Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

Sodium dehydroacetate-induced disorder of coagulation function in broiler chickens and the protective effect afforded by vitamin K.

Sodium dehydroacetate (S-DHA) is used widely as a preservative in several products, including poultry feed. The anticoagulation effect of 200 mg/kg S-DHA in rats has been reported to accompany a reduction in hepatic expression of vitamin K epoxide reductase complex 1 (VKORC1). Poultry and mammals have different physiology and coagulation systems, and species differences in VKORC1 expression have been found. The effect of S-DHA on blood clotting of poultry has not been studies deeply. S-DHA was given to yellow-plumage broilers (YBs) as single and multiple administrations. Vitamin K3 (VK3) was injected into YBs 2 wk after S-DHA administration. Then, the prothrombin time (PT), partial activated prothrombin time (APTT), plasma levels of vitamin K (VK), factor IX (FIX), and S-DHA, and hepatic expression of VKORC1 were obtained. Chicken hepatocellular carcinoma (LMH) cells were also exposed to S-DHA, and the cell activity, VK level, and FIX level were measured. S-DHA prolonged the PT or APTT significantly, decreased levels of VK and FIX in blood, and inhibited hepatic expression of VKORC1. The maximum changes were 1.15-fold in the PT, 1.42-fold in the APTT, 0.8-fold in the VK level, 0.7-fold in the FIX level, and 0.35-fold in VKORC1 expression compared with controls. The cell activity, VK level, FIX level, and VKORC1/VKORC1L1 expression of LMH cells were reduced significantly at S-DHA doses of 2.0 to 10.0 mM. Prolongation of the PT/APTT and lower levels of VK/FIX in YBs or the lower cell activity and VK/FIX levels in LMH cells induced by S-DHA therapy were resisted significantly by VK3 treatment. We demonstrated that S-DHA could induce a disorder in coagulation function in YBs or in LMH cells via reduction of VKORC1/VKORC1L1 expression, and that VK could resist this anticoagulation effect.

Isolation and identification of a novel canine parvovirus type 2c strain in domestic cats in Dalian, China.

Canine parvovirus (CPV) and feline panleukopenia virus (FPV) are highly contagious and cause severe enteric diseases, with high mortality rates in dogs and cats. In the present study, we isolated and identified a novel CPV-2c strain (FPV-DL04 strain) from 18 cats with gastroenteritis symptoms and a positive parvovirus PCR test result in Dalian, China. Molecular characterization, sequence analysis, and phylogeny determination were performed on the VP2 gene of this strain. The results showed that the FPV-DL04 strain had 99.4% homology with the CPV-2c CN/HN1708 strain, and both strains had S297A and A300G key mutation sites. Interestingly, we also found that the DL04 strain has a A5G mutation site, but no F267Y and Y324I mutation sites. This study provided new important findings regarding the evolution of parvovirus infection in domestic cats in China.

Sex Metabolic Differences and Effects on Blood Coagulation Among Rats Exposed to Sodium Dehydroacetate.

Sodium dehydroacetate (Na-DHA), a fungicide used in food, feed, cosmetics, and medicine, has been found to cause coagulation aberration accompanied by the inhibition of vitamin K epoxide reductase (VKOR) in the liver in rats. VKOR complex 1 (VKORC1) and VKORC1 like-1 (VKORC1L1) are two homologous VKOR proteins. Little information is available on the effect of Na-DHA on VKORC1L1 in the liver or VKORC1/VKORC1L1 in extrahepatic tissue and sex differences in Na-DHA metabolism. In the present study, after administration of 200 mg/kg Na-DHA by gavage, significant inhibition of VKORC1 or VKORC1L1 expression in tissues, as well as prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT), were observed. The PT/APTT in the Na-DHA-exposed males were 1.27- to 1.48-fold/1.17- to 1.37-fold, while the corresponding values in the Na-DHA-exposed females were 1.36- to 2.02-fold/1.20- to 1.70-fold. Serum or tissue Na-DHA concentrations were significantly higher in females than in males. The pharmacokinetic parameters (t1/2, Cmax, AUC0∼24 h, and MRT0∼24 h) of Na-DHA in female rats were significantly higher than those in male rats. Furthermore, cytochrome P450 (CYP) activity was investigated using the cocktail probe method. The results revealed that Na-DHA exhibited an inductive effect on CYP1A2, 2D1/2, and 3A1/2 activities by changing the main pharmacokinetic parameters of probe drugs in male rats. However, no significant change in CYP2E1 activity was found. There were sex differences in the metabolism and coagulation in rats exposed to Na-DHA. The lower metabolism and higher blood Na-DHA concentration in females may be the reasons for higher coagulation sensitivity in female rats.

Preparation of single-chain antibody against VP3 protein of duck hepatitis virus type 1 by phage display technology.

To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction. Finally, the library for anti-VP3 scFv was screened by four rounds of adsorption-elution-enrichment with the purified VP3 protein. The characters of binding ability, specificity and neutralization of soluble antibodies expressed were evaluated by ELISA. The results showed 7 VP3-specific scFvs have been screened and identified with high both sensitivity and specificity for binding DHAV-1. To our knowledge, this is the first report for VP3-specific scFv of DHAV-1 and potentially promising application used in prevention and treatment of duck viral hepatitis.