LIGHT (TNFSF14) inhibits glucose uptake of adipocytes by downregulating GLUT4 expression via AKT signaling pathway.

Glucose homeostasis of adipocytes could be regulated by immune-adipose crosstalk. In order to investigate the effects of Lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells (LIGHT) on glucose metabolism, we performed the present study. Our results showed that LIGHT deficiency improved glucose tolerance and enhanced glucose consumption of inguinal white adipose tissue (iWAT) under high fat diet. Consistently, Light overexpression could inhibit glucose uptake during the process of white adipogenesis. Mechanistically, LIGHT interacted with lymphotoxin-ß receptor (LTßR) to attenuate AKT pathway leading to downregulation of glucose transporter-4 (GLUT4) expression, which resulted in glucose uptake inhibition. In summary, our findings revealed LIGHT-LTßR-AKT-GLUT4 axis as a regulator of glucose uptake in adipose tissue, which suggested the pivotal role of LIGHT in maintaining glucose homeostasis.

Evaluation of toxoplasmosis in pregnant women using dot-immunogold-silver staining with recombinant Toxoplasma gondii peroxiredoxin protein.

BACKGROUND: Toxoplasma gondii infection endangers human health and affects animal husbandry. Serological detection is the main method used for epidemiological investigations and diagnosis of toxoplasmosis. The key to effective diagnosis of toxoplasmosis is the use of a standardized antigen and a specific and sensitive detection method. Peroxiredoxin is an antigenic protein and vaccine candidate antigen of T. gondii that has not yet been exploited for diagnostic application. METHODS: In this study, recombinant T. gondii peroxiredoxin protein (rTgPrx) was prepared and used in dot-immunogold-silver staining (Dot-IGSS) to detect IgG antibodies in serum from mice and pregnant women. The rTgPrx-Dot-IGSS method was established and optimized using mouse serum. Furthermore, serum samples from pregnant women were analyzed by rTgPrx-Dot-IGSS. RESULTS: Forty serum samples from mice infected with T. gondii and twenty negative serum samples were analyzed. The sensitivity and specificity of rTgPrx-Dot-IGSS were 97.5 and 100%, respectively, equivalent to those of a commercial ELISA kit for anti-Toxoplasma IgG antibody. Furthermore, 540 serum samples from pregnant women were screened with a commercial ELISA kit. Eighty-three positive and 60 negative serum samples were analyzed by rTgPrx-Dot-IGSS. The positive rate was 95.18%, comparable to that obtained with the commercial ELISA kit. CONCLUSIONS: The Dot-IGSS method with rTgPrx as an antigen might be useful for diagnosing T. gondii infection in individuals.

Beam splitter and router via an incoherent pump-assisted electromagnetically induced blazed grating.

We propose a scheme for a beam splitter and a beam router via an electromagnetically induced blazed grating in a four-level double-Λ system driven by an intensity-modulated coupling field and an incoherent pump field. The blazed grating relies on the incoherent pump process, which helps in inducing large refractivity with suppressed absorption or even gain. Consequently, the weak probe beam can be effectively deflected with high diffraction efficiency, and, meanwhile, its energy is amplified. When using an intensity mask with two symmetric domains in the coupling field, the presented blazed grating provides the possibility of a symmetric beam splitter. The diffraction efficiency and diffraction order of the gratings are sensitive to the intensity of the coupling field, and, thus, the gratings can function as a tunable asymmetric beam splitter or a beam router, which distributes the probe field into different spatial directions. Therefore, the proposed scheme may have potential applications in optical communication and networking.

[Prokaryotic Expression and Immunoreactivity Analysis on Profilin of Toxoplasma gondii].

OBJECTIVE: To clone and express the profilin (PRF) gene of Toxoplasma gondii, and analyze the immunoreactivity. METHODS: Total RNA was extracted from tachyzoites of T. gondii RH strain. The coding region of TgPRF was amplified with a pair of specific primers. PCR product was digested with double restriction enzyme and ligated into pET30a(+) vector. The recombinant pET30a(+)-TgPRF plasmid was transformed into E. coli DH5α with positive clones confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were purified with Ni-NTA affinity chromatography and analyzed by SDS-PAGE. Western blotting with rabbit anti-T. gondii serum was used to analyze its antigenicity. RESULTS: The product of RT-PCR was with 492 bp. pET30a-TgPRF was confirmed by the double restriction enzyme digestion, PCR and sequencing. SDS-PAGE analysis showed that the expressed product was a soluble protein with a relative molecular weight of 35,000. Western blotting assay revealed that rTgPRF was recognized by rabbit anti-T. gondii serum. CONCLUSION: TgPRF gene has been expressed in prokaryotic expression system and shows immunoreactivity.

[Cloning, expression and immunogenicity analysis of malate dehydrogenase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the malate dehydrogenase (MDH) gene of Toxoplasma gondii, and analyze the immunogenicity. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii (GenBank accession No. AY650028). The coding region of TgMDH was amplified with a pair of specific primers. The product of RT-PCR was digested with double restriction enzyme and ligated into pET30a (+) vector. The recombinant pET30a (+)-TgMDH plasmid was transformed into E. coli DH5alpha. The positive clones were confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity. RESULTS: The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a(+)-TgMDH was confirmed by the double restriction enzyme digestion, PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE, followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T. gondii serum. CONCLUSION: TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.

[Protection effect of baicalin-regulated IKKα on inflammatory reaction of human oral keratinocytes].

PURPOSE: To observe the regulation of baicalin on IKKα mediated MASPIN in Human oral keratinocytes (HOKs) inflammatory reaction, this study was to explore the molecular regulation mechanism of baicalin on oral mucosal inflammation. METHODS: HOKs were stimulated by lipopolysaccharide (LPS) to mimic the inflammatory response of oral mucosal inflammation in vitro. CCK-8 assay was used to detect the toxicity of baicalin to HOKs; then different concentrations of baicalin were pre-treated to LPS-stimulated HOKs, enzyme-linked immunosorbent assays (ELISA) was used to detect the secretion of IL-6 and TNF-α in LPS-stimulated HOKs; reverse transcription polymerase chain reaction(RT-PCR) and Western blot assay were used to detect the regulatory effects of baicalin on gene and protein expression levels of IKKα mediated MASPIN in LPS-stimulated HOKs. SPSS 21.0 software package was used for statistical analysis of the data. RESULTS: HOKs stimulated by 10 µg/mL LPS successfully simulated the inflammatory environment of oral mucosal inflammation. The concentration of baicalin between 1 µg/mL and 20 µg/mL had no toxic effect on HOKs. With the increasing concentration of baicalin, the expression of MASPIN increased gradually, while the expression of IKKα and inflammatory factors decreased gradually(P＜0.05). CONCLUSIONS: Baicalin can decrease the expression of inflammatory factors in LPS-stimulated HOKs, down-regulate IKKα and up-regulate MASPIN.

iTRAQ-Based Phosphoproteomic Analysis of Toxoplasma gondii Tachyzoites Provides Insight Into the Role of Phosphorylation for its Invasion and Egress.

The invasion and egress are two key steps in lytic cycle vital to the propagation of Toxoplasma gondii infection, and phosphorylation is believed to play important roles in these processes. However, the phosphoproteome of T. gondii at these two stages has not been characterized. In this study, we profiled the phosphoproteome of tachyzoites at the stages of "just invading" (JI) and "prior to egress" (PE) based on iTRAQ quantitative analysis, in which a total of 46 phosphopeptides, 42 phosphorylation sites, and 38 phosphoproteins were detected. In the comparison of PE vs. JI, 10 phosphoproteins were detected with their phosphorylation level significantly changed, and four of them were demonstrated to be significantly down-regulated at the transcriptional level. Bioinformatic analysis of these identified phosphoproteins suggested that phosphorylation-mediated modulation of protein function was employed to regulate the pathway of toxoplasmosis and metabolism and cellular processes correlated with tachyzoite's binding, location, and metabolism, and thus play vital roles in the parasite lytic cycle. Moreover, cytoskeletal network (CN)-associated Inner Membrane Complex (IMC1, IMC4, IMC6 and IMC12), Intravascular Network (IVN)-related GRAs (GRA2, GRA3, GRA7 and GRA12), and Parasitophorous Vacuole Membrane (PVM)-localized ROP5 were shown to be enriched at the central nodes in the protein interaction network generated by bioinformatic analysis, in which the phosphorylation level of IMC4, GRA2, GRA3, and GRA12 were found to be significantly regulated. This study revealed the main cellular processes and key phosphoproteins crucial for the invasion and egress of T. gondii, which will provide new insights into the developmental biology of T. gondii in vitro and contribute to the understanding of pathogen-host interaction from the parasite perspective.

Integrated analysis of miRNAs and transcriptomes in Aedes albopictus midgut reveals the differential expression profiles of immune-related genes during dengue virus serotype-2 infection.

Mosquito microRNAs (miRNAs) are involved in host-virus interaction, and have been reported to be altered by dengue virus (DENV) infection in Aedes albopictus (Diptera: Culicidae). However, little is known about the molecular mechanisms of Aedes albopictus midgut-the first organ to interact with DENV-involved in its resistance to DENV. Here we used high-throughput sequencing to characterize miRNA and messenger RNA (mRNA) expression patterns in Aedes albopictus midgut in response to dengue virus serotype 2. A total of three miRNAs and 777 mRNAs were identified to be differentially expressed upon DENV infection. For the mRNAs, we identified 198 immune-related genes and 31 of them were differentially expressed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses also showed that the differentially expressed immune-related genes were involved in immune response. Then the differential expression patterns of six immune-related genes and three miRNAs were confirmed by real-time reverse transcription polymerase chain reaction. Furthermore, seven known miRNA-mRNA interaction pairs were identified by aligning our two datasets. These analyses of miRNA and mRNA transcriptomes provide valuable information for uncovering the DENV response genes and provide a basis for future study of the resistance mechanisms in Aedes albopictus midgut.

Expression of Toll-like receptor (TLR) 2 and TLR4 in the livers of mice infected by Clonorchis sinensis.

INTRODUCTION: Clonorchis sinensis is one of the most important foodborne pathogens in humans, and can cause biliary diseases such as gallstones, cholecystitis, cholangitis, and cholangiocarcinoma. Toll-like receptors (TLRs) as sensors are crucial to initiating both innate and adaptive immune defenses against pathogens. However, little is known about the hepatic expression of TLRs of hosts induced by C. sinensis infection. METHODOLOGY: In the present study, the expression and distribution of TLR2 and TLR4 were investigated in a mouse model of clonorchiasis on days 28, 56, 84, and 112 post-infection (PI) using real-time quantitative reverse transcription polymerase chain reaction (PCR) and immunohistochemically staining, respectively. The levels of cytokines that are mediated by TLR2 and TLR4 were also evaluated using a cytometric bead array. RESULTS: Results showed that the transcripts of TLR2 and TLR4 were upregulated on day 28 PI in C. sinensis-infected mice compared with non-infected ones (p < 0.01). In addition, their proteins were strongly immunohistochemically positive in the cytoplasm and membrane of endothelial cells, fibroblasts, and biliary epithelium cells of C. sinensis-infected mice. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were increased with activation of TLR2 and TLR4. CONCLUSIONS: The expression of TLR2 and TLR4 is upregulated against C. sinensis infection, which suggests that TLR2 and TLR4 might be involved in immune responses during C. sinensis infection.

[Isolation and identification of Toxoplasma gondii strains from cats in Xuzhou region].

OBJECTIVE: To isolate Toxoplasma gondii (T. gondii) strains from stray cats and explore their prevalence in Xuzhou City. METHODS: The sera of 41 stray cats were collected to detect the antibodies of T. gondii by using a commercial enzyme-linked immunosorbent (ELISA) kit. The tissues including the heart, brain and tongue from these cats were digested by acid pepsin solution and inoculated to Kunming mice. These suspicious isolates were subsequently identified by a specific PCR method. RESULTS: A total of 11 strains were isolated from 41 stray cats, which were confirmed by the PCR results. Moreover, 17 cats (41.5%) were found to be positive with the antibodies of T. gondii. CONCLUSION: A high prevalence of T. gondii infection was found in Xuzhou City, which indicates that the stray cats infected with T. gondii would be an important infection source that may infect humans and other animals in this area.