Weifeng Mao

Anisakiasis in China: the first clinical case report.

Anisakiasis is a parasitic disease acquired by humans when ingesting raw or undercooked fish infected with L3 larvae of the nematode genus Anisakis or Pseudoterranova. Here we report the first case of human anisakiasis in China. The patient, male, 56 years old, Dalian citizen, was admitted into the hospital with vomiting, peripheral umbilicus and abdominal distension, and frequent mucous diarrhea. The patient was examined using an electronic gastroscope, which displayed a parasite residing in the stomach, and subsequently gastroscope-assisted surgery was implemented. A white round worm was removed from the patient and stained. It was identified as L3 larvae of Anisakis. After the removal of the L3 larvae of Anisakis, the inflammation symptoms disappeared. As the first report of clinical case of Anisakis infection in China, the morphology of L3 Anisakis larvae from the patient is described and discussed. We conclude that anisakiasis should be considered in patients who have a habit of eating raw fish and who display associated symptoms.

Visualization of Marek's disease virus in vitro using enhanced green fluorescent protein fused with US10.

Marek’s disease virus (MDV) is an highly cell-associated avian alphaherpesvirus. Although viral replication is supported in chicken embryo fibroblasts (CEF) or duck embryo fibroblasts, identification of MDV-infected cells is quite cumbersome especially during the early stages of virus replication when plaques can be difficult to recognize. To visualize MDV replication in infected cells and characterize MDV US10 in vitro, rMd5-US10-EGFP, a recombinant MDV, was generated that expresses enhanced green fluorescent protein (EGFP) as a tagged protein fused with US10 at the C-terminal end. The expression of US10-EGFP was detected in infected CEF using fluorescent microscopy and the expression intensity was quantified using flow cytometry analysis. In addition, confocal microscopic analysis provided information on subcellular localization of US10-EGFP in virus-infected cells. In conclusion, rMd5-US10-EGFP virus can be used to help monitor virus activity in vitro.

Cloning and functional characterization of chicken stem cell antigen 2.

Stem cell antigen 2 (SCA2) is a Ly6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 was expressed in E. coli, purified, and a polyclonal antibody developed. Utilizing the polyclonal antibody, SCA2 is a 13 kDa cell surface protein anchored by a glycosyl-phosphatidylinositol (GPI) moiety. SCA2 is expressed in connective tissues of thymus and bursa based on immunohistochemistry, immunoprecipitation, and western blots. In bursal follicles, SCA2 is specifically expressed on the cortical-medullary epithelial cells (CMEC) surrounded by MHC class II presenting cells. Expression profiles of bursal cells induced by contact with SCA2-expressing cells shows down-regulation of numerous genes including CD79B, B cell linker (BLNK), spleen tyrosine kinase (SYK), and gamma 2-phospholipase C (PLCG2) that are involved in the B cell receptor (BCR) and immune response signaling pathways. These results suggest chicken SCA2 plays a role in regulating B lymphocytes.

Inhibition of Rad51 sensitizes breast cancer cells with wild-type PTEN to olaparib

PTEN is a tumor suppressor gene well characterized as a phosphatase. However, more evidences demonstrate PTEN functions in DNA repair independent of its phosphatase activity, which affects the efficacy of DNA damage anti-tumoral drugs in treating cancer cells with PTEN variations. Using BT549 breast cancer cells, we studied the roles of PTEN in DNA repair and in sensitization of breast cancer cells to olaparib, a poly(ADP-ribose) polymerase (PARP) inhibitor. Comet assay showed PTEN promoted DNA repair. PTEN-deficient BT549 cells are sensitive to olaparib, which shows the synthetic lethality between PTEN and PARP1. We expressed PTEN in BT549 cells and found PTEN-proficient BT549 cells resist to olaparib. Western blot showed that PTEN up-regulated Rad51 expression, suggesting PTEN promotes DNA repair through Rad51-dependnent homologous recombination. We used 5μM olaparib or 5μM RI-1, a Rad51 inhibitor, to treat PTEN-proficient BT549 cells respectively. The immunofluorescent analysis showed the combination of olaparib and RI-1 induced more than 4-fold of γH2AX foci than either of them. MTT assay showed 5μM RI-1 did not change the survival of PTEN-proficient BT549 cells, however, this dose of RI-1 sensitized PTEN-proficient BT549 cells to olaparib. Consequently, these results demonstrate that inhibition of Rad51 can sensitize BT549 cells with wild type PTEN to olaparib, which would contribute to using PARP inhibitors in individual treatment of breast cancer patients with PTEN variations.

Berberine activates caspase-9/cytochrome c-mediated apoptosis to suppress triple-negative breast cancer cells in vitro and in vivo

Berberine (BBR) is an isoquinoline alkaloid isolated from Cotridis rhizoma and exhibits multiple biological roles including anti-microbe, anti-inflammation and anti-tumor activities. In this study, two triple-negative breast cancer cell (TNBC) lines, MDA-MB-231 and BT549, were used to investigate the effect of BBR on growth of TNBC in vitro and in vivo. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the viability of cells treated with BBR. After 48h treatments, a 50% inhibitory concentration (IC50) of BBR to BT549 and MDA-MB-231 cells are at 16.575±1.219μg/ml and 18.525±6.139μg/ml respectively. BBR reduced colony formation of BT549 and MDA-MB-231 cells. The wound-healing assay showed BBR decreased breast cancer cell migrations (P<0.01). AnnexinV-PI staining assay confirmed BBR induced cellular apoptosis. The expressions of caspase-3, caspase-9, Bcl-2 and Bax were detected by western blot, which showed BBR activated caspase-3, 9 and Bax, but down-regulated Bcl-2 expression. BBR promoted the release of cytochrome c through the immunofluorescent analysis (P<0.01). We also found BBR increased the level of cellular γH2AX and increased the expression of Ligase4, which suggests BBR induces the double-strand breaks (DSB). These results thus demonstrated that BBR induced DSB, subsequently increased the release of cytochrome c and eventually triggered the caspase9-dependent apoptosis. In addition, we used a MDA-MB-231 mouse-xenograftmodel to evaluate the effect of BBR on tumor growth. BBR suppressed tumor growth and increased caspase-9 levels in xenograft tumors through immunohistochemistry analysis (P<0.01). Taken together, these results demonstrate that BBR activates caspase-9/cytochrome c-mediated apoptosis to inhibit the growth of TNBC breast cancer cells in vitro and in vivo.

Nuclear PTEN interferes with binding of Ku70 at double-strand breaks through post-translational poly(ADP-ribosyl)ation.

PTEN is a tumor suppressor gene characterized as a phosphatase that antagonizes the phosphatidylinositol 3-kinase signaling pathway in the cytoplasm. Nuclear PTEN plays roles in chromosomal stability, in which the double-strand breaks (DSB) repair mediated by homologous recombination (HR) and non-homologous end joining (NHEJ) is critical. Herein, the role of nuclear PTEN in DSB repair and the underlying molecular mechanism was investigated in this study. Using human breast cancer BT549 and MDA-MB-231 cell lines, we reveal a specific feature of PTEN that controls poly(ADP-ribosyl)ation of Ku70 and interferes with binding of Ku70 at DSB. Plasmid-based end joining and reporter assays showed that nuclear PTEN restrained NHEJ efficacy. Electrophoretic mobility shift assays showed that nuclear PTEN impaired Ku70 complex binding to DSB by 3-fold. Co-immunoprecipitation assay showed PTEN regulated poly(ADP-ribosyl)ation of Ku70 instead of directly interacting with Ku70, while PTEN promoted the poly(ADP-ribosyl)ation of PARP1 and induced the degradation of PARP1 in PTEN-WT cells exposed to DSB agents. Of note, the role of PTEN in DSB repair mostly depends on its nuclear localization rather than its phosphatase activity. As a result, the absence of nuclear PTEN rather than phosphatase-negative PTEN confers cell hypersensitivity to anti-tumor DNA damage drugs. This finding contributes to understanding the effect of PTEN in repair of DSB and using defined anti-tumor DSB drugs to treat tumor cells with aberrant PTEN.

Evaluation of recombinant CXCL8(3–73)K11R/G31P in muscle fibrosis and Trichinella larvae encapsulation in a murine model of trichinellosis

Trichinella spiralis (T. spiralis) larvae in raw or inadequately cooked meat can cause chronic infections in a wide range of hosts including humans. During the development inside the skeletal muscles, T. spiralis larvae infect muscle cells accompanying with the infiltration of host inflammatory cells, eventually create a new type of cell known as nurse cell developing a surrounding vascular network to support the larvae development. Controlling of host inflammatory responses and angiogenesis influences both the nurse cell differentiation and the parasite larvae development. CXCL8 is a chemokine that acts on G-protein coupled receptors, of which activation contributes to fibrosis and angiogenesis. CXCL8(3-73)K11R/G31P (G31P) has been reported as a CXCL8 analogue. The aim of this study is to investigate the effect of G31P in inflammatory responses and the development of T. spiralis larvae in muscle tissues of mice infected with T. spiralis. The level of inflammatory factors and the morphology of T. spiralis larvae in infected tissues were investigated through ELISA and electron-microscopy analysis. G31P up-regulated IFN-γ and down-regulated CXCL8 level, and impaired the encapsulation of T. spiralis larvae in vivo. The results showed that G31P influenced the development of T. spiralis larvae in muscle tissues.

Curcumin sensitizes lymphoma cells to DNA damage agents through regulating Rad51-dependent homologous recombination

Curcumin is a natural compound isolated from the rhizome of Curcuma longa. It possesses anti-tumor activity through arresting cell cycles and promoting cell apoptosis. However, the effect of curcumin on DNA damage is not well defined. In this study, we investigated the effect of curcumin on inducing DNA damage and on sensitizing lymphoma cells to anti-tumoral DNA damage drugs. Western blot showed curcumin induced γ-H2AX foci in CH12F3 lymphoma cells, which suggests curcumin induces DNA breaks. In addition, curcumin decreased the expression of Rad51, which suggests curcumin induces DNA damage through regulating Rad51-dependant homologous recombination. Rad51-dependant homologous recombination is a vital DNA repair pathway for cancer cells to resist anti-tumoral DNA damage drugs, therefore, we studied the effect of curcumin on the sensitizing lymphoma cells to various chemotherapeutic drugs. We found low level of curcumin (5μM) sensitized lymphoma cells to anti-tumoral DNA damage agents including cisplatin, methyl methanesulfonate, hydroxyurea and camptothecin. We also found curcumin sensitized CH12F3 lymphoma cells to DNA-PK and PARP inhibitors. Flow cytometry analysis showed curcumin promoted apoptosis and western blot analysis confirmed curcumin activated caspase3-dependent apoptosis. Taken together, these results demonstrate that curcumin induces DNA damage through regulating Rad51-dependant homologous recombination and triggers caspase3-dependent apoptosis, more importantly, curcumin sensitizes lymphoma cells to various DNA damage drugs. Consequently, curcumin would be a potent agent for sensitizing lymphoma cells to anti-tumoral chemotherapeutic agents.

Triptolide induces DNA breaks, activates caspase‑3‑dependent apoptosis and sensitizes B‑cell lymphoma to poly(ADP‑ribose) polymerase 1 and phosphoinositide 3‑kinase inhibitors

Triptolide is the primary compound isolated from Tripterygium wilfordii, which has been reported to inhibit nucleotide excision repair as well as exhibit anti-inflammatory and antitumor activities. However, the action of triptolide in DNA breaks remains unknown. The present study investigated the effects of triptolide in the induction of DNA breaks and apoptosis in a murine B-cell lymphoma cell line, CH12F3. An MTT assay revealed that X-ray repair cross-complementing protein 1 (XRCC1)-/- CH12F3 cells were more sensitive to 6 nM triptolide compared with the wild-type CH12F3 cells, which suggests that low levels of triptolide induce DNA breaks in a manner that is dependent on the XRCC1-mediated repair pathway. Flow cytometric analysis identified that 50 nM triptolide increased the phospho-histone H2AX level, demonstrating that triptolide induces double-strand breaks. Western blot analysis revealed that triptolide up-regulated Rad51 and nuclear proliferating cell nuclear antigen. Annexin V/propidium iodide staining identified that triptolide promoted apoptosis and western blot analysis confirmed that triptolide activated caspase-3-dependent apoptosis. The results of the present study also demonstrated that 5 nM triptolide sensitized CH12F3 lymphoma cells to poly(ADP-ribose) polymerase 1 and phosphoinositide 3-kinase inhibitors, suggesting that triptolide may be a potent antitumor drug for sensitizing lymphoma cells to chemotherapeutic agents.