Phosphoproteomic Analysis Reveals the Predominating Cellular Processes and the Involved Key Phosphoproteins Essential for the Proliferation of Toxoplasma gondii.

PURPOSE: To explore the essential roles of phosphorylation in mediating the proliferation of T. gondii in its cell lytic life. METHODS: We profiled the phosphoproteome data of T. gondii residing in HFF cells for 2 h and 6 h, representing the early- and late-stages of proliferation (ESP and LSP) within its first generation of division. RESULTS: We identified 70 phosphoproteins, among which 8 phosphoproteins were quantified with the phosphorylation level significantly regulated. While only two of the eight phosphoproteins, GRA7 and TGGT1_242070, were significantly down-regulated at the transcriptional level in the group of LSP vs. ESP. Moreover, GO terms correlated with host membrane component were significantly enriched in the category of cellular component, suggesting phosphoprotein played important roles in acquiring essential substance from host cell via manipulating host membrane. Further GO analysis in the categories of molecular function and biological process and pathway analysis revealed that the cellular processes of glucose and lipid metabolism were regulated by T. gondii phosphoproteins such as PMCAA1, LIPIN, Pyk1 and ALD. Additionally, several phosphoproteins were enriched at the central nodes in the protein-protein interaction network, which may have essential roles in T. gondii proliferation including GAP45, MLC1, fructose-1,6-bisphosphate aldolase, GRAs and so on. CONCLUSION: This study revealed the main cellular processes and key phosphoproteins crucial for the intracellular proliferation of T. gondii, which would provide clues to explore the roles of phosphorylation in regulating the development of tachyzoites and new insight into the mechanism of T. gondii development in vitro.

Toxoplasma gondii rhoptry protein (TgROP18) enhances the expression of pro-inflammatory factor in LPS/IFN-γ-induced murine BV2 microglia cells via NF-κB signal pathway.

Toxoplasma gondii, an opportunistic pathogenic protozoan, exhibits a strong predilection to infect the brain, causing severe neurological diseases, such as toxoplasmic encephalitis (TE), in immunocompromised patients. Microglia, the resident immune cells in the brain, is reported to play important roles in regulating the neuroinflammation mediated by T. gondii infection. Here we demonstrated that the tachyzoites of T. gondii RH strain could significantly upregulate the expression levels of microglial M1 phenotype markers including IL-1ß, IL-6, TNF-α, iNOS and IL18 in activated murine BV2 microglia cells, which were regulated by T. gondii rhoptry protein 18 (TgROP18). Moreover, we found that TgROP18 could enhance the expression of M1 phenotype markers in activated murine BV2 microglia cells via activating NF-κB signal pathway. Additionally, TgROP18 was suggested to interact with the host p65 in activated murine BV2 microglia cells and induce the phosphorylation of p65 at S536. In summary, the present study demonstrated that TgROP18 could promote the activated microglia to polarize to M1 phenotype and enhanced the expression of pro-inflammatory factors via activating NF-κB signal pathway, which could contribute to elucidating the mechanism underlying the neuroinflammation mediated by activated microglia in the brain with T. gondii infection.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

[Effects of long-term fertilization on organic nitrogen fractions in aquic brown soil]. / 长期施肥对潮棕壤有机氮组分的影响.

The purpose of present research was to investigate how different fertilization regimes altered soil organic nitrogen fractions and their inter-annual dynamics based on a series of long-term experiment (initiated at 1990), including: CK (non-fertilization); M (recycled pig manure); NPK (chemical fertilizer NPK); NPK + M (recycled pig manure with chemical fertilizer NPK). The results showed that soil organic nitrogen components under the different fertilization treatments presented contrastive patterns from the establishment the experiments to 2015. Generally, acid hydrolysable organic nitrogen content increased year by year. The amino acid nitrogen content under CK and NPK treatments consistently declined, although amino acid nitrogen for M and NPK+M treatments showed a increasing trend. These phenomena were probably ascribed to the utilization of soil amino acids by microbes. From 1990 to 2015, NPK treatment substantially elevated the content of acid-released ammonium nitrogen by 31.1% compared with CK (mean value across the experiment), and for the treatments using organic manure (M and NPK+M), the contents of all fractions of soil organic nitrogen increased. Notably, the increase magnitudes for NPK+M were more dramatic than those of M. These results demonstrated that combined use of organic and inorganic fertilizers could more effectively elevate soil organic nitrogen, subsequently helping to improve the capacity of soil nitrogen supply and enhance the soil fertility.