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Objective To investigate the cell composition and the transcriptional characteristics in microenvironments of hepatic tissues in mice at late stage of Echinococcus multilocularis infection at a single-cell level. Methods Peri-lesion and paired distal hepatic specimens were collected from two BALB/c mice (6 to 8 weeks old) infected with E. multilocularis for single-cell RNA sequencing. The Seurat package in the R software was employed for quality control of data, multi-sample integration and correction of batch effects, and uniform manifold approximation and projection (UMAP) algorithm was used for cell clustering. Cell types were annotated using classical marker genes. Differentially expressed genes were screened in each cell type through differential gene expression analysis, and the biological roles of cells were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results A total of 43 710 cells from peri-lesion and distal hepatic tissues of E. multilocularis-infected mice were analyzed, and were classified into 11 cell types, including neutrophils, T cells, macrophages, granulocyte-monocyte progenitor cells, B cells, plasma cells, basophils, hepatic stellate cells, endothelial cells, hepatocytes, and platelets. T cells were the largest population of immune cells in the microenvironment of hepatic tissues, including five CD4+ T cell subsets, two CD8+ T cell subsets and phosphoantigen-reactive γδT cells. The proportions of CD4+ helper T cells and cytotoxic CD4+ T cells decreased and the proportion of T helper 2 (Th2) cells increased in peri-lesion tissues relative to distal hepatic tissues. In addition, the differentially expressed genes in Th2 cells were associated with negative regulation of the immune system, and the highly expressed genes in cytotoxic CD4+ T cells correlated with activation of the immune system. Conclusions Single-cell RNA sequencing deciphers the cell composition and distribution in microenvironments of hepatic tissues from mice infected with E. multilocularis, and the increased proportion of Th2 cells in peri-lesion hepatic tissues may be associated with formation of immunosuppressive microenvironments.
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Echinococcosis is an age-old disease that causes serious damage to the animal husbandry and the human health perennially. As a newly discovered species of Echinococus, E. shiquicus has the potential public health significance and could be a potential parasitic zoonosis. In this review, its etiology, life cycle, epidemiology, detection and diagnoses, public health etc. are discussed or summarized. Also, a series of comparisons among E. granulosus, E. multilocularis and E. shiquicus are made.
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This review focuses on biological characteristics of Echinococcus f elidis including molecular genetic markers , species status ,host coverage ,geographical distribution ,epidemiological implications ,phylogeny ,and evolution .The molecular genetic markers are involved in mitochondrial cox1 (cytochrome C oxidase subunit 1) and nad1 (nicotinamide adenine dinucle-otide dehydrogenase subunit 1) genes ,nuclear protein-coding gene sequences such as elp (ezrin-radixin-moesin-like protein) , e f1a (elongation factor 1 alpha) ,pepck (phosphoenolpyruvate carboxykinase ) ,pold (DNA polymerase delta ) ,and ribosome RNA gene sequences such as ITS1 and 18S rRNA .The establishment of species status is based on distinctly discriminated mor-phological characteristics such as hooks on the rostrum with apparent rugae ,the special definitive host (lions) ,and divergence of DNA sequences ,etc .between E . f elidis and other Echinococcus species .In brief ,the review has provided researchers and ex-perts in the field of echinococcosis with fundamental background knowledge and guidelines for future research directions ,clinical and epidemiological investigations ,and prevention and control of echinococcosis .
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Echinococcosis (hydatid disease) caused by the metacestodes of Echinococcus granulosus tapeworm is a seri-ous zoonotic infection ,which leads to a large amount of economic loss in human and animals .It needs to be prevented urgently . The EG95 protein is highly conserved and crucial for survival and development of E .granulosus in the host ,which means that it is one of the potential candidate antigens for vaccines characterized so far .Great effort has been made to construct and ex-press the recombinant EG95 (rEG95) gene in various kinds of expression systems in order to obtain an efficient defined antigen . This review will summarize research progress on expression of rEG95 in different vector systems .
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To obtain the recombinant 18 kD protein with high purity and normal bioactivity of Cysticercus cellulosae (rCE18), E. coli cells with the rCE18 were disrupted ultra-sonically, and the inclusion bodies were washed with a solution containing 0.2% deoxycholic acid sodium (DOC)and 2% DOC, respectively. Then they were denatured with 0.9% sodium lauroyl sarcosine (SKL) followed by dialysis and gel filtration to refold and purify the target protein. At the same time, this method was compared with GST-FF affinity chromatography and recovering from SDS-PAGE gel. Biological activity of purified rCE18 was analyzed with indirect ELISA, and the purity of the products was identified using SDS-PAGE. The purity of refolded inclusion bodies exceeded 60% and the total recovery of activated protein rCE18 was about 41.3%. The specificity of rCE18 reached up to 97.2% using indirect ELISA. An effective way for purifying and refolding rCE18 expressed in E. coli as inclusion bodies was established, rCE18 with higher purity and activity was obtained, which has the potential for developing diagnosis methods of porcine cysticercosis.
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Animais , Antígenos de Helmintos , Genética , Alergia e Imunologia , Cromatografia em Gel , Cysticercus , Genética , Alergia e Imunologia , Metabolismo , Escherichia coli , Genética , Metabolismo , Corpos de Inclusão , Metabolismo , Renaturação Proteica , Proteínas Recombinantes , Genética , Alergia e ImunologiaRESUMO
Objective To analyze the relationship between genetic variability and evolution among Trypanosoma brucei (including T. b. brucei, T. b. rhodesiense and T. b. gambiense), T. evansi and T. equiperdum isolates. Methods Genomic DNAs of 26 trypanosome isolates were amplified by a mobile genetic elements (MGE)-PCR technique and cluster analysis was performed based on the molecular profiles with Neighbor-Joining method. Results The genetic variability among trypanosome isolates examined was obvious with an average genetic distance of 41.2% (ranged from 0 to 100%). Similarity coefficient among T. brucei isolates was 41.15% which was lower than that between T. evansi and T. equiperdum isolates. The closest relationship was found between T. evansi and T. brucei isolates with a similarity coefficient of 62.94%. The genetic variability between T. b. rhodesiense and T. b. brucei isolates was higher than that among T. b. gambiense isolates. Conclusion Species and subspecies in Trypanozoon displayed a higher genetic variability; T. equiperdum isolates collected from China and from South America, and T. evansi isolates from China and from South America, should have a similar origin.
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Objective To clone the genes coding for cysteine proteases (CPs, TvCPs) from Trichomonas vaginalis and to analyze their genetic variations with the related sequences from NCBI database (GenBank) and T. vaginalis Genome Project database from The Institute for Genomic Research (TIGR). Method TvCP genes were amplified using PCR, and inserted into vector pET28b or pBS-T. The recombinant plasmids were then transformed to Escherichia coli BL21 or Top10 strain. The recombinant plasmids were used for sequencing. Homologous TvCP genes were blasted based on NCBI GenBank and TIGR T. vaginalis Genome Project database. The sequences of cloned TvCP genes were aligned and clustered by Clustal X (1.83 version) with retrieved sequences. Comparisons of amino acids among cathepsin L-like TvCPs, human L-like cathepsins and papaya papain were performed using DNAstar software, and their phylogenic tree was constructed based on neighbor-joining method using Clustal X. Results Two TvCP3 clones and one TvCP2 had a high identity of more than 99% with their responding TvCPs. Three clones of TvCP4 genes, GZ-CP4-clone 1-3, belonged to two members of a family showing a high percentage identity of more than 97.5% with the sequences of TvCP4 genes from databases (GenBank and TIGR) both at amino acid and nucleotide levels. Nine homologous TvCP4 pro-enzymes with 304 amino acids and other two members with deletions of N-terminal sequence existed in T. vaginalis sharing a similarity of 62.3-96.7% amino acids, which may evolve by means of gene replication and deletion. TvCP1-4, TvCP12, TvCP25 and CP65 had an identity of 61-88.2% at amino acid levels. So far, all reported sequences of C1 family from T. vaginalis belonged to capanthesin L-like subfamily with the same enzymatic active sites, conserved cysteine residues and similar structural features such as ERFNIN-like motif in pro-enzyme region, suggesting that they might result from gene duplication and mutations. Conclusion TvCPs belong to cathepsin L-like family with genetic diversity, but they have the same active amino acid residues, cysteine residues and similar structural characteristics, suggesting that they may derive from one ancestor.
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Objective To construct the recombinant secretion type BCG-Eg95 vaccine of Echinococcus granulosus (rsBCG-Eg95). Methods BCG-Ag85B signal sequence with 117 bp and Eg95 gene with 471 bp were amplified from the genome of BCG and pGEX-4T-Eg95 by PCR,respectively. BCG-Ag85B signal coding gene and Eg95 gene were cloned into E. coli-BCG shuttle-vector pMV261 to get the recombinant plasmid pSMEg95,which was confirmed by restriction endonuclease digestion,PCR amplification and gene sequencing. These recombinant plasmids were introduced into BCG by electroporation for the construction of rsBCG-Eg95 vaccine. The rsBCG-Eg95 positive clones were screened by Kan+ and identified by PCR amplification. Results BCG-Ag85B signal sequence coding gene and Eg95 coding gene were successfully cloned into pMV261,which was confirmed by restriction endonuclease digestion,PCR amplification and sequencing of the plasmid pSMEg95. The plasmids were introduced into BCG and confirmed as the recombinant secreting BCG-Eg95 vaccine of E. granulosus (rsBCG-Eg95). Conclusion The recombinant secretion type BCG-Eg95 vaccine (rsBCG-Eg95) of E. granulosus with BCG-Ag85B signal sequence and Eg95 gene has been constructed.