Mosquito densovirus significantly reduces the vector susceptibility to dengue virus serotype 2 in Aedes albopictus mosquitoes (Diptera: Culicidae).

BACKGROUND: Dengue virus (DENV) is a major public health threat, with Aedes albopictus being the confirmed vector responsible for dengue epidemics in Guangzhou, China. Mosquito densoviruses (MDVs) are pathogenic mosquito-specific viruses, and a novel MDV was previously isolated from Ae. albopictus in Guangzhou. This study aims to determine the prevalence of MDVs in wild Ae. albopictus populations and investigate their potential interactions with DENV and impact on vector susceptibility for DENV. METHODS: The prevalence of MDV in wild mosquitoes in China was investigated using open access sequencing data and PCR detection in Ae. albopictus in Guangzhou. The viral infection rate and titers in MDV-persistent C6/36 cells were evaluated at 12, 24, 48, 72, 96, and 120 h post infection (hpi) by indirect immunofluorescence assay (IFA) and real time quantitative PCR (RT-qPCR). The midgut infection rate (MIR), dissemination rate (DR), and salivary gland infection rate (SGIR) in various tissues of MDV-infected mosquitoes were detected and quantified at 0, 5, 10, and 15 days post infection (dpi) by RT-PCR and RT-qPCR. The chi-square test evaluated dengue virus serotype 2 (DENV-2) and Aedes aegypti densovirus (AaeDV) infection rates and related indices in mosquitoes, while Tukey's LSD and t-tests compared viral titers in C6/36 cells and tissues over time. RESULTS: The results revealed a relatively wide distribution of MDVs in Aedes, Culex, and Anopheles mosquitoes in China and an over 68% positive rate. In vitro, significant reductions in DENV-2 titers in supernatant at 120 hpi, and an apparent decrease in DENV-2-positive cells at 96 and 120 hpi were observed. In vivo, DENV-2 in the ovaries and salivary glands was first detected at 10 dpi in both monoinfected and superinfected Ae. albopictus females, while MDV superinfection with DENV-2 suppressed the salivary gland infection rate at 15 dpi. DENV-2 titer in the ovary and salivary glands of Ae. albopictus was reduced in superinfected mosquitoes at 15 dpi. CONCLUSIONS: MDVs is widespread in natural mosquito populations, and replication of DENV-2 is suppressed in MDV-infected Ae. albopictus, thus reducing vector susceptibility to DENV-2. Our study supports the hypothesis that MDVs may contribute to reducing transmission of DENV and provides an alternative strategy for mosquito-transmitted disease control.

Aal-circRNA-407 regulates ovarian development of Aedes albopictus, a major arbovirus vector, via the miR-9a-5p/Foxl axis.

Aedes albopictus shows a rapid global expansion and dramatic vectorial capacity for various arboviruses, thus posing a severe threat to global health. Although many noncoding RNAs have been confirmed to play functional roles in various biological processes in Ae. albopictus, the roles of circRNA remain a mystery. In the present study, we first performed high-throughput circRNA sequencing in Ae. albopictus. Then, we identified a cysteine desulfurase (CsdA) superfamily gene-originated circRNA, named aal-circRNA-407, which was the third most abundant circRNA in adult females and displayed a fat body highly expressed manifestation and blood feeding-dependent onset. SiRNA-mediated knockdown of circRNA-407 resulted in a decrease in the number of developing follicles and a reduction in follicle size post blood meal. Furthermore, we demonstrated that circRNA-407 can act as a sponge of aal-miR-9a-5p to promote the expression of its target gene Foxl and eventually regulate ovarian development. Our study is the first to report a functional circRNA in mosquitoes, expanding our current understanding of important biological roles in mosquitoes and providing an alternative genetic strategy for mosquito control.

Blockage of MDM2-mediated p53 ubiquitination by yuanhuacine restrains the carcinogenesis of prostate carcinoma cells by suppressing LncRNA LINC00665.

Prostate cancer (PCa) is a challenging issue for men's health worldwide due to its uncontrolled proliferation and high metastatic potential. Increasing evidence has supported plant extracts and natural plant derivatives as promising antitumor therapy with less toxic side effects. Yuanhuacine is an active component isolated from Daphne genkwa and can effectively suppress the tumorigenesis of several cancers. However, its role in PCa remains unclear. In this study, yuanhuacine dose-dependently inhibited the proliferation and induced apoptosis of PCa cells. Moreover, yuanhuacine also restrained the invasion and migration of PCa cells. Mechanically, yuanhuacine decreased the ubiquitination and degradation of p53 protein, and ultimately increased p53 levels, which was regulated by inhibiting the phosphorylation and total protein levels of mouse double minute 2 (MDM2). Moreover, elevation of MDM2 reversed the suppressive efficacy of yuanhuacine in PCa cell viability, invasion, and migration. The network pharmacologic and bioinformatics analysis confirmed that MDM2 might be a common target of D. genkwa and LINC00665. Furthermore, yuanhuacine inhibited LINC00665 expression. Upregulation of LINC00665 reversed yuanhuacine-mediated inhibition in MDM2 protein expression and suppressed p53 levels by enhancing its ubiquitination in yuanhuacine-treated cells. Importantly, the inhibitory effects of yuanhuacine on cell viability and metastatic potential were offset after LINC00665 elevation. Together, the current findings highlight that yuanhuacine may possess tumor-suppressive efficacy by inhibiting LINC00665-mediated MDM2/p53 ubiquitination signaling. Therefore, this study indicates that yuanhuacine may be a promising candidate for the treatment of PCa.

Densovirus Oil Suspension Significantly Improves the Efficacy and Duration of Larvicidal Activity against Aedes albopictus.

Aedes albopictus is the sole vector for various mosquito-borne viruses, including dengue, chikungunya, and Zika. Ecofriendly biological agents are required to reduce the spread of these mosquito-borne infections. Mosquito densoviruses (MDVs) are entomopathogenic mosquito-specific viruses, which can reduce the capacity of isolated vectors and decrease mosquito-borne viral disease transmission. However, their variable pathogenicity restricts their commercial use. In the present study, we developed a series of novel larvicide oil suspensions (denoted Bacillus thuringiensis (Bti) oil, Ae. albopictus densovirus (AalDV-5) oil, and a mixture of AalDV-5+Bti oil), which were tested against Ae. albopictus larvae under experimental semi-field and open-field conditions. The effect of AalDV-5 on non-target species was also evaluated. The combined effect of AalDV-5+Bti was greater than that of individual toxins and was longer lasting and more persistent compared with the laboratory AalDV-5 virus strain. The virus was quantified on a weekly basis by quantitative polymerase chain reaction (qPCR) and was persistently detected in rearing water as well as in dead larvae. Wildtype densovirus is not pathogenic to non-target organisms. The present findings confirm the improved effect of a mixed microbial suspension (AalDV-5+Bti oil) larvicide against Ae. albopictus. The development and testing of these products will enable better control of the vector mosquitoes.

The influence of LncRNA H19 polymorphic variants on susceptibility to cancer: A systematic review and updated meta-analysis of 28 case-control studies.

OBJECTIVE: Although myriad researches upon the associations between LncRNA H19 polymorphic variants (rs2839698 G>A, rs217727 G>A, rs2107425 C>T, rs2735971 A>G and rs3024270 C>G) and the susceptibility to cancer have been conducted, these results remained contradictory and perplexing. Basing on that, a systematic review and updated meta-analysis was performed to anticipate a fairly precise assessment about such associations. METHODS: We retrieved the electronic databases EMBASE, PubMed and Web of Science for valuable academic studies before February 28, 2021. Ultimately, 28 of which were encompassed after screening in this meta-analysis, and the available data was extracted and integrated. The pooled odds ratios (ORs) with 95% confidence intervals (CIs) was used to evaluate such associations. For multi-level investigation, subgroup analysis derived from source of controls together with genotypic method was preformed. RESULTS: Eventually, 28 articles altogether embodying 57 studies were included in this meta-analysis. The results illuminated that LncRNA H19 polymorphisms mentioned above were all irrelevant to cancer susceptibility. Nevertheless, crucial results were found concentrated in population-based control group when subgroup analysis by source of controls were performed in H19 mutation rs2839698 and rs2735971. Meanwhile, in the stratification analysis by genotypic method, apparent cancer risks were discovered by TaqMan method in H19 mutation rs2107425 and rs3024270. Then, trial sequential analysis demonstrated that the results about such associations were firm evidence of effect. CONCLUSION: Therefore, this meta-analysis indicated that LncRNA H19 polymorphisms were not associated with the susceptibility to human cancer. However, after the stratification analysis, inconsistent results still existed in different genotypic method and source of control. Thus, more high-quality studies on cancer patients of different factors were needed to confirm these findings.

Up-Regulation of LINC00665 Facilitates the Malignant Progression of Prostate Cancer by Epigenetically Silencing KLF2 Through EZH2 and LSD1.

This study aimed to explore the function of LINC00665 on the proliferation and metastasis of prostate cancer (PCa), and the potential regulatory mechanisms were also investigated. The expression level of LINC00665 in 50 pairs of PCa tissues and adjacent ones was studied by qRT-PCR, and the associations between LINC00665 and clinicopathological characteristics of PCa patients were analyzed. Control group (sh-NC) and LINC00665 knock-down group (sh-LINC00665) were set in 22RV1 and DU145 cells, respectively. The biological functions of LINC00665 in PCa cell lines were assessed by CCK-8, EdU, Transwell assays, and the nude mouse xenograft model was used to evaluate the tumorigenicity in vivo. In addition, qRT-PCR, Western Blot, RIP and ChIP assays were also used to determine the regulation mechanism of LINC00665 in PCa cell lines. In this study, our results showed that LINC00665 expression level in PCa cancer tissues was significantly up-regulated, compared with that in adjacent ones. Besides, similar results were found in PCa cell lines. Knock-down of LINC00665 significantly attenuated the proliferation and migration ability in 22RV1 and DU145 cells, compared to sh-NC. Mechanically, LINC00665 could interact with EZH2 and LSD1, recruiting them to KLF2 promoter region to inhibit its transcription. Moreover, the tumor-suppressive effects mediated by sh-LINC00665 were significantly reversed through the down-regulation of KLF2. Also, the suppression of LINC00665 impaired tumor growth of PCa in vivo. In summary, LINC00665 exerted the oncogenic functions in PCa cell lines by epigenetically silencing KLF2 expression by binding to EZH2 and LSD1, illuminating a novel mechanism of LINC00665 in the malignant progression of PCa and furnishing a prospective therapeutic biomarker to combat PCa.

Experimental reconstruction of double-stranded break repair-mediated plastid DNA insertion into the tobacco nucleus.

The mitochondria and plastids of eukaryotic cells evolved from endosymbiotic prokaryotes. DNA from the endosymbionts has bombarded nuclei since the ancestral prokaryotes were engulfed by a precursor of the nucleated eukaryotic host. An experimental confirmation regarding the molecular mechanisms responsible for organelle DNA incorporation into nuclei has not been performed until the present analysis. Here we introduced double-stranded DNA breaks into the nuclear genome of tobacco through inducible expression of I-SceI, and showed experimentally that tobacco chloroplast DNAs insert into nuclear genomes through double-stranded DNA break repair. Microhomology-mediated linking of disparate segments of chloroplast DNA occurs frequently during healing of induced nuclear double-stranded breaks (DSB) but the resulting nuclear integrants are often immediately unstable. Non-Mendelian inheritance of a selectable marker (neo), used to identify plastid DNA transfer, was observed in the progeny of about 50% of lines emerging from the screen. The instability of these de novo nuclear insertions of plastid DNA (nupts) was shown to be associated with deletion not only of the nupt itself but also of flanking nuclear DNA within one generation of transfer. This deletion of pre-existing nuclear DNA suggests that the genetic impact of organellar DNA transfer to the nucleus is potentially far greater than previously thought.

Serum metabolomic analysis of human upper urinary tract urothelial carcinoma.

To find potential serum biomarkers for upper tract urothelial carcinomas (UTUCs) via (1)H nuclear magnetic resonance ((1)H NMR)-based metabolomic analysis. Serum specimens collected from 34 healthy individuals and 39 patients with UTUCs were subjected to (1)H NMR-based metabolomic analysis. Principal component and orthogonal partial least squares discriminant analyses were used to analyse the data. Compared with serum samples from healthy subjects, samples from UTUC patients showed elevated levels of lactate and creatinine as well as decreased levels of glucose, glutamine and taurine. Serum low-density lipoprotein/very low-density lipoprotein, valine and glycoprotein levels showed decreasing trends whereas serum polyunsaturated fatty acids and 3,7-dimethyluric acid level presented increasing trends in UTUC patients. (1)H NMR-based metabolomic analysis of serum enhances the current understanding of the mechanisms involved in UTUC development. The present analysis may be a valuable tool for UTUC detection.

[Identification of MicroRNA-like Viral Small RNAs from AaeDV].

Several virus families have been shown to encode microRNAs (miRNAs), which have roles in the infection and replication of viruses in host cells. These virus-encoded miRNAs are identified in double-stranded DNA virus (dsDNA virus) and in several RNA virus families, but not in single-stranded DNA virus (ssDNA virus). We used a bioinformatics approach based on VMir, miRNAFold and MaturePred software to predict virus-encoded miRNA-like small RNAs from the genome of a ssDNA virus: Aedes aegypti densovirus (AaeDV). Northern blotting and stem-loop reverse transcription-polymerase chain reaction (RT-PCR) were used to detect predicted small RNAs. A miRNA-like small RNA termed "AaeDVMD" was identified by stem-loop RT-PCR from predicted candidates. This is the first report demonstrating that a ssDNA virus can encode miRNA-like small RNAs. These data will aid further exploration of the interaction between the AaeDV and its mosquito host.

Effects of TSP-1-696 C/T polymorphism on bladder cancer susceptibility and clinicopathologic features.

Thrombospondin-1 (TSP-1) is a glycoprotein that plays a major role in bladder cancer. We investigated the relationship between the distribution of the TSP-1 -696 C/T polymorphism (rs2664139) and the clinical features of bladder cancer. TaqMan assay was used to determine the genotype among the 609 cases and 670 controls in a Chinese population. Logistic regression was used to assess the association between the polymorphism and bladder cancer risk. Compared with the CT/TT genotypes, the CC genotype was associated with a significantly increased risk of bladder cancer (adjusted odds ratio [OR] 1.43, 95% CI 1.01-2.04), which was more prominent among the male participants (OR 1.82, 95% CI 1.20-2.76). The polymorphism was associated with a higher risk of developing grade 3 (OR 1.84, 95% CI 1.00-3.36), multiple-tumor (OR 1.81, 95% CI 1.08-3.02), and large-tumor (OR 1.94, 95% CI 1.22-3.10) bladder cancers. These observations suggest that the TSP-1 -696 C/T polymorphism may contribute to bladder cancer susceptibility in the Chinese population.

Association between NFKB1 -94ins/del ATTG Promoter Polymorphism and Cancer Susceptibility: An Updated Meta-Analysis.

Nuclear factor- κ B is associated with the pathogenesis of numerous malignancies, and the functional polymorphism -94ins/del ATTG (rs28362491) in the human NFKB1 gene is associated with cancer risk. Previous studies on the association between the -94ins/del ATTG polymorphism and cancer risk reported conflicting results. To clarify this relationship, we performed a meta-analysis of 21 case-control studies involving 6127 cases and 9238 controls. We used pooled odds ratios (ORs) with their 95% confidence intervals (95% CIs) to assess the association. We found that the NFKB1 promoter -94ins/del ATTG polymorphism was significantly associated with cancer risk in four genetic models (ins/ins versus del/del, OR = 1.47, 95% CI = 1.11-1.93; dominant model, OR = 1.26, 95% CI = 1.03-1.53; recessive model, OR = 1.26, 95% CI = 1.05-1.51; ins allele versus del allele, OR = 1.19, 95% CI = 1.05-1.35). Stratified analyses revealed a significant association between the polymorphism and ovarian, oral, and prostate cancers. Similar results were determined in an Asian population and not in a Caucasian population. Thus, our results suggested that the polymorphism can contribute to cancer risk. Moreover, the polymorphism can exert race- and cancer-specific effects on cancer risk. Further large-scale and functional studies are necessary to elucidate this possible effect.

TSP-1-1223 A/G Polymorphism as a Potential Predictor of the Recurrence Risk of Bladder Cancer in a Chinese Population.

Backgrounds. TSP-1 is a glycoprotein that functions in the biology of bladder cancer. We investigated the relationship between the distribution of TSP-1-1223 A/G polymorphism (rs2169830) and the clinical characteristics of bladder cancer. Materials and Methods. TaqMan assay was performed to determine the genotype of 609 cases and 670 control subjects in a Chinese population. Logistic regression was used to assess the association between the polymorphism and the risk of bladder cancer. Quantitative real-time polymerase chain reaction was performed to determine TSP-1 mRNA expression. Survival curves were generated using the Kaplan-Meier method. Results. No significant differences were detected in the genotype frequencies of healthy control subjects and patients with bladder cancer. By contrast, the time until the first recurrence differed significantly between genotypes (P = 0.017). The expression of TSP-1 mRNA in bladder cancer tissues was lower in patients with an AG genotype than in those with an AA genotype. The lowest expression was observed in patients with a GG genotype. Conclusions. In conclusion, TSP-1-1223 A/G polymorphism may contribute to the recurrence of bladder cancer in Chinese population.

Association of the glutathione s-transferase m1, t1 polymorphisms with cancer: evidence from a meta-analysis.

BACKGROUND: Glutathione S-transferases (GSTs) are a family of multifunctional enzymes that are involved in the metabolism of many xenobiotics, including a wide range of environmental carcinogens. While the null genotypes in GSTM1 and GSTT1 have been implicated in tumorigenesis, it remains inconsistent and inconclusive. Herein, we aimed to assess the possible associations of the GSTM1 and GSTT1 null genotype in cancer risks. METHODS: A meta-analysis based on 506 case-control studies was performed. Odds ratios (OR) with corresponding 95% confidence intervals (CIs) were used to assess the association. RESULTS: The null genotypes of GSTM1 and GSTT1 polymorphisms were associated with a significantly increased risk in cancer (for GSTM1: OR = 1.17; 95%CI = 1.14-1.21; for GSTT1: OR = 1.16; 95%CI = 1.11-1.21, respectively). When the analysis was performed based on their smoking history, the risk associated of GSTM1 null and GSTT1 null genotypes with cancer is further increased (for GSTM1: OR = 2.66; 95%CI = 2.19-3.24; for GSTT1: OR = 2.46; 95%CI = 1.83-3.32, respectively). CONCLUSIONS: These findings indicate that GSTM1 and GSTT1 polymorphisms may play critical roles in the development of cancer, especially in smokers.

Functional promoter -94 ins/del ATTG polymorphism in NFKB1 gene is associated with bladder cancer risk in a Chinese population.

BACKGROUND: A functional -94 insertion/deletion polymorphism (rs28362491) in the promoter of the NFKB1 gene was reported to influence NFKB1 expression and confer susceptibility to different types of cancer. This study aims to determine whether the polymorphism is associated with risk of bladder cancer. MATERIALS AND METHODS: TaqMan assay was used to determine genotype among 609 cases and 640 controls in a Chinese population. Logistic regression was used to assess the association between the polymorphism and bladder cancer risk, and quantitative real-time polymerase chain reaction was used to determine NFKB1 mRNA expression. RESULTS: Compared with the ins/ins/ins/del genotypes, the del/del genotype was associated with a significantly increased risk of bladder cancer [adjusted odd ratio (OR) = 1.92, 95% confidence interval (CI) = 1.42-2.59]. The increased risk was more prominent among subjects over 65 years old (OR = 2.37, 95% CI= 1.52-3.70), male subjects (OR = 1.97, 95% CI = 1.40-2.79) and subjects with self-reported family history of cancer (OR = 3.59, 95% CI = 1.19-10.9). Furthermore, the polymorphism was associated with a higher risk of developing non-muscle invasive bladder cancer (OR= 2.07, 95% CI= 1.51-2.85), grade 1 bladder cancer (OR = 2.40, 95% CI = 1.68-3.43), single tumor bladder cancer (OR = 2.04, 95% CI = 1.48-2.82) and smaller tumor size bladder cancer (OR = 2.10, 95% CI= 1.51-2.92). The expression of NFKB1 mRNA in bladder cancer tissues with homozygous insertion genotype was higher than that with deletion allele. CONCLUSIONS: In conclusion, the -94 ins/del ATTG polymorphism in NFKB1 promoter may contribute to the etiology of bladder cancer in the Chinese population.

[Antibacterial activity of recombinant thanatin expressed by E.coli ER2566].

OBJECTIVE: To express antibacterial peptide thanatin in the prokaryotic expression system and test its antibacterial activity. METHODS: The DNA sequence coding for the 21 peptides of thanatin was synthesized using the preferred genetic codes of E. coli, cloned into pTYB11 plasmid, and transformed into E.coli ER2566. The expression of thanatin fused with intein was induced by IPTG in E.coli, and intein-thanatin specifically bound to the column through intein tag was cleaved overnight at 4 degrees celsius; in DTT/cysteine buffer. RESULTS: The cleaved thanatin was eluted with a protein concentration of 245 microg/ml in the first 4 ml. The purified thanatin had showed strong antibacterial activities against G- bacteria such as Shigella flexneri, Klebsiella pneumoniae, Shigella snnei, Escherichia coli O157, toxin producing Escherichia coli, Pseudomonas aeruginosa, and fungi such as Candida albicans, with especial potency in killing drug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and extended-spectrum beta-lactamases (ESBL)-producing E.coli. Eighty strains of drug-resistant (ESBL-producing) and 30 strains of sensitive E. coli were used for anti-bacterial assay, and no significant differences in the antibacterial activity of thanatin were found between the sensitive and drug-resistant E. coli (P>0.05). CONCLUSION: The recombinant thanatin obtained shows strong antibacterial activity against drug-resistant and sensitive bacteria, and can be a potential substitute for routine antibiotics in the treatment of G- bacterial infections.

[Effect of cinobufagin on nuclear factor-kappaB pathway in HepG2 cells].

OBJECTIVE: To investigate the effect of cinobufagin on nuclear factor-kappaB (NF-kappaB) pathway of liver cancer cell line HepG2. METHODS: Dual-luciferase cis-reporting system was used to detect the relative value of pNF-kappaB-TA-luc upon tumor necrosis factor-alpha (TNF-alpha) stimulation of NF-kappaB pathway. Western blotting was used to detect the protein level of NF-kappaB p65, and RT-PCR was used to detect the gene transcription level of intercellular adhesion molecule-1 (ICAM-1), a target downstream gene of NF-kappaB. RESULTS: At the concentration of 0.25 and 0.5 microg/ml, cinobufagin significantly lowered the relative value of luciferase (P<0.05). The results of Western blotting showed that cinobufagin significantly suppressed the protein expression of NF-kappaB p65. The transcription level of ICAM-1 was reduced by different doses of cinobufagin. CONCLUSION: The anti-cancer effect of cinobufagin may be related to its activity in inhibiting the activation of NF-kappaB pathway.

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

OBJECTIVE: To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). METHODS: Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. RESULTS: MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. CONCLUSION: The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

[Construction of the life cycle of Angiostrongylus cantonensis in laboratory].

OBJECTIVE: To construct the life cycle of Angiostrongylus cantonensis (A.cantonensis) in laboratory condition. METHODS: SD rats were infected orally with the third-stage larvae of A.cantonensis collected from Jiangmen, Guangdong province. Six weeks after infection, the first-stage larvae were isolated from fresh feces of the rats by using Baermann funnel to infect 25 second-generation white jade snails raised in laboratory at the daily dose of 300 000 for 3 consecutive days. Three weeks later, the snails were dissected for counting the third-staged larvae of A.cantonensis, and those positive for A.cantonensis infection were fed directly to 10 fasting rats. The serum samples of the rats were then collected 2 weeks later for examination of specific antibodies using ELISA. The feces of the infected rats were examined microscopically after 6 weeks, and the brain, heart and lungs of the infected rats were dissected to observe the larvae at 3, 5, and 8 weeks, respectively. RESULTS: The 3-stage larvae of A.cantonensis were found in the second-generation snails 3 weeks after infection. The positivity rate of serum specific antibodies was 100% in the 10 rats 2 weeks after feeding of the infected snails. The 1-stage larvae were detected in the feces of the rats 6 weeks after infection, and the fourth-stage larvae were found in the brain of the rats at 3 weeks, while adult worm and eggs were found in the heart and lungs of the infected rats at 5 and 8 weeks. CONCLUSION: The successful establishment of human colon carcinoma cell line with PRL-3 gene knock-down provide a basis for investigation of the role of PRL-3 gene in the metastasis of human colorectal carcinoma.

[Construction of a yeast model for screening Aedes albopictus ecdysone agonist pesticides].

OBJECTIVE: To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. METHODS: The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene--green fluorescence protein (GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site (pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcription level of GFP in the tebufenozide affected yeast and the control. RESULTS: In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tebufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tebufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. CONCLUSION: The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.