[Observation of the Feline Intestinal Epithelial Cell Infected with Toxoplasma gondii].

Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenase XI/dispase I. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

[Advances in epigenetic researches of Toxoplasma gondii].

Toxoplasma gondii undergoes a complex life cycle that involves multiple development stages, hosts and environments. The ability to transform from one stage to another and adapt to changing environments demands precise regulation of gene expression. Bioinformatic surveys of the sequenced genomes of T. gondii revealed a peculiar absence of DNA-binding transcription factors that are well-conserved from yeast through humans, but a wealth of epigenetic machinery present in T. gondii. Evidence from reports demonstrates that remodeling of the chromatin structure particularly through post-translational modifications of histones, such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, is potentially a major process that coordinates regulation of its gene expression. In addition, no-coding RNAs may play an important role in modulating gene expression of T. gondii. These results provide reliable foundations for prevention of toxoplasmosis by revealing its pathogenic mechanism.

The regulation effect of WNT-RAS signaling in hypothalamic paraventricular nucleus on renal fibrosis.

BACKGROUND: Abnormal activation of wnt/ß-catenin signaling and renin-angiotensin system is known to play a vital role in the development and progression of CKD. We hypothesized that abnormal expression of central wnt/ß-catenin signaling and renin-angiotensin system (WNT-RAS signaling) was tightly involved in CKD. METHODS: We established sham-operated and 5/6 nephrectomized (5/6 NX) rat model and blocked the central wnt signaling by intracerebroventricular injection of adeno-associated virus vector which can overexpress target gene DKK1. The central and renal expression level of wnt/ß-catenin signaling and RAS and renal injury were assessed. RESULTS: The expression levels of the main wnt/ß-catenin signaling components in both brain and kidney of 5/6NX rats, such as wnt3a and active-ß-catenin, were elevated obviously and the up-regulation were inhibited by central blockade of the wnt signaling. Furthermore, the expression of the major components of RAS in both brain and kidney in 5/6NX rats, such as angiotensinogen (AGT), angiotensin converting enzyme (ACE-1), angiotensin II AT1-receptor (AT1R), was significantly up-regulated and the up-regulated expression was inhibited by central blockade of the wnt singling. Notably, central blockade of the wnt signaling improved renal function as indicated by decreased serum creatinine and 24 h urinary protein, and attenuated interstitial fibrosis as indicated by reduced Sirius red staining and expression of Fibronectin, Collagen-I and α-SMA. CONCLUSION: These data suggest that the central WNT-RAS signaling plays a significant role in the development and progression of CKD.

Urinary Matrix Metalloproteinase-7 and Prediction of AKI Progression Post Cardiac Surgery.

AIMS: Early detection of patients at high risk for progressive acute kidney injury (AKI) after cardiac surgery remains a major challenge. We aim to evaluate the utility of urinary matrix metalloproteinase-7 (uMMP-7) and other reported biomarkers for predicting AKI progression during postoperative hospital stay. METHODS: We conducted a prospective, multicenter cohort study in 121 adult patients with stage 1 or 2 AKI after cardiac surgery. uMMP-7 and other well-reported biomarkers (uIL-18, uNGAL, and UACR) were measured at time of AKI clinical diagnosis. The primary outcome is the progression of AKI after cardiac surgery, defined as worsening of AKI stage (stage 1 to either stage 2 or stage 3 or from stage 2 to stage 3). RESULTS: A level of uMMP-7 > 7.8 µg/g Cr at time of AKI diagnosis conveyed an 8-fold risk of AKI progression as compared to those with uMMP-7 < 2.7 µg/g after adjusting for clinical risk factors. The performance of uMMP-7 for predicting progressive AKI was good with an AUC of 0.80. The combination of uMMP-7 and IL-18 produces the greatest AUC for predicting progressive AKI. Addition of uMMP-7 to the clinical risk factor model significantly improved risk reclassification for AKI progression. CONCLUSIONS: uMMP-7, measured at time of AKI clinical diagnosis, is a novel biomarker for predicting the progression of AKI after cardiac surgery. Adding uMMP-7 to the clinical risk factor model may be used as a noninvasive approach to identify a subpopulation that is at high risk for progressive AKI after cardiac surgery.

Efficient genome engineering of Toxoplasma gondii using the TALEN technique.

BACKGROUND: Aromatic amino acid hydroxylase 2 (AAH2) is a bradyzoite-specific upregulated protein that may alter host behaviour by altering the host dopaminergic pathway. To better understand the role of the parasite's AAH2 in host-parasite interactions, we generated an AAH2 fluorescent marker strain of T. gondii using the TALEN technique. METHODS: We generated an AAH2 fluorescent marker strain of T. gondii, which was designated PRU/AAH2-eGFP, using the TALEN technique. This strain stably expressed pyrimethamine resistance for screening and expressed enhanced green fluorescent protein (eGFP)-tagged AAH2 in the bradyzoite stage. The bradyzoite conversion of PRU/AAH2-eGFP was observed both in vitro and in vivo. The fluorescence localization of AAH2 in mouse models of chronic infection was observed by a Bruker in vivo imaging system. RESULTS: Transgenic T. gondii was successfully generated by the TALEN system. The eGFP-tagged AAH2 could be detected by in vivo imaging. CONCLUSIONS: This study verified the feasibility of using TALEN technology for T. gondii research and provided an in vivo imaging method for in vivo research of bradyzoite-stage proteins.

Numb Depletion Promotes Drp1-Mediated Mitochondrial Fission and Exacerbates Mitochondrial Fragmentation and Dysfunction in Acute Kidney Injury.

AIMS: Mitochondrial fragmentation is a crucial mechanism contributing to tubular cell apoptosis during acute kidney injury (AKI). However, the mechanism of modulating mitochondrial dynamics during AKI remains unclear. Numb is a multifunction adaptor protein that is expressed in renal tubules. The aim of the present study was to evaluate the role of Numb in mitochondrial dysfunction during AKI. RESULTS: The expression of Numb was upregulated in both ischemia-reperfusion- and cisplatin-induced AKI. Depletion of Numb from proximal tubules (PT-Nb-KO) exacerbated AKI shown as more severe renal tubular damage and higher serum creatinine than wild-type mice. Numb depletion alone significantly increased mitochondrial fragmentation without altering mitochondrial mass and function, including adenosine triphosphate production, mitochondrial membrane potential, oxygen consumption, and reactive oxygen species production. However, mitochondrial fragmentation and dysfunction were significantly aggravated after cisplatin exposure in PT-Nb-KO mice. Mechanistically, Numb depletion triggered dynamin-related protein 1 (Drp1) recruitment to mitochondria by increasing the phosphorylation of Drp1 at serine 656 residue (human Drp1 ser637). Inhibiting the activity of Rho-associated coiled-coil containing protein kinase (ROCK) by Y-27632 attenuated phosphorylation of Drp1 ser656 and mitochondrial fragmentation in Numb-deficient cells. Administration of mdivi-1, a pharmacological inhibitor of Drp1, restored mitochondrial morphology, attenuated cisplatin-induced tubular injury, and renal dysfunction in PT-Nb-KO mice. Innovation and Conclusion: Our data suggest that Numb depletion promotes mitochondrial fragmentation by promoting the phosphorylation of Drp1 Ser637 and thus exacerbates cisplatin-induced mitochondrial dysfunction and tubular cell apoptosis. These findings add a novel insight into modulating mechanism of mitochondrial dynamics during AKI.

Wnt/ß-catenin/RAS signaling mediates age-related renal fibrosis and is associated with mitochondrial dysfunction.

Renal fibrosis is the common pathological feature in a variety of chronic kidney diseases. Aging is highly associated with the progression of renal fibrosis. Among several determinants, mitochondrial dysfunction plays an important role in aging. However, the underlying mechanisms of mitochondrial dysfunction in age-related renal fibrosis are not elucidated. Herein, we found that Wnt/ß-catenin signaling and renin-angiotensin system (RAS) activity were upregulated in aging kidneys. Concomitantly, mitochondrial mass and functions were impaired with aging. Ectopic expression of Klotho, an antagonist of endogenous Wnt/ß-catenin activity, abolished renal fibrosis in d-galactose (d-gal)-induced accelerated aging mouse model and significantly protected renal mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species. In an established aging mouse model, dickkopf 1, a more specific Wnt inhibitor, and the mitochondria-targeted antioxidant mitoquinone restored mitochondrial mass and attenuated tubular senescence and renal fibrosis. In a human proximal tubular cell line (HKC-8), ectopic expression of Wnt1 decreased biogenesis and induced dysfunction of mitochondria, and triggered cellular senescence. Moreover, d-gal triggered the transduction of Wnt/ß-catenin signaling, which further activated angiotensin type 1 receptor (AT1), and then decreased the mitochondrial mass and increased cellular senescence in HKC-8 cells and primary cultured renal tubular cells. These effects were inhibited by AT1 blocker of losartan. These results suggest inhibition of Wnt/ß-catenin signaling and the RAS could slow the onset of age-related mitochondrial dysfunction and renal fibrosis. Taken together, our results indicate that Wnt/ß-catenin/RAS signaling mediates age-related renal fibrosis and is associated with mitochondrial dysfunction.

Characterization of protein arginine methyltransferase of TgPRMT5 in Toxoplasma gondii.

BACKGROUND: Protein arginine methylation is a prevalent post-translational modification. The protein arginine methyltransferase family (PRMT) is involved in many cellular processes in eukaryotes, including transcriptional regulation, epigenetic regulation, RNA metabolism, and DNA damage repair. Toxoplasma gondii, an opportunistic protozoan parasite, encodes five conserved PRMTs. PRMT5 is thought to be responsible for substantial PRMT activity in T. gondii; however, it has not yet been characterized. METHODS: We tagged the 3' end of the endogenous TgPRMT5 genomic locus with sequence encoding a 3X hemagglutinin (HA) epitope. IFA and WB were performed to check the expression and subcellular localization of TgPRMT5 in tachyzoites and bradyzoites. In vitro methylation assays were performed to determine whether endogenous TgPRMT5 has arginine methyltransferase activity. RESULTS: IFA and WB results showed that T. gondii PRMT5 (TgPRMT5) was localized in the cytoplasm in the tachyzoite stage; however, it shifts largely to the nuclear compartment in the bradyzoite stage. The in vitro methylation showed that TgPRMT5 has authentic type II PRMT activity and forms monomethylarginines and symmetric dimethylarginines. CONCLUSIONS: We determined the expression and cellular localization of TgPRMT5 in tachyzoites and bradyzoites and confirmed its type II PRMT activity. We demonstrated the major changes in expression and cellular localization of TgPRMT5 during the tachyzoite and bradyzoite stages in T. gondii. Our findings suggest that TgPRMT5 protein may be involved in tachyzoite-bradyzoite transformation.

[A colloidal gold immunochromatographic assay for detecting p38 antigen of Schistosome japonicum].

OBJECTIVE: To establish a colloidal gold immunochromatographic assay (GICA) for detecting Schistosome japonicum (Sj). METHODS: Eight monoclonal antibodies (mAbs) against Sj p38 antigen (1A6, 3C4, 3D12, 6F10, 6G12, 9H6, 9G7 and A5H) prepared previously were purified by protein-G affinity chromatography. The affinity constant (K(aff)) was determined by indirect enzyme-linked immunosorbent assay. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and the mAb pairs with high affinity and stability were identified according to their optical density at 450 nm (OD450). Four mAbs (1A6, 6G12, 9H6 and 9G7) were chosen for colloidal gold labeling. GICA was then performed by further optimization of the labeling and the conditions were determined. The sera of mice at different infection stages were examined with GICA dipstick, with the sera collected before infection and those of Toxoplasma gondii-infected mice as negative controls. RESULTS: The purity of the 8 mAbs was higher than 95% with K(aff) ranging from 2.8x10(-10) to 1x10(-8) mol/L. 9G7 coating (2.5 mg/ml) as the capture antibody and detection with 1A6 (diluted at 1:4) as the labeling antibody was determined as the best reaction model. With this combination, the positivity rates of the detection were 40%, 50%, 60% and 80% for mouse sera collected at 3, 4, 5 and 6 weeks after Schistosome japonicum infection, respectively, without positive results for the negative control samples. CONCLUSION: GICA established in this study is characterized by simplicity, rapidity and good sensitivity, and the prepared rSjP38 dipstick can test the circulating antigen SjP38 in early stage of infection.

[Soluble expression and characterization of the immunoreactive multiepitope antigen of Toxoplasma gondii in E.coli].

OBJECTIVE: To obtain soluble expression product of immunoreactive recombinant multiepitope antigen of Toxoplasma gondii from E.coli. METHODS: The gene encoding the multiple epitopes (MEG) of Toxoplasma gondii was amplified by PCR from the original plasmid containing MEG gene and cloned into the prokaryotic soluble expression vector pET32a. After identification by enzyme digestion and sequencing, the positive recombinant plasmid pET32a-MEG was transformed into BL21(DE3), which was induced with IPTG for expression of the target antigen. The relative molecular mass, solubility and antigenicity of the expression products were analyzed by SDS-PAGE and Western blotting. RESULTS: The recombinant expression plasmid pET32a-MEG was successfully constructed and the highly efficient expression of the antigen was achieved after IPTG induction of E.coli. Improvement of the induction condition increased the expression product which accounted for about 28% of the total bacterial protein. The target protein, with good solubility and a relative molecular mass of about 31 000, was purified by immobilized metal affinity chromatography (Ni-NTA resin) and could be well recognized by mouse and rabbit antisera derived by infection of the animals with Toxoplasma gondii B36 and RH, respectively. CONCLUSION: The recombinant multiepitope antigen has good antigenicity and potential value in diagnosis and vaccine development of toxoplasmosis.

[Establishment of rabbit model of renal allograft transplantation using microsurgical technique].

OBJECTIVE: To establish rabbit model of renal allograft transplantation with reduced complications and high survival rate using microsurgical technique. METHODS: Twelve healthy adult rabbits were randomly divided into 2 groups of equal number, one as donor group and the other recipient. The left kidneys of the donor rabbits were removed followed by immediate reperfusion with 4 degrees celsius H-CA solution, before they were transplanted into the recipient rabbits with their left kidneys excised and end-to-end anastomosis of the renal arteries, veins and ureter respectively performed with microsurgical technique. Another 12 normal rabbits received operations to temporarily block the right renal arteries and veins, serving as control group, in which 11 completed the experiment. RESULTS: No thrombosis or stricture occurred at the site of anastomosis in rabbits with renal allograft transplantation, and the survival rate reached 91.7% (11/12). CONCLUSION: This rabbit model of renal allograft transplantation has markedly fewer complications with improved survival rate, thus providing a more practical and reliable model for experimental and clinical studies of renal transplantation.

[Preliminary study of cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation].

OBJECTIVE: To study the changes in the constituents of the cercaria antigen of Schistosoma japonicum before and after ultraviolet irradiation. METHODS: The cercaria of Schistosoma japonicum were exposed to ultraviolet light (UV) irradiation at a dose of 400 mgrW/cm2 for 1 min, and the UV-irradiated cercaria antigen (UVCA) and normal cercaria antigen (NCA) were simultaneously analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. RESULTS: At least 2 antigens with relative molecular mass (Mr) of 212 000 and 82 000 were identified in UVCA but not in NCA by SDS-PAGE analysis, and the concentrations of the antigens with Mr of 116 000, 26 000 and 16 000 in UVCA were significant higher than those in NCA. On the other hand, the antigenic molecule with Mr of 67 000 in NCA was recognized by serum from pigs vaccinated with UV-attenuated cercariae, but not by serum from pigs with Schistosoma japonicum infection. Antigens with Mr of 79 000 and 94 000 were apparently more strongly reactive with the former porcine serum than with the latter. CONCLUSION: The results suggest that all the novel antigens arising from or increased by UV exposure, or antigens specifically recognized by serum from pigs vaccinated by UV-attenuated carcariae may be the principal factors in the highly protective immunity provoked by irradiated cercariae.

[Construction of the plant expression vectors containing the multiepitope gene of Toxoplasma gondii].

OBJECTIVE: To construct the plant expression vectors containing the multiepitope gene of Toxoplasma gondii (TGMG). METHODS: 1. TGMG was subcloned into pBAC55 vector to construct the intermediate plasmid pB35MG. The E35S/TGMG/NOS3' fragment was cleaved from pB35MG and ligated into the plant binary vector pCAMBIA2300 to construct the plant expression vector pC35MG. 2. Tomato fruit-specific E81. 1 promoter was introduced to pB35MG to construct pB35E1MG vector. The E35SE81. 1/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pC35E1MG. 3. Tomato fruit-specific E82.2 promoter was inserted to pB35MG to construct pBE2MG vector. The E82.2/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pCE2MG. The insert gene TGMG in the vectors pB35MG, pC35E1MG and pCE2MG were confirmed by sequencing. 4. pC35MG, pC35E1MG and pCE2MG were introduced into Agrobacterium tumefaciens strain LBA4404 competent cell. RESULTS: Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments. And the sequencing results were confirmed correct. CONCLUSION: The TGMG intermediate vectors pB35MG, pB35E1MG and pBE2MG and the plant expression vectors pC35MG, pC35E1MG and pCE2MG were constructed successfully, and the three plant expression vectors were introduced into Agrobacterium tumefaciens.

[A hydroponic cultivation system for rapid high-yield transient protein expression in Nicotiana plants under laboratory conditions].

OBJECTIVE: To develop a hydroponic Nicotiana cultivation system for rapid and high-yield transient expression of recombinant proteins under laboratory conditions. METHODS: To establish the hydroponic cultivation system, several parameters were examined to define the optimal conditions for the expression of recombinant proteins in plants. We used the green fluorescent protein (GFP) and the geminiviral plant transient expression vector as the model protein/expression vector. We examined the impact of Nicotiana species, the density and time of Agrobacterium infiltration, and the post-infiltration growth period on the accumulation of GFP. The expression levels of GFP in Nicotiana leaves were then examined by Western blotting and ELISA. RESULTS: Our data indicated that a hydroponic Nicotiana cultivation system with a light intensity of 9000 LX/layer, a light cycle of 16 h day/8 h night, a temperature regime of 28 degrees celsius; day/21 degrees celsius; night, and a relative humidity of 80% could support the optimal plant growth and protein expression. After agroinfiltration with pBYGFPDsRed.R/LBA4404, high levels of GFP expression were observed in both N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants cultured with this hydroponic cultivation system. An optimal GFP expression was achieved in both Nicotiana species leaves 4 days after infiltration by Agrobacterium with an OD(600) of 0.8. At a given time point, the average biomass of N. tobaccum (cv. Yuyan No.5) was significantly higher than that of N. benthamiana. The leaves from 6-week-old N. benthamiana plants and 5-week-old N. tobaccum (cv. Yuyan No.5) plants could be the optimal material for agroinfiltration. CONCLUSION: We have established a hydroponic cultivation system that allows robust growth of N. benthamiana and N. tobaccum (cv. Yuyan No.5) plants and the optimal GFP expression in the artificial climate box.

Diagnosis of human toxoplasmosis by using the recombinant truncated surface antigen 1 of Toxoplasma gondii.

The goal of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) using a truncated surface antigen 1 (SAG1) gene of Toxoplasma gondii for the diagnosis of human toxoplasmosis. The truncated SAG1 gene was highly expressed in Escherichia coli. An ELISA kit based on the purified recombinant truncated SAG1 (rtSAG1) was developed, which was used to detect antibodies against T. gondii in human sera. The results showed that the infection of T. gondii could be detected sensitively and specifically by this serologic method. The positive concordance between rtSAG1-ELISA and Western blot, the gold standard, was 93.9% (31/33). However, the positive concordance between the commercial available ELISA Kit 1 (Haitai, Zhuhai, China) and ELISA Kit 2 (DiaSorin ETI-TOXOK-M reverse Plus, Italy) with Western blot was 79.5% (31/39) and 91.2% (31/34), respectively. Comparatively, the positive concordance of ELISA Kit 1 and 2 with Western blot was lower than rtSAG1-ELISA, in particular, the ELISA Kit 1 (P < 0.01), which indicated that the rtSAG1 protein could be used as the diagnostic antigen for human toxoplasmosis.