dendritic cells. Understanding both the mechanism of action and the host targets of Tg14-3-3 effector is important because of their importance to the parasite's virulence. The aim of the present study was to explore the function of Tg14-3-3 by utilizing the yeast two-hybrid system (Y2HS) to identify novel Tg14-3-3 interactors/substrates in host cells. A human cDNA library was screened using Tg14-3-3 as the bait. Tg14-3-3 (RH strain, Type I) was cloned into the pGBKT7 vector and expressed in the Y2HGold yeast strain. The bait protein expression was validated by Western blotting analysis, auto-activation, and toxicity investigation compared with control (Y2HGold yeast strain transformed with empty pGBKT7 vector). Two positive Tg14-3-3 interactors identified by this screening, hCG1821272 and eIF5B (eukaryotic translation initiation factor 5B), were isolated and characterized. This approach made it possible to gain a better understanding of the function of Tg14-3-3 in regulating host proteins involved in key cellular processes, such as translational initiation and cell migration.

[Effects of different concentrations of nitrogen,phosphorus and potash on Glycyrrhiza uralensis and its relationship with soil element content].

With annual Glycyrrhiza uralensis seedlings as experimental material, using "3414" optimal regression design and applied fertilizer, through the sampling of G. uralensis at harvest, root fresh weight and content of active components were measured in Lanzhou, Bayan Nur city, Chifeng, Jiuquan. Combined with NPK content in soil, potted experiments were used to study the effects of different nitrogen and phosphorus ratios on the dry matter accumulation and accumulation of active components of G. uralensis. The results reported as follows: the optimum fertilizer treatment in Lanzhou, Bayan Nur city, Chifeng, Jiuquan was N1P2K1,N2P2K1,N1P1K2 and N2P1K2, respectively. The efforts of single fertilizer on the fresh root weight acted as parabolic type.There was no significant effect of fertilizer treatment on the accumulation of active components of G. uralensis. Furthermore, in terms of nitrogen and phosphorus, the type of fertilizers that restricted the growth of the region was the type of elements with lower content in the soil. The optimal fertilizer usage was in inverse proportion to content of elements in soil. When the content of phosphorus in soil was low, nitrogen fertilizer and potash fertilizer showed positive interaction with phosphorus fertilizer, whereas, they showed negative interaction.

[Identification of Taenia solium Insulin Receptor TsIR-1316 and Expression of Its Ligand Binding Domain].

Objective: To characterize the structure of insulin receptor of Taenia solium（TsIR-1316） and express its ligand binding domain (LBD). Methods: Primers for TsIR-1316 were designed according to the genomic data of T. solium, and the TsIR-1316 gene was amplified by PCR. The nucleotide and amino acid sequences of TsIR-1316 were aligned using BLASTN and BLASTP, and the putative signal peptide and structure domains were predicted. The LBD fragment of TsIR-1316 was cloned into the pET-30a（+） vector and expressed. The expressed proteins were purified, separated by SDS-PAGE and analyzed with Western blotting using cysticercus cellulosae-positive serum and TsIR-LBD-immunized rabbit serum. Results: The open reading frame of TsIR-1316 was 5 196 bp, encoded a protein of 1 732 amino acids which had a typical conserved domain of tyrosine kinase family, was 84% homologous with Echinococcus multilocularis, and had a "V"-shaped tertiary structure. As expected, SDS-PAGE showed that the expressed protein had a band at Mr 59 000. Western blotting showed that the recombinant protein had specific reactions with cysticercus cellulosae positive serum and TsIR-LBD immunized rabbit serum, resulting in a specific band at M(r) 59 000. Conclusion: The TsIR-1316 gene was successfully cloned and identified. The expressed protein of TsIR-1316 LBD can be recognized by cysticercus cellulosae positive serum, which suggests a good antigenicity of this protein.

[Effects of different concentrations of nitrogen and phosphorus on growth and active components of Salvia miltiorrhiza].

With annual Salvia miltiorrhiza seedlings as experimental material, using "3414" optimal regression design recommended by the Ministry of Agriculture and regularly watered with nutrient solution, through the dynamic sampling of S. miltiorrhiza in different growing stages, and the growth index, dry weight of plant root and content of active components were measured. The potted experiments were applied to study the effects of different nitrogen and phosphorus ratios on the growth, dry matter accumulation and accumulation of active components of S. miltiorrhiza, in order to explore a compatible fertilization method of nitrogen and phosphorus ratio that are suitable for production and quality of S. miltiorrhiza. The results reported as follows：①High concentrations of nitrogen fertilizer was beneficial to dry matter accumulation of S. miltiorrhiza aerial parts, and low concentration of nitrogen fertilizer transferred the dry matter accumulation to underground, and N1P1 could make the transfer ahead of time;②Regression analysis showed that in the early growth stage (before early July), we could use the nitrogen and phosphorus as basic fertilizer at a concentration of 1.521,0.355 g•L⁻¹ respectively to promote the growth of S. miltiorrhiza and at a concentration of 2.281,0.710 g•L⁻¹ respectively to promote the dry matter accumulation of root (after mid-August);③Five kinds of active components of S. miltiorrhiza decreased with the increase of nitrogen concentration, and increased with the increase of the concentration of phosphate fertilizer. Nitrogenous fertilizer, phosphate fertilizer in N-P=2∶3 ratio was more suitable for the accumulation of salvianolic acids, in N-P=1∶2 ratio was more suitable for the accumulation of tanshinone.

[Identification, Expression and Antigenicity Analysis of Serpin B6 of Taenia solium].

Objective: To identify and express serpin B6 of Taenia solium (Tsserpin B6) and explore its possible use as a diagnostic antigen. Methods: Primers for Tsserpin B6 were designed according to T. solium genome and transcriptome data. The Tsserpin B6 gene was amplified from the total RNA of T. solium cysticercus and subsequently analyzed by bioinformatics. Multiple amino acid sequence alignments of Tsserpin B6 and other parasites serpins were created using the Clustal X1.83. Phylogenetic analyses were performed using the MEGA 6.0. The recombinant expression vector pET-30a-Tsserpin B6 was constructed and expressed in E. coli strain BL21 （DE3）. The expressed proteins were purified, isolated by SDS-PAGE, and analyzed by Western blotting using pig serum infected with T. solium cysticerci. Results: The complete reading frame of Tsserpin B6 was 1 131 bp and encoded a protein of 376 amino acids. The encoded protein had a conservative reactive center loop and distinctive domains of NEEGAE and FTVDHPFLF, and harbored 9 potential linear B cell epitopes. The expressed products of Tsserpin B6 mainly existed as an inclusion body, and reacted with pig serum infected with T. solium, resulting in a specific band at the Mr 53 000. Conclusion: The Tsserpin B6 gene was successfully cloned, and its expressed products can be recognized by pig serum infected with T. solium.

[Stability analysis of allelopathic effects of Panax notoginseng on main crops by AMMI model].

This paper is aimed to study the differences of allelopathic effects of Panax notoginseng under different allelopathic chemicals resources and selection of appropriate rotation crops. The additive main effects and multiplicative interaction ( AMMI) model had been used to evaluate the stability of allelopathic effects of P. notoginseng on the varieties of corn, wheat and rice properly. The model could use not only to evaluate the stability of non-regional trial data but also explore the interaction between the rotation crop genotypes and donor substances more efficiently. Meanwhile, correspondence analysis can be used in the AMMI to evaluate genotype stability and donor substances. Ejingza No. 1 (g6) had stronger allelopathic effects with high stability, but Yunrui No. 1 (g9) which was appropriate rotation crop genotype, had weaker allelopathic effects with high stability. These findings will aid in choosing appropriate rotation crops and establishing proper rotation system.

[Influence of Different Growing Years on Accumulation of Flavonoids and Saponins in Astragali Radix].

OBJECTIVE: To study the effects of different growing years on the accumulation of flavonoids and saponins in Astragali Radix, in order to offer evidence to determine the most appropriate harvest time. METHODS: HPLC method was developed for the determination of the contents of flavonoids and saponins. RESULTS: The contents of flavonoids and saponins in different growing years of Astragali Radix existed obvious differences, the longer growing, the content was relatively higher. CONCLUSION: With the increase of growing years, the contents of flavonoids and saponins are roughly rising year by year, the harvest time has a great influence on the accumulation of the compositions.

[Effects of plant growth regulator uniconazole on plant morphology and biomass allocation of Salvia miltiorrhiza].

In this study, we use pot experiment to evaluate the effect of plant growth regulator on plant morphology and biomass allocation of Salvia miltiorrhiza. Different concentrations of uniconazole were supplied to S. miltioohiza by means of foliar spray. Height, breadth and stem diameter were measured dynamically, the biomass of leaf, stem, flower and fruit, root biomass and biomass ratio were also examined at the harvest time. Owing to the treatment, plant morphology showed significant changes, the height had been greatly reduced and the breadth decreased largely. Meanwhile, the biomass allocation changed too. The biomass ratio of leaf and stem had been notably reduced while the biomass ratio of root had been increased remarkably. It appears that foliar application of uniconazole during vigorous growth period in S. miltioohiza has dramatic effect on dwarfing plant and improving resistant to lodging. This measure could also be applied to condensed cultivation of S. miltioohiza to increase production.

[Allelopathic effects of aqueous extracts from Panax notoginseng on three maize varieties (Zea mays)].

It has been showed that there were obvious obstacle effects of Panax notoginseng replanting. Crop rotation was the main effective technique to overcome the obstacle. To find a reasonable crop rotation system for P. notoginseng, aqueous extracts from root, stem and leaf of P. notoginseng were analyzed for allelopathic effect on three maize varieties (which are often grown in regions where P. notoginseng grown). The main results were as follows: (1) Allelopathic effect of P. notoginseng stem and leaf extracts on the three other tested plants was stronger than that of root extracts; (2) Corn was more vulnerable to the effects of allelochemicals at seedling stage than at germination stage, and the corn root was more sensitive than aerial part to allelochemicals; (3) Lusan No. 3 and Yunrui No. 1 showed resistance to P. notoginseng allelopathy, with respective comprehensive sensitivity indexes (M3) of - 0.089 3 and -0.159 2, while Bainuo No. 1 is sensitive at M3 = -0.261 0. It then can be concluded that Lusan No. 3 and Yunrui No. 1 may be an alternative rotation plants for overcoming P. notoginseng continuous cropping obstacle.

[Influence of different original processing methods on quality of Salvia Miltiorrhizae Radix et Rhizoma from Shandong].

In this paper the contents of rosmarinic acid, salvianolic acid B, crytotanshinone, tanshinone II(A) in samples of different original processed Salvia Miltiorrhizae Radix et Rhizoma were determined by HPLC. Different processing methods have varied influences on four active ingredients in Salvia Miltiorrhizae Radix et Rhizoma. Sun-drying reduced the content of crytotanshinone, tanshi-none II(A) and rosmarinic acid, integralsamples were better than those cut into segments. Oven dry method had great influence on water--soluble ingredients, high temperature (80-100 degrees C) could easily cause big loss of rosmarinic acid and salvianolic acid B. The role of traditional processing method "fahan: was complicated, the content of rosmarinic acid decreased, crytotanshinone and tanshinone II(A) increased, and salvianolic acid B showed no difference after "fahan". Drying in the shade and oven dry under low temperatrure (40-60 degrees C) were all effective to keep active ingredients of Salvia Miltiorrhizae Radix et Rhizoma, and, there was no difference between integral samples and samples cut into segments. Therefore, considering comprehensively the content of active ingredients in Salvia Miltiorrhizae Radix et Rhizoma, and processing costing etc., shade-drying or oven dry underlow temperature (40-60 degrees C) should be the most suitable original processing method.

[Genotype, environment and their interactions of major bioactive components in 2-year licorice (Glycyrrhiza uralensis) population].

OBJECTIVE: This study aimed at analyzing the effect of genotype (G), environment (E) and their interactions (G x E) on the major bioactive components of 2-year licorice (Glycyrrhiza uralensis) population, in order to provide a theoretical basis for the licorice breeding with high content of bioactive components and quality improvement. METHOD: Four genotype licorice populations were transplanted under four different environments by using complete randomized block design with three replicates, and four major bioactive components, including glycyrrhizin (GL), total saponins (TS), liquiritin (LQ) and total flavonoids (TF) were determined by UV and by HPLC. RESULT: The major bioactive components of licorice were influenced by genotype and environment, and the genotype had more effect on all of the bioactive components. The contents of GL and LQ were codetermined by genotype and environment factors. CONCLUSION: There exist different selective effects on different growth region for quality breeding in cultivated population of licorice.