RAMP1 Protects Hepatocytes against Ischemia-reperfusion Injury by Inhibiting the ERK/YAP Pathway.

Background and Aims: Hepatic ischemia-reperfusion injury (HIRI) is a prevalent complication of liver transplantation, partial hepatectomy, and severe infection, necessitating the development of more effective clinical strategies. Receptor activity-modifying protein 1 (RAMP1), a member of the G protein-coupled receptor adapter family, has been implicated in numerous physiological and pathological processes. The study aimed to investigate the pathogenesis of RAMP1 in HIRI. Methods: We established a 70% liver ischemia-reperfusion model in RAMP1 knockout (KO) and wild-type mice. Liver and blood samples were collected after 0, 6, and 24 h of hypoxia/reperfusion. Liver histological and serological analyses were performed to evaluate liver damage. We also conducted in-vitro and in-vivo experiments to explore the molecular mechanism underlying RAMP1 function. Results: Liver injury was exacerbated in RAMP1-KO mice compared with the sham group, as evidenced by increased cell death and elevated serum transaminase and inflammation levels. HIRI was promoted in RAMP1-KO mice via the induction of hepatocyte apoptosis and inhibition of proliferation. The absence of RAMP1 led to increased activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway and yes-associated protein (YAP) phosphorylation, ultimately promoting apoptosis. SCH772984, an ERK/MAPK phosphorylation inhibitor, and PY-60, a YAP phosphorylation inhibitor, reduced apoptosis in in-vitro and in-vivo experiments. Conclusions: Our findings suggest that RAMP1 protects against HIRI by inhibiting ERK and YAP phosphorylation signal transduction, highlighting its potential as a therapeutic target for HIRI and providing a new avenue for intervention.

Case Report: Primary hepatic neuroendocrine tumor: two cases report with literature review.

Background & Aims: Primary hepatic neuroendocrine tumors (PHNETs) are rare malignant liver tumors that present diagnostic challenges owing to their rarity and absence of specific clinical features. This study aimed to investigate the characteristics of this rare liver tumor to enhance our understanding of the disease, improve diagnostic accuracy, and explore standardized diagnostic and treatment approaches. Case description: During physical examination, two elderly women, aged 64 and 74 years, were found to have liver masses. 18F-FDG Positron Emission Tomography-Computed Tomography (18F-FDG PET-CT) and Ga68-DOTATATE PET-CT scans of both individuals revealed multiple liver masses that were initially suspected to be hepatic neuroendocrine tumors. Subsequent puncture pathology confirmed the diagnosis of neuroendocrine tumors. Furthermore, in Case 1, the tumor was also detected by 18F-FDG PET-CT in the lung, suggesting a metastatic tumor, in conjunction with liver immunohistochemistry and imaging findings. Laboratory tests revealed no significant abnormalities in liver function or autoimmune liver disease indicators, and there was no evidence of viral hepatitis infection. However, partial hepatectomy was not indicated for cases with distant metastasis or multiple space-occupying lesions. Individualized treatment approaches have been developed for such situations. A large portion of the tumor underwent Transarterial Embolization (TAE), and targeted combination chemotherapy or endocrine therapy was administered based on the pathological results. During regular follow-ups a 13 and 12 months, the tumor remained stable. The patients' quality of life was good, and their psychological well-being was healthy. They led active lifestyles, demonstrated a thorough understanding of their disease and its progression, and actively cooperated during the follow-up process. Conclusion: Our findings suggest that a combination of serological, radiological, and immunohistochemical examinations can aid in the diagnosis of PHNET. In addition, we determined that TAE combined with drug therapy could be an effective method for controlling PHNET progression. Regular postoperative follow-ups are important for monitoring the prognosis and tumor progression status of patients with PHNET.

Retraction Notice to: LINC01234/MicroRNA-31-5p/MAGEA3 Axis Mediates the Proliferation and Chemoresistance of Hepatocellular Carcinoma Cells.

[This retracts the article DOI: 10.1016/j.omtn.2019.10.035.].

Case Report: Cetuximab in Combination With Chemotherapy for the Treatment of Multifocal Hepatic Metastases From Colorectal Cancer Guided by Genetic Tests.

Hepatic metastases were reported in up to 70% of colorectal cancer patients, among which multifocal hepatic metastasis represents one of the complications that lead to poor prognosis. The majority of the patients carrying multifocal hepatic metastases required pharmaceutical treatments to reduce the tumor size prior to surgical resection. However, the clinical responses to pharmaceutical agents were difficult to predict due to the heterogeneous nature of the multifocal tumors. Here, we report a case with multifocal hepatic metastases from colorectal cancer that was resistant to the primary chemotherapy and Bevacizumab plus chemotherapy, but responded to the combined therapy of Cetuximab and FOLFOX. Genetic tests had revealed that the tumor was highly metastatic due to the mutations of the WNT signaling pathway, and the metastatic tumors might be sensitive to Cetuximab. Consistent with the molecular characterizations, the metastatic tumors continue to emerge after chemotherapy, and rapidly relapsed in great numbers after liver resection. However, the combined therapy of Cetuximab and FOLFOX guided by the genetic tests significantly reduced the size and number of metastatic tumors. To conclude, deciphering the mutation profiles of multifocal metastatic tumors may guide the determination of treatment tactics, which may benefit the patients with non-resectable advanced carcinoma.

LINC01234/MicroRNA-31-5p/MAGEA3 Axis Mediates the Proliferation and Chemoresistance of Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is a prevalent malignancy characterized by aggressiveness and poor prognosis; however, the molecular mechanism remains to be fully identified. Based on the analysis of The Cancer Genome Atlas (TCGA) database, melanoma-associated antigen A3 (MAGEA3) and long non-coding RNA (lncRNA) LINC01234 were upregulated in HCC and associated with poor prognosis of HCC. We investigated the mechanism of how MAGEA3 and LINC01234 influenced HCC cellular functions and cisplatin resistance. MAGEA3 depletion inhibited proliferation, invasion, and cisplatin resistance of HepG2 cells and Huh7 cells in vitro, reduced resistance-associated protein 2 (MRP2), MRP3, and multidrug resistance protein 1 (MDR-1) expression, and elevated ALB expression. RNA pull-down and RIP assays identified the binding of LINC01234 and MAGEA3 to microRNA-31-5p (miR-31-5p). LINC01234 could restore MAGEA3 expression by binding to miR-31-5p. Furthermore, we delivered plasmids into HepG2 cells and Huh7 cells to alter the expression of LINC01234 and miR-31-5p. When miR-31-5p was downregulated, the proliferation and invasion of HepG2 cells and Huh7 cells were enhanced and the cisplatin-induced apoptosis was inhibited, while LINC01234 knockdown could diminish the effects caused by miR-31-5p depletion. In summary, these data highlight the vital role of MAGEA3/LINC01234/miR-31-5p axis in the HCC progression and chemoresistance of HCC cells.

Long Non-coding RNA FENDRR Acts as a miR-423-5p Sponge to Suppress the Treg-Mediated Immune Escape of Hepatocellular Carcinoma Cells.

Long non-coding RNAs (lncRNAs) have been known to partake in the development and the immune escape of hepatocellular carcinoma (HCC). The initial microarray analysis of GSE115018 expression profile revealed differentially expressed lncRNA fetal-lethal non-coding developmental regulatory RNA (FENDRR) in HCC. Therefore, this study's main purpose was to explore the mechanism of tumor suppressor lncRNA FENDRR in regulating the immune escape of HCC cells. Notably, it was further validated through this study that lncRNA FENDRR competitively bound to microRNA-423-5p (miR-423-5p), and miR-423-5p specifically targeted growth arrest and DNA-damage-inducible beta protein (GADD45B). The effects that lncRNA FENDRR and miR-423-5p have on the cell proliferation and apoptosis, the immune capacity of regulatory T cells (Tregs), and the tumorigenicity of HCC cells were examined through overexpressing or the knocking down of lncRNA FENDRR and miR-423-5p both in vitro and in vivo. Subsequently, lncRNA FENDRR and GADD45B were revealed to have poor expressions in HCC. Meanwhile, miR-423-5p was highly expressed in HCC. Importantly, overexpressed lncRNA FENDRR and downregulated miR-423-5p diminished cell proliferation and tumorigenicity, and promoted apoptosis in HCC cells, thus regulating the immune escape of HCC mediated by Tregs. Taken conjointly, lncRNA FENDRR inhibited the Treg-mediated immune escape of HCC cells by upregulating GADD45B by sponging miR-423-5p.

MicroRNA-194 inhibits cell invasion and migration in hepatocellular carcinoma through PRC1-mediated inhibition of Wnt/ß-catenin signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is a commonly occurring malignancy accompanied by significant mortality rates. More recently, extensive investigations into microRNA (miRNA) expression profiles have been conducted to identify their ability to inhibit tumors. Thus, this study explored the role of miR-194 in epithelial-mesenchymal transition (EMT), cell invasion and migration through Wnt/ß-catenin signaling pathway by binding to protein regulator of cytokinesis 1 (PRC1) in HCC. METHODS: Initially, HCC related microarray data were retrieved and analyzed, and regulatory miRNAs of PRC1 were predicted accordingly. Next, the roles of miR-194, PRC1, and Wnt/ß-catenin signaling pathway in HCC were determined, with relationship between PRC1 and miR-194 being verified subsequently. The role of miR-194 in cell EMT, migration, proliferation and invasion was evaluated through gain- and loss- function studies. Finally, tumor xenograft in nude mice was induced to assess tumor growth of HCC. RESULTS: miR-194 affected HCC development in Wnt/ß-catenin signaling pathway with putative binding sites to PRC1. MiR-194 could target PRC1. MiR-194 was downregulated while PRC1 was upregulated in HCC tissues. Additionally, miR-194 elevation and PRC1 silencing could suppress EMT, growth, proliferation, invasion, and migration in HCC cells by inactivating Wnt/ß-catenin signaling pathway. CONCLUSION: Taken together, this study demonstrated that miR-194 inhibited EMT, cell invasion and migration through inactivation of PRC1-dependent Wnt/ß-catenin signaling pathway.

Integrator complex subunit 6 (INTS6) inhibits hepatocellular carcinoma growth by Wnt pathway and serve as a prognostic marker.

BACKGROUND: Integrator complex subunit 6 (INTS6) was found to play a tumour suppressing role in certain types of solid tumours. In this study, we wanted to determine the expression level of INTS6 in hepatocellular carcinoma (HCC) and evaluate its clinical characteristics and mechanisms in HCC patients (Lui and Lu, European Journal of Cancer, 51:S94, 2015). METHODS: First, we used a microarray analysis to explore the mRNA expression levels in HCC and paired normal liver tissues; second, we used qRT-PCR to measure the INTS6 mRNA levels in a cohort of 50 HCC tissues and adjacent normal liver tissues; third, we used Western blot analyses to detect the INTS6 protein levels in 20 paired HCC and normal liver tissues; fourth, we used immunohistochemistry to determine the INTS6 expression levels in 70 archived paraffin-embedded HCC samples. Finally, we investigated the suppressive function of INTS6 in the Wnt pathway. RESULTS: Herein, according to the microarray data analysis, the expression levels of INTS6 were dramatically down-regulated in HCC tissues vs. those in normal liver tissues (p<0.05). qRT-PCR and Western blot analyses showed that the INTS6 mRNA and protein expression was significantly down-regulated in tumour tissues compared to the adjacent normal liver tissues (p<0.05). Immunohistochemical assays revealed that decreased INTS6 expression was present in 62.9% (44/70) of HCC patients. Correlation analyses showed that INTS6 expression was significantly correlated with serum alpha-fetoprotein levels (AFP, p =0.004), pathology grade (p =0.005), and tumour recurrence (p =0.04). Kaplan-Meier analysis revealed that patients with low INTS6 expression levels had shorter overall and disease-free survival rates than patients with high INTS6 expression levels (p =0.001 and p =0.001). Multivariate regression analysis indicated that INTS6 was an independent predictor of overall survival and disease-free survival rates. Mechanistically, INTS6 increased WIF-1 expression and then inhibited the Wnt/ß-catenin signalling pathway. CONCLUSION: The results of our study show that down-regulated INTS6 expression is associated with a poorer prognosis in HCC patients. This newly identified INTS6/WIF-1 axis indicates the molecular mechanism of HCC and may represent a therapeutic target in HCC patients.

Pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) as a novel plasma-based biomarker for hepatocellular carcinoma screening.

In this study, we aimed to determine whether the pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) in plasma could be used as a novel approach to screen for and detect hepatocellular carcinoma (HCC). We explored the clinical role of INTS6P1: First, the expression level of INTS6P1 was measured in a cohort of 33 HCC tissue samples and adjacent normal liver tissue, next, the INTS6P1 expression was detected in the culture medium and tumor cells in a cellular experiment, and last, the diagnostic performance of INTS6P1 was examined in an independent cohort of 100 people. The expression level of INTS6P1 was remarkably downregulated in the HCC tissues compared with that in the normal liver tissues (p = 0.0066). In plasma, the INTS6P1 levels were significantly decreased in HCC patients compared with non-HCC patients (p < 0.01). Additionally, we inferred that INTS6P1 might be a prospective biomarker for screening HCC patients in which the serum-AFP levels were lower than 20 ng/ml by the area under the curve-receiver operating characteristic (AUC-ROC) analysis (p < 0.05). Pseudogene INTS6P1 could be used as a novel HCC plasma-based biomarker and might improve the accuracy of HCC screening.

Immunoproteomic analysis of Schistosoma japonicum schistosomulum proteins recognized by immunoglobulin G in the sera of susceptible and non-susceptible hosts.

The aim of this study was to search for immunogenic schistosomula proteins in the hope of identifying novel intervention targets. Schistosomula proteins were analyzed by immunoproteomic which the probes were sera derived from BALB/c mice (susceptible hosts) and Microtus fortis (resistant hosts). A total of 116 immunoreactive proteins recognized by 10 days post-infected BALB/c mice, M. fortis sera, and uninfected M. fortis sera were selected for further analysis. Finally, 95 protein spots were identified by mass spectrometry (MS) analysis. Bioinformatics analysis showed that the differentially identified immunogenic proteins participated mainly in cytoskeleton organization, cell motility, energy metabolism, responses to stimuli, and protein folding. Many of these proteins were the tegument or excretory-secretory products of schistosomes reported in previous studies. Among of them, Schistosoma japonicum DnaJ (Hsp40) homologue (SjDnaJ) was successfully expressed and the purified recombinant product was evaluated by immunoprotective experiment. After immunization of BALB/c mice with recombinant SjDnaJ, it could induce 34.5% and 48.9% reductions in the numbers of worms and eggs in the liver. These results contribute to a better understanding of the molecular mechanisms underlying the host-parasite relationship and provide a major dataset to facilitate the further development of new vaccine candidates and/or diagnostic markers for schistosomiasis. BIOLOGICAL SIGNIFICANCE: Schistosomiasis is caused by parasitic blood-dwelling flukes in tropical and subtropical areas, and it is one of the world's most prevalent tropical diseases. The lack of effective vaccine and reliable diagnostic methods make this disease difficult to control. In China, S. japonicum can infect more than 40 different susceptible mammals for this parasite. However, M. fortis is the only known mammal where the schistosome cannot develop and it exhibits no significant pathological effects. Many studies' results showed that native antibodies against S. japonicum are present in M. fortis that may have important anti-schistosomiasis roles during the infection process. The aim of this study was to search for immunogenic schistosomula proteins in the hope of identifying novel intervention targets. We present a comparative immunoproteomics analysis of the proteins recognized by susceptible and resistant host antibodies before and 10-days after infections. The results of this analysis will be helpful for identifying the key molecules required for the survival and development of schistosomes. At the same time, the study contributes to a better understanding of the molecular mechanisms underlying the host-parasite relationship associated with schistosomes and they also provide a major dataset to facilitate the further development of new diagnostic assays and/or vaccine candidates for schistosomiasis.

Retinoic acid receptor-related receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma.

Retinoic acid receptor-related receptor alpha (RORalpha) has been proven to play a tumor suppressive role in certain types of solid tumors. However, the clinical characteristic of RORalpha has not been reported by far. This study investigated the expression of RORalpha in hepatocellular carcinoma (HCC) and evaluated its relationship with clinical parameters and prognosis in HCC patients. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to detect RORalpha expression levels in 20 paired HCC and corresponding adjacent non-cancerous tissues. Immunohistochemistry was performed on 100 archived paraffin-embedded HCC samples. Statistical analyses evaluated the correlations between RORalpha expression and clinicopathological features. qRT-PCR showed that RORalpha mRNA expression was significantly down-regulated in tumors compared to the adjacent non-cancerous tissues, and Western blots found that RORalpha protein expression was also reduced in tumor tissues. Immunohistochemical assays revealed that decreased RORalpha expression was present in 65 % of HCC patients. Correlation analyses showed that RORalpha expression was significantly correlated with serum alpha fetoprotein (AFP, p = 0.005), pathology grade (p < 0.001), tumor recurrence (p = 0.008), and vascular invasion (p < 0.001). Kaplan-Meier analysis revealed that patients with low RORalpha expression levels had a shorter overall and disease-free survival than patients with high expression (p < 0.001 and p = 0.002, respectively). Multivariate regression analysis indicated that RORalpha was an independent predictor for overall survival and disease-free survival. In conclusion, the results of our study showed that down-regulated RORalpha expression was associated with poorer prognosis in HCC patients. RORalpha may be a new potential prognostic marker for HCC patients.

Multi-strain infections and 'relapse' of Leucocytozoon sabrazesi gametocytes in domestic chickens in southern China.

Leucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to the poultry industry. Although the prevalence and distribution of two Leucocytozoon species (L. sabrazesi and L. caulleryi) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from seven sites in four provinces of China to identify Leucocytozoon infection, characterized parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (coxIII) and cytochrome b (cytb) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of L. sabrazesi strains. Monitoring gametocytemia in blood samples from infected chickens over time showed 'relapse' or persistence of low-level gametocytemia for 4-5 months, which could be explored as an in vivo model for testing drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of L. sabrazesi and for disease control.

Proteomic analysis of tegument-exposed proteins of female and male Schistosoma japonicum worms.

The interplay between sexes is a prerequisite for female growth, reproductive maturation, and egg production, and the basis of schistosome pathopoiesis and propagation. The tegument is in direct contact with the host environment and its surface membranes are particularly crucial for schistosome survival in the definitive host. In this study, a streptavidin-biotin affinity purification technique combined with LC-MS/MS was used to analyze putative tegument-exposed proteins in female and male adult Schistosoma japonicum worms. In total, 179 proteins were identified in females and 300 in males, including 119 proteins common to both sexes, and 60 female biased and 181 male biased proteins. Some (e.g., serpin and CD36-like class B scavenger receptor) were involved in host-schistosome interactions, while some (e.g., gynecophoral canal protein) were important in the interplay between sexes. Gene Ontology analysis revealed that proteins involved in protein glycosylation and lysosome were highly expressed in females, while proteins involved in intracellular signal transduction, regulation of actin filament polymerization, and proteasome core complex were highly expressed in males. These results might elucidate physiological differences between the sexes. Our study provides new insights into schistosome growth and sexual maturity in the final host and permits the screening of vaccine candidates or drug targets for schistosomiasis.

A retinoid X receptor (RXR1) homolog from Schistosoma japonicum: its ligand-binding domain may bind to 9-cis-retinoic acid.

Retinoid X receptor (RXR) is an important member of the nuclear receptor superfamily of ligand-activated transcription factors that are present in all major groups of metazoans. A full-length cDNA encoding RXR, an orthologue of SmRXR1 in platyhelminth Schistosoma japonicum (SjRXR1) was identified and characterized. The SjRXR1 cDNA is 2806 bp long, and contains an open reading frame encoding a 745 amino acid protein. The deduced SjRXR1 protein sequence which was aligned with RXR proteins from other species revealed a highly conserved DNA binding domain (DBD) and moderately conserved ligand binding domain (LBD). The gene structure of SjRXR1 was analyzed and showed that it consists of seven exons spanning 18.4 kbp. The relative mRNA expression of SjRXR1 was evaluated in six different S. japonicum developmental stages in the final host (days 7-42 post-infection) and showed higher expression at days 21 and 35. In an in vitro study the transcription of SjRXR1 mRNA was shown to increase almost 3-fold and the SjRXR1 protein expression was also upregulated at the 48 h time point by treating the S. japonicum with 5.0 µM 9-cis-retinoic acid (RA). Flow cytometry analysis demonstrated that the percentage of HeLa cells expressing SjRXR1LBD-Myc fusion protein is approximately 11%. Over-expression of SjRXR1LBD-Myc in HeLa cells may result in the inhibition of innate apoptosis of this cancer cell line induced by 9-cis-RA. Our studies suggested that the retinoid signaling pathways may be conserved in the platyhelminth. The full cDNA sequence of SjRXR1 reported here has been submitted to the GenBank with accession no. JX111997.

Gene gun bombardment with DNA-coated golden particles enhanced the protective effect of a DNA vaccine based on thioredoxin glutathione reductase of Schistosoma japonicum.

Schistosomiasis, caused by infection with Schistosoma species, remains an important parasitic zoonosis. Thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR) plays an important role in the development of the parasite and for its survival. Here we present a recombinant plasmid DNA vaccine, pVAX1/SjTGR, to estimate its protection against S. japonicum in BALB/c mice. The DNA vaccine administrated by particle bombardment induced higher protection than by intramuscular injection. All animals vaccinated with pVAX1/SjTGR developed significant specific anti-SjTGR antibodies than control groups. Moreover, animals immunized by gene gun exhibited a splenocyte proliferative response, with an increase in IFN- γ and IL-4. The recombinant plasmid administrated by gene gun achieved a medium protective efficacy of 27.83-38.83% (P < 0.01) of worm reduction and 40.38-44.51% (P < 0.01) of liver egg count reduction. It suggests that different modes of administering a DNA vaccine can influence the protective efficacy induced by the vaccine. Interestingly, from the enzymatic activity results, we found that worms obtained from pVAX1/SjTGR-vaccinated animals expressed lower enzymatic activity than the control group and the antibodies weakened the enzymatic activity of SjTGR in vitro, too. It implies that the high-level antibodies may contribute to the protective effects.

Characterization and expression of the Schistosoma japonicum thioredoxin peroxidase-2 gene.

We analyzed proteins that were differentially expressed by 10-day-old schistosomula from 3 different hosts and determined that a functional thioredoxin peroxidase-2 gene has an important antioxidant role in Schistosoma japonicum , which we investigated further. A full-length cDNA encoding the S. japonicum thioredoxin peroxidase-2 (SjTPx-2) had an open reading frame of 681 bp that encoded 226 amino acids with a signal peptide of 24 amino acids. A cDNA encoding SjTPx-2 without the signal peptide sequence was isolated from 42-day-old schistosome cDNAs. Real-time quantitative RT-PCR analysis revealed that SjTPx-2 was upregulated in 7- and 13-day-old schistosomes, while the expression level in females was around 2-fold higher than that in male worms at 42 days. SjTPx was subcloned into pET28a(+) and expressed as both inclusion bodies and supernatant in Escherichia coli BL21 (DE3) cells. Western blotting showed that the recombinant SjTPx-2 (rSjTPx-2) was immunogenic. The purified recombinant protein could form disulfide-bonded dimers and it had peroxidase activity in vitro. An immunoprotection experiment in BALB/c mice showed that vaccination with recombinant SjTPx-2 could induce 31.2% and 34.0% reductions in the numbers of worms and eggs in the liver, respectively. This study suggests that SjTPx-2 may be an important antioxidative enzyme in scavenging ROS, and it may be a potential vaccine candidate or new drug target for schistosomiasis.

[Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum].

OBJECTIVE: To clone and preliminarily analyze the full-length cDNA encoding retinoid X receptor 2 (RXR2) from Schistosoma japonicum. METHODS: The rapid amplification cDNA ends (RACE)was applied to get a full-length cDNA encoding retinoid X receptor 2 from S. japonicum (SjRXR2). The transcription of SjRXR2 was detected by real-time PCR. By bioinformatical technology, the gene structure was analyzed and the antibody epitope was predicted. The polyclonal antibodies were raised in mice immunized with the synthesis peptide. Western blot was applied to detect its expression in the worm. RESULTS: The full-length cDNA of SjRXR2 was 5 960 bp and contained an open reading frame encoding a 1 435 amino acid which had a predicted molecular weight 159 kDa. Bioinformatical analysis indicated that SjRXR2 had a highly conserved DNA binding domain (DBD) and a moderate conserved ligand binding domain (LBD). The relative mRNA (s) of SjRXR2 with higher expressions at Day 21 and 42 were evaluated in five different S. japonicum developmental stages. The Western blot analysis showed that polyclonal antibodies were able to specifically recognize the protein with molecular around 150 kDa from the extract of S. japonicum. CONCLUSION: A full-length cDNA encoding retinoid X receptor 2 (RXR2) from S. japonicum is obtained which provides preliminary information for further investigation of SjRXR2 functions in S. japonicum.

Molecular cloning and characterization of glutamine synthetase, a tegumental protein from Schistosoma japonicum.

Glutamine synthetase catalyzes the synthesis of glutamine, providing nitrogen for the production of purines, pyrimidines, amino acids, and other compounds required in many pivotal cellular events. Herein, a full-length cDNA encoding Schistosoma japonicum glutamine synthetase (SjGS) was isolated from 21-day schistosomes. The entire open reading frame of SjGS contains a 1,095-bp coding region corresponding to 364 amino acids with a calculated molecular weight of 40.7 kDa. NCBIP blast shows that the putative amino acid of SjGS contains a classic ß-grasp domain and a catalytic domain of glutamine synthetase. The relative mRNA expression of SjGS was evaluated in 7-, 13-, 21-, 28-, 35-, and 42-day worms of S. japonicum in the final host and higher expression at day 21, and 42 worms were observed. This protein was also detected in worm extracts using Western blot. Immunofluorescence studies indicated that the SjGS protein was mainly distributed on tegument and parenchyma in 28-day adult worms. The recombinant glutamine synthetase with a molecular weight of 45 kDa was expressed in Escherichia coli and purified in its active form. The enzyme activity of the recombinant protein was 3.30 ± 0.67 U.µg-1. The enzyme activity was highly stable over a wide range of pH (6-9) and temperature (25-40 °C) under physiological conditions. The transcription of SjGS was upregulated in praziquantel-treated worms at 2-, 4-, and 24-h posttreatment compared with the untreated control. As a first step towards the clarification of the role of glutamine synthetase in schistosome species, we have cloned and characterized cDNAs encoding SjGS in S. japonicum, and the data presented suggest that SjGS is an important molecule in the development of the schistosome.

Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice.

BACKGROUND: Thyroid hormones (TH) modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs). The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRß) and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. METHODS: The full-length cDNA sequence of SjTHRß, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. RESULTS: The SjTHRß cDNA was cloned, verified by 5' and 3' Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3'end, suggesting the transcript is full-length. SjTHRß is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRß was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRß-DBD) and ligand binding domain (SjTHRß-LBD) were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRß (rSjTHRß). Western blotting revealed that anti-rSjTHRß-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA) analysis showed that rSjTHRß-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRß-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs reduction)against schistosome infection. Enzyme-linked immunosorbent assay showed that mice vaccinated with recombinant SjTHRß-LBD (rSjTHRß-LBD) generated increased levels of specific IgG, IgG1 and IgG2a antibody. Bio-plex analysis demonstrated that rSjTHRß-LBD induced considerably higher levels of T helper 1 cytokines (IL-2, IL-12 and TNF-α) than T helper 2 cytokines (IL-10, IL-4), suggesting that rSjTHRß-LBD vaccination could stimulate mixed Th1/Th2 types with Th1 dominant immune responses. CONCLUSIONS: Our study presented here identified SjTHRß as a new schistosome THR that might play an important role in host-parasite interaction and be a vaccine candidate for schistosomiasis.

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells.

AIM: To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis. METHODS: Male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 d and were treated with Gardenia jasminoides by gavage. The effects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells (LX-2) in vitro. RESULTS: Treatment with Gardenia jasminoides decreased serum alanine aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 146.6 ± 15 U/L vs 77 ± 6.5 U/L, P = 0.0007) and aspartate aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 188 ± 35.2 U/L vs 128 ± 19 U/L, P = 0.005) as well as hydroxyproline (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 438 ± 40.2 µg/g vs 228 ± 10.3 µg/g liver tissue, P = 0.004) after BDL. Furthermore, Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor ß1 (TGF-ß1), collagen type I (Col I) and α-smooth muscle actin (α-SMA). Gardenia jasminoides significantly suppressed the upregulation of TGF-ß1, Col I and α-SMA in LX-2 exposed to recombinant TGF-ß1. Moreover, Gardenia jasminoides inhibited TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.