Xinbing Yu

The draft genome of the carcinogenic human liver fluke Clonorchis sinensis.

BackgroundClonorchis sinensis is a carcinogenic human liver fluke that is widespread in Asian countries. Increasing infection rates of this neglected tropical disease are leading to negative economic and public health consequences in affected regions. Experimental and epidemiological studies have shown a strong association between the incidence of cholangiocarcinoma and the infection rate of C. sinensis. To aid research into this organism, we have sequenced its genome.ResultsWe combined de novo sequencing with computational techniques to provide new information about the biology of this liver fluke. The assembled genome has a total size of 516 Mb with a scaffold N50 length of 42 kb. Approximately 16,000 reliable protein-coding gene models were predicted. Genes for the complete pathways for glycolysis, the Krebs cycle and fatty acid metabolism were found, but key genes involved in fatty acid biosynthesis are missing from the genome, reflecting the parasitic lifestyle of a liver fluke that receives lipids from the bile of its host. We also identified pathogenic molecules that may contribute to liver fluke-induced hepatobiliary diseases. Large proteins such as multifunctional secreted proteases and tegumental proteins were identified as potential targets for the development of drugs and vaccines.ConclusionsThis study provides valuable genomic information about the human liver fluke C. sinensis and adds to our knowledge on the biology of the parasite. The draft genome will serve as a platform to develop new strategies for parasite control.

Nestin expression in different tumours and its relevance to malignant grade.

Background: Nestin, an intermediate filament (IF) protein, is expressed in proliferating progenitor cells of developmental and regenerating tissues, and is identified as a neuroepithelial precursor cell marker. Recently, nestin was detected in some neoplasms such as glioma, ependymoma, melanoma, rhabdomyosarcoma, gastrointestinal stromal tumour (GIST), and testicular stromal tumour. Moreover, the expression intensity of nestin exhibited significant correlation with the malignant grade of glioma. Aims: To detect the expression of nestin in different tumours and to analyse the relationship between the expression of nestin and the malignant grade of the tumours. Methods: Formalin-fixed and paraffin-embedded surgical samples of neoplastic tissues were obtained from the Department of Pathology of Sun Yat-sen University. Histological analysis and immunohistochemical staining for nestin were performed. Histoscores were analysed by semi-quantitative evaluation. Results: Nestin was expressed predominantly in the cytoplasm of angiosarcoma, pancreatic adenocarcinoma and GIST samples, and some tumour cells expressed in the nucleus. There was a statistically significant difference between the histoscore of nestin in high malignant GIST (2.2366 (0.6920)) and that in low malignant GIST (1.3783 (0.4268)) (p = 0.003); and also between that in high malignant angiosarcoma (1.9188 (0.2069)) and that in low malignant angiosarcoma (0.6474 (0.3273)) (p = 0.000). Cavernous angioma did not express nestin. The histoscore of nestin in high malignant pancreatic adenocarcinoma (7/14) was 1.1767 (0.4676), and that in low malignant pancreatic adenocarcinoma (3/8) was 0.6577 (0.0056) (no significant difference, p = 0.112). Conclusions: Results suggest that the expression of nestin may play an important role in the development of some neoplasms such as GIST and angiosarcoma.

The Carcinogenic Liver Fluke, Clonorchis sinensis: New Assembly, Reannotation and Analysis of the Genome and Characterization of Tissue Transcriptomes

Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.

Proteomic identification of differently expressed proteins responsible for osteoblast differentiation from human mesenchymal stem cells

Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.

Clonorchis sinensis enolase: Identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products

Enolase plays a key role in energy metabolism and development of most organisms. We isolated a gene encoding enolase from Clonorchis sinensis (C. sinensis) adult cDNA library and expressed the recombinant protein in Escherichia coli. C. sinensis enolase (Csenolase) was identified as both an excretory/secretory product and a tegumental component of C. sinensis by western blot analysis. The transcriptional level of Csenolase was examined at adult worm, metacercaria, cercaria and egg of C. sinensis, and results showed that Csenolase is transcribed at the four life stages of C. sinensis while showing a significant higher expression level at the stage of adult worm. Immunohistochemical localization indicated that Csenolase was specifically deposited on the tegument of adult worm and cyst wall of metacercaria. Ligand blot assay revealed a specific characteristic of dose-dependent plasminogen-binding activity of Csenolase and kinetic parameters were explored using 2-phospho-D-glycerate (2-PGA) as the primary substrate by monitoring the conversion of nicotinamide-adenine dinucleotide (NADH) into nicotinamide adenine dinucleotide (NAD). In addition, Csenolase exhibited active enzyme activity in catalytic reactions while the anti-Csenolase serum inhibited the enzyme activity. In vitro incubation experiments revealed that Csenolase might play key roles in the growth of the parasites. In conclusion, Csenolase is an important glycolytic enzyme required for the development of C. sinensis, and may be a potential vaccine candidate and drug target against C. sinensis infection.

Generation of functional hepatocytes from mouse induced pluripotent stem cells

Induced pluripotent stem cells are derived from somatic cells by forced expression of several transcriptional factors. Induced pluripotent stem cells resemble embryonic stem cells in many aspects, such as the expression of certain stem cell markers, chromatin methylation patterns, embryoid body formation and teratoma formation. Therefore, induced pluripotent stem cells provide a powerful tool for study of developmental biology and unlimited resources for transplantation therapy. Here we reported the successful induction of mouse induced pluripotent stem cells and a simple and efficient process for generation of functional hepatocytes from mouse induced pluripotent stem cells by sequential addition of inducing factors. These induced pluripotent stem cell‐derived hepatocytes, just as mouse embryonic stem cell‐derived hepatocytes, expressed hepatic lineage markers including CK7, CK8, CK18, CK19, alpha‐fetoprotein, albumin, Cyp7a1, and exhibited functional hepatic characteristics, including glycogen storage, indocyanine green (ICG) uptake and release, low‐density lipoprotein (LDL) uptake and urea secretion. Although we observed some variations in the efficiency of hepatic differentiation between induced pluripotent stem cells and common mouse embryonic stem cell lines, our results indicate that mouse induced pluripotent stem cells can efficiently differentiate into functional hepatocytes in vitro, which may be helpful for the study of liver development and regenerative medicine. J. Cell. Physiol. 222: 492–501, 2010.

Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis

A gene encoding a homologue of phospholipase A(2) was identified from the Clonorchis sinensis adult cDNA plasmid library. The deduced amino acid sequence including a signal peptide that has 28-46% identity with secretory phospholipase A(2), group III (group III sPLA(2)) of other species. It also has typical features of group III sPLA(2)s including 10 cysteines, the key residues of the Ca(2+) loop and catalytic site. The recombinant protein encoded by this gene expressed in Escherichia coli showed a product of about 34kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A(2) of C. sinensis (CsGIIIsPLA(2)) was an excretory-secretory product (ES product). The enzyme activity of the recombinant protein was determined using phosphatidylcholine as substrates. The result revealed that the protein was a Ca(2+)-dependent PLA(2). Both MTT test and cell cycle analysis of LX-2 showed a higher percentage of cells are in proliferation phase. Semi-quantitative RT-PCR experiments demonstrated an up-regulated expression of collagen III in these cells after incubation with the recombinant protein. We also identified that the recombinant CsGIIIsPLA(2) could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA(2) on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA(2) might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis.

Experimental model in rats for study on transmission dynamics and evaluation of Clonorchis sinensis infection immunologically, morphologically, and pathologically

This study aims to gain a better insight into the transmission patterns and immunologic profile of Clonorchis sinensis infection and make a headway on the pathogenesis regarding cholangiocarcinoma and hepatic lesions. Experimental models orally infected by C.sinensis metacercariae were constructed in rats. Immunological assays were performed to measure serum level of IgA, IgE, IgG1, IgG2a, IFN-γ, and IL-4. Infection parameters were assessed by worm recovery rate, eggs per gram faece and worm size. Pathological sections with livers were managed with immunofluorescence, hematoxylin, eosin, and Massons trichrome staining to evaluate the hepatic pathological changes. Interestingly, rats infected with only one C.sinensis metacercariae even gained a high worm recovery rate of 83.3% compared with rats infected with more metacercariae. Serological changes according to different infection doses indicated that immune response presented a tendency to Th2 type by expressing transient high level of IgG1, IL-4, and IgE. Hepatic tissues appeared inflammatory and fibrotic, revealed by different stainings. Intrahepatic bile ducts displayed cholangiectasis and proliferation with excreted/secreted antigen histologically located. C.sinensis, as a fish-borne zoonosis, presented novel transmission patterns which explained high infection rate in endemic areas; infection rate of C.sinensis was frequency-dependent and dose-related. Humoral immunity played a prevalent role in resisting to C.sinensis based on the rat models. C.sinensis infection played an undoubted role in biliary and hepatic diseases.

Surface display of Clonorchis sinensis enolase on Bacillus subtilis spores potentializes an oral vaccine candidate

Clonorchis sinensis (C. sinensis) infections remain the common public health problem in freshwater fish consumption areas. New effective prevention strategies are still the urgent challenges to control this kind of foodborne infectious disease. The biochemical importance and biological relevance render C. sinensis enolase (Csenolase) as a potential vaccine candidate. In the present study, we constructed Escherichia coli/Bacillus subtilis shuttle genetic engineering system and investigated the potential of Csenolase as an oral vaccine candidate for C. sinensis prevention in different immunization routes. Our results showed that, compared with control groups, both recombinant Csenolase protein and nucleic acid could induce a mixed IgG1/IgG2a immune response when administrated subcutaneously (P<0.001), intraperitoneally (P<0.01) and intramuscularly (P<0.001) with worm reduction rate of 56.29%, 15.38% and 37.42%, respectively. More importantly, Csenolase could be successfully expressed as a fusion protein (55kDa) on B. subtilis spore indicated by immunoblot and immunofluorescence assays. Killed spores triggered reactive Th1/Th2 immune response and exhibited protective efficacy against C. sinensis infection. Csenolase derived oral vaccine conferred worm reduction rate and egg reduction rate at 60.07% (P<0.001) and 80.67% (P<0.001), respectively. The shuttle genetic engineering system facilitated the development of oral vaccine with B. subtilis stably overexpressing target protein. Comparably vaccinal trails with Csenolase in different immunization routes potentialize Csenolase an oral vaccine candidate in C. sinensis prevention.

A novel biomimetic composite scaffold hybridized with mesenchymal stem cells in repair of rat bone defects models.

In this study, the in vivo bone-regenerative potential of a novel bioactive glass-collagen-hyaluronic acid-Phosphatidylserine (BG-COL-HYA-PS) composite scaffold hybridized with mesenchymal stem cells (MSCs) was investigated in a rat bone defect model. HrGFP-labeled MSCs were cultured for 2 weeks on the BG-COL-HYA-PS scaffold before implantation into the defect. A cell-free scaffold and an untreated defect were used as controls. The regeneration process was evaluated by histology, X-ray, and mechanical rigidity experiments at different time points post-implantation. The results revealed that BG-COL-HYA-PS scaffold exhibited a low inflammatory response and foreign body response within 3 weeks. At week 6, those responses disappeared following the resorption of scaffolds and the formation of new bone. Compared with the pure scaffold or empty group, the introduction of MSCs into the porous scaffold dramatically enhanced the efficiency of the new bone formation and biomechanical property of the femur. In addition, the transplanted MSCs could survive for up to 3 weeks or longer. The results demonstrated that the BG-COL-HYA-PS scaffold was biocompatible and osteoconductive and the transplanted MSCs with the scaffold enhanced the healing of the bone defect.