csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis.

BACKGROUND: Clonorchis sinensis (CS) is classified as a group 1 carcinogen and can cause intrahepatic cholangiocarcinoma (ICC). CS extracellular vesicles (CsEVs) play important roles in mediating communication between parasitic helminths and humans. Ferroptosis is a novel cell death mechanism that is mainly induced by lipid peroxidation and iron overload. However, the role of CsEVs in the regulation of ferroptosis in ICC remains unclear. This study aimed to explore the role of CS-secreted miR-96-5p (csi-miR-96-5p) delivered by CsEVs in ICC progression and ferroptosis. METHODS: Tissue samples were collected from ICC patients with CS infection (CS-ICC) or without CS infection (NC-ICC). The levels of csi-miR-96-5p and PTEN gene were determined by quantitative polymerase chain reaction (qPCR) and western blotting, and survival analysis was performed. CsEVs were isolated and identified by ultracentrifugation and transmission electron microscopy. Lentiviruses were used to establish stable cell lines with csi-miR-96-5p mimic expression, PTEN overexpression (PTEN-EXO) and PTEN CRISPR/Cas9-based knockout (PTEN-KO) and their respective negative controls. Cell proliferation was assessed by performing Cell Counting Kit-8 assays in vitro and in a tumor xenograft model in vivo, and cell migration was assessed by performing Transwell assays. Erastin is used to induce ferroptosis. Ferroptosis levels were evaluated using biomarkers. RESULTS: High csi-miR-96-5p and low PTEN expression was observed in CS-ICC tissues and was associated with poor overall survival. csi-miR-96-5p was highly enriched in CsEVs and was taken up by ICC cells. csi-miR-96-5p mimics or PTEN-KO significantly promoted the growth and migration of ICC cells in vitro and in vivo, whereas PTEN-EXO exerted the opposite effect. Mechanistically, csi-miR-96-5p mimics or PTEN-KO inhibited erastin-induced ferroptosis, including reducing the accumulation of Fe2+, lipid reactive oxygen species, and malondialdehyde, increasing the GSH/GSSG ratio and levels of SLC7A11 and GPX4, whereas PTEN-EXOs exerted the opposite effect. CONCLUSIONS: csi-miR-96-5p delivered by CsEVs reduced ferroptosis by regulating the expression of the PTEN/SLC7A11/GPX4 axis, thereby promoting ICC proliferation and migration. For the first time to our knowledge, we found that CS miRNAs could promote tumor development through ferroptosis.

Gene expression profiling analysis of ovarian cancer.

As a gynecological oncology, ovarian cancer has high incidence and mortality. To study the mechanisms of ovarian cancer, the present study analyzed the GSE37582 microarray. GSE37582 was downloaded from Gene Expression Omnibus and included data from 74 ovarian cancer cases and 47 healthy controls. The differentially-expressed genes (DEGs) were screened using linear models for microarray data package in R and were further screened for functional annotation. Next, Gene Ontology and pathway enrichment analysis of the DEGs was conducted. The interaction associations of the proteins encoded by the DEGs were searched using the Search Tool for the Retrieval of Interacting Genes, and the protein-protein interaction (PPI) network was visualized by Cytoscape. Moreover, module analysis of the PPI network was performed using the BioNet analysis tool in R. A total of 284 DEGs were screened, consisting of 145 upregulated genes and 139 downregulated genes. In particular, downregulated FBJ murine osteosarcoma viral oncogene homolog (FOS) was an oncogene, while downregulated cyclin-dependent kinase inhibitor 1A (CDKN1A) was a tumor suppressor gene and upregulated cluster of differentiation 44 (CD44) was classed as an 'other' gene. The enriched functions included collagen catabolic process, stress-activated mitogen-activated protein kinases cascade and insulin receptor signaling pathway. Meanwhile, FOS (degree, 15), CD44 (degree, 9), B-cell CLL/lymphoma 2 (BCL2; degree, 7), CDKN1A (degree, 7) and matrix metallopeptidase 3 (MMP3; degree, 6) had higher connectivity degrees in the PPI network for the DEGs. These genes may be involved in ovarian cancer by interacting with other genes in the module of the PPI network (e.g., BCL2-FOS, BCL2-CDKN1A, FOS-CDKN1A, FOS-CD44, MMP3-MMP7 and MMP7-CD44). Overall, BCL2, FOS, CDKN1A, CD44, MMP3 and MMP7 may be correlated with ovarian cancer.

[The binding property of different regions in Duffy-binding-like domain alpha of Plasmodium falciparum erythrocyte membrane protein 1 to heparin].

OBJECTIVE: To clone and express Plasmodium falciparum erythrocyte membrane protein 1 DBLalpha (PfEMP1-DBLalpha) and three fragments genes, and screen the strongest affinity sequence with the red blood cell surface receptors-heparin or heparin sulfate in the structure of PfEMP1-DBLalpha. METHODS: The sequence of PfEMP1-DBLalpha1245 was optimized according to the characteristics of E. coli codon, synthesized, and divided into three fragments (DBLaA, DBLalphaB, and DBLalphaC) by PCR. Full-length gene and three gene fragments were subcloned into PGEX-4T-1 vector, and transformed into E. coli BL21 and then induced with IPTG for expression. The recombinant protein was purified from bacterial lysates using glutathione-Sepharose 4B. Heparin affinity test and glycosaminoglycan (GAG) inhibition test were used to analyze the affinity between recombinant protein and heparin. RESULTS: Four recombinant proteins(DBLalpha1245, DBLalphaA, DBLalphaB, and DBLalphaC) were expressed as solubility and the relative molecular weight (M(r) 73 600, M(r) 41 600, M(r) 42 500, and M(r) 41 500) were conformed to the prediction size. Heparin affinity test and GAG inhibition test showed that the four recombinant proteins were binded to the heparin-Sepharose, but not for the GST control. DBLalphaC (Q285-Y415) had the strongest affinity to heparin. CONCLUSION: The strongest affinity sequence with heparin or heparin sulfate in the structure of PfEMP1-DBLalpha is Q285-Y415, which plays a role in binding of Plasmodium falciparum infected red blood cells to the peripheral red blood cells.

Analysis of var genes cloned from a Plasmodium falciparum isolate in China.

OBJECTIVE: To analyse the var gene repertoire and characterise the chondroitin sulphate A (CSA)-binding activity of the Duffy-binding like (DBL) domains encoded by the var2csa gene of a Plasmodium falciparum (P. falciparum) isolate in Hainan Province, China. METHODS: The sequences of var DBL1 regions were PCR-amplified, sequenced and the sequence characteristics was bioinformatically analysed. Recombinant proteins encoded by the var2csa genes were expressed and purified. The binding activities of the recombinant proteins to CSA receptor was detected by ELISA assays. RESULTS: Fifty six unique DBL α sequences were obtained, and the sequences represented similar diversity to the var genes of the genome parasite 3D7. There are two var2csa genes in the P. falciparum isolated from Hainan Province. Unlike in other falciparum parasites such as HB3, the two var2csa genes are more diverged. The receptor-binding capacity of DBL-5ε and DBL-6ε domains of HN var2CSA was studied. CONCLUSIONS: This work represented the diversity of var genes of a P. falciparum isolate in China.

[Serological detection of Cryptosporidium spp. infection in outpatients in Changchun].

CP23 gene of Cryptosporidium parvum was expressed in Escherichia coli, and the recombinant protein was purified. Its immunoreactivity was analyzed by Western blotting. Serum samples were collected from outpatients of different ages from August to November, 2010 in Changchun. Indirect ELISA was established to detect the anti-CP23 IgG in sera. Western blotting analysis indicated that the recombinant CP23 protein was recognized by sera from Cryptosporidium panum-infected calves and positive human sera, but not recognized by sera of mice infected with Schistosoma japonicum, sera from falciparum malaria patients and negative human sera. The overall anti-CP23 IgG positive rate was 3.2% (65/2 046). The seropositive rate was 2.7% (28/1 036) in men and 3.7% (37/1 010) in women (P > 0.05). The seropositive rates were significantly different among age groups (P < 0.05), and the age group of 71-80 had the highest positive rate (8.6%, 13/152).

[Cloning and expression of var2csa DBL domains from Plasmodium falciparum hainan isolate and functional analysis of the recombinant protein].

OBJECTIVE: To clone and express three VAR2CSA duffy antigen-binding ligand (DBL) domains (DBL4/ 5/6) encoded by var2csa gene of a Hainan isolate of Plasmodium falciparum, and study the difference of chondroitin sulfate A (CSA)-binding activity among them. METHODS: Three DBL domains was amplified by PCR and cloned into the vector pMD18-T. The recombinant plasmids were identified by enzyme digestion and sequencing, and then subcloned into the prokaryotic expression vector pET-22b. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was purified with His GraciTrap kit and identified by SDS-PAGE and Western blotting. CSA-binding activity of the three recombinant DBL domains was assayed by ELISA. RESULTS: The target genes were amplified with the length of 996 bp, 859 bp and 894 bp. The constructed recombinant plasmids were identified by enzyme digestion and DNA sequencing. The recombinant proteins (DBL4/5/6) were purified, the relative molecular mass of DBLfA, DBL5 and DBL6 was Mr 439 800, Mr 34,500 and Mr 36,000, respectively. The purified protein has been confirmed with immunogenicity by Western blotting. The results of adhesion experiment indicated that A405 values of DBL5 domain with different concentration were significantly higher than that of DBLA and DBL6. CONCLUSION: The three recombinant proteins (DBLA/5/6) of VAR2CSA DBL domains were expressed, and DBL5 domain has high binding affinity with CSA.

[Polysaccharide and molecular pathogenesis of Plasmodium falciparum].

In the interaction of Plasmodium falciparum with human cells, sporozoite adheres to the receptor of the liver endothelial cell, then invades to liver. Merozoite binds to the surface of red blood cells, and invades to erythrocyte. The adhesion of membrane protein of the infected erythrocytes to the surface molecules of vascular endothelial cell in the vital organs leads to the obstruction of blood circulation eventually. The adhesion is mediated by interaction between parasite-derived ligands and the negative charged polysaccharides on the surface of host cells. This review is to discuss the molecular mechanisms in the host-parasite interactions.

[Screening for relevant proteins involved in adhesion of Cryptosporidium parvum sporozoites to host cells].

The Cryptosporidium parvum T7 phage display library was screened by using Caco-2 cells. Five specific gene fragments were identified by blasting sequences in GenBank, one of which encoding the CP2 protein was previously identified as a surface molecule of sporozoites and involved in parasite invasion. The others are hypothetic proteins with unknown functions. Bioinformatic analysis of these proteins indicated that they may be involved in the host-parasite interactions.

[Research and perspectives in parasitology].

This article reviews the recent achievements in parasitology including new diagnostic techniques, molecular mechanism of parasitic pathogenesis, drug resistance, antigenic variation, parasite genomics and proteomics. The perspective development in the area is also discussed.

[Construction and screening of recombinant fowlpox virus expressing Eimeria tenella F2 hybrid strain SO7 gene].

OBJECTIVE: To construct the recombinant fowlpox virus expressing Eimeria tenella F2 hybrid strain SO7 gene. METHODS: A recombinant expression plasmid pUTA-SO7 was constructed by inserting the SO7 gene of Eimeria tenella F2 hybrid strain into downstream of a hybrid poxvirus promoter which was flanked by the TK gene of fowlpox virus (FPV). The constructed pUTA-SO7 was firstly transfected into chicken embryo fibroblast cells(CEF) pre-infected with FPV strain 282E4 by using liposome, then the viruses resulted from the transfection were selected for 2 passages by culturing in CEF cells with MEM medium containing 40 mg/L 5-bromo-2-deoxy-uridine (BrdU). The selected viruses were plaque-purified in CEF cultured with MEM medium without BrdU. RESULTS Polymerase chain reaction (PCR), indirect immunofluorescence assay and Western blotting showed that S07 gene was expressed in recombinant fowlpox virus. CONCLUSION: The recombinant FPV (rFPV) expressing the SO7 gene has been obtained.

[Cloning and sequence analysis of a partial gene of Trichomonas vaginalis dsRNA virus].

A pair of degenerate primers were designed following the published nucleotide sequence of Trichomonas vaginalis virus(U08999, NC003873, NC003824, NC003834). Using the total nucleic acids extracted from the Trichomonas vaginalis as template, RT-PCR was performed with the primers to obtain a fragment of the TVV. The product was cloned, sequenced and compared with the sequences available in the GenBank. The size of the amplified gene was 1 454bp, which shares 82.9% sequence identity with the Trichomonas vaginalis virus T1.

[Development of hybrid strains from geographic isolates of Eimeria tenella and their immunoprotection].

OBJECTIVE: To cultivate hybrid strains from three geographic isolates of Eimeria tenella and to explore the possibility of developing vaccine candidates. METHODS: Three parental strains were selected from five geographic isolates of E. tenella through immune experiment, and hybrid strains were cultivated. The genomic DNA of the three parental strains and their filial generation were analyzed by random amplified polymorphic DNA (RAPD) technique with 30 optimization primers screened from 200 primers, the hybrid strains were isolated from the filial generation by RAPD. Chicken were inoculated with hybrid strains, and challenged with different strains to compare the immunogenicity and immunoprotection. RESULTS: Immunogenicity and immunoprotection of the three strains isolated from Guangzhou, Baoding and Changchun were stronger than those of other strains. Hybridization was performed to cultivate hybrid strain. Two hybrid strains were isolated from Changchun x Baoding and Guangzhou x F1 by RAPD. The result of immune experiment proved that immunoprotecion of F1 and F2 were higher than their parental strains. CONCLUSION: Two hybrid strains have been cultivated from the three geographic isolates of E. tenella, with the immunogenicity of their parental strains. Chicken immunized by F2 strain have shown strong resistance against the infection of the geographic strains, with an average protection rate of 84%.

[Immunoprotection induced by recombinant plasmids pVAX1-Mzp5-7 of Eimeria tenella].

OBJECTIVE: To observe the immunoprotection of recombinant plasmids of lambda Mzp5-7 to chicken challenged with Eimeria tenella (E. t) oocysts. METHODS: All the chickens were immunized with the recombinant plasmids by different inoculation pathways, three times on d7, d14 and d21, and challenged with E. t oocysts. The response of specific humoral and cell immunity were detected by the immunological methods such as the specific antibody response, the ratio of CD4+/CD8+ T cells. RESULTS: The results showed that the recombinant plasmids can induce immune response. The specific immune responses were strengthened with the increase of immunization times. The difference of the immunity indexes among experiment groups was not significant, while the ratio of CD4+/CD8+ T cells and antibody titer were significantly higher in experiment groups than those in the control groups. The number of oocysts shed in experiment groups decreased and the shedding duration was shorter significantly. The body weight of the chicken in experiment groups increased significantly and the lesion in cecum was slighter than that of control groups. CONCLUSION: The recombinant plasmids of lambda Mzp5-7 show an immunoprotection to chicken challenged with E. t oocysts.

[Cloning of a species-specific gene fragment from Cryptosporidium parvum and the development of diagnostic PCR primers].

OBJECTIVE: To develop a pair of diagnostic PCR primers for Cryptosporidium parvum. METHODS: A species-specific gene fragment of C. parvum was obtained through RAPD analysis. After the fragment was isolated, purified, cloned and sequenced, a pair of primers FF was designed and synthesised based on the sequence. With the primers, the anticipated fragment in size of 603 bp was amplified by PCR from 2 American strains and 4 Chinese strains of C. parvum. The samples of 35 rabbits feces and 55 human feces were detected by PCR with primers FF and 021, the latter was a species-specific diagnostic primer reported by Morgan. RESULTS: All six strains amplified by the primers FF showed same detection rate with 021. Sensitivity test indicated that DNA of 1 oocyst per gram of feces could be detected by the PCR. CONCLUSION: The primers FF showed high specificity and sensitivity, and can be used for diagnosing Crytosporidium parvum infection.

[Cloning and sequencing of the gp23 gene encoding a surface antigen on sporozoites of Cryptosporidium parvum].

OBJECTIVE: To clone and sequence the gp23 gene encoding a surface antigen on the sporozoites of Cryptosporidium parvum. METHODS: Genomic DNA was isolated from oocysts of Cryptosporidium parvum. The gp23 gene was amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector and sequenced by the methods of dideoxy-mediated chain termination. RESULTS AND CONCLUSION: The gp23 gene was 345 bp in length and included an open reading frame encoding a protein of 114 amino acid residues. The homology of the nucleotide and amino sequences of the gp23 gene was 97.3% and 98.2% to that in the GenBank, respectively. The gp23 gene cloned contained 6 nucleotides more than that in the GenBank.