RBBP6 aggravates the progression of ovarian cancer by targeting PIK3R6.

OBJECTIVE: RBBP6 is identified to be a cancer-associated gene by bioinformatics analysis. This study aims to explore the role of RBBP6 in regulating proliferation and metastasis in ovarian cancer, thus providing theoretical references for ovarian cancer treatment. PATIENTS AND METHODS: Differential expressions of RBBP6 in ovarian cancer and normal ones were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The relationship between RBBP6 and prognosis in ovarian cancer patients was analyzed. The interaction between RBBP6 and PIK3R6 was detected by bioinformatics analysis and Dual-Luciferase reporter assay. Moreover, regulatory effects of RBBP6 and PIK3R6 on proliferative and migratory potentials in A2780 and CAOV3 cells were examined by Cell Counting Kit-8 (CCK-8) and transwell assay, respectively. Finally, tumorigenicity assay was conducted in nude mice to illustrate the in vivo regulations of PBBP6 and PIK3R6 on ovarian cancer growth. RESULTS: RBBP6 was upregulated in ovarian cancer tissues than normal ones. RBBP6 was irrelevant to age, tumor size and tumor node metastasis (TNM) staging in ovarian cancer patients, but correlated to lymphatic metastasis and distant metastasis. RBBP6 was abundantly expressed in ovarian cancer cells, and among the tested cell lines, CAOV3 and A2780 expressed the highest level of RBBP6. Knockdown of RBBP6 attenuated in vitro proliferative and migratory potentials in CAOV3 and A2780 cells. PIK3R6 was the target gene binding RBBP6, which was positively regulated by RBBP6. Overexpression of PIK3R6 could abolish the inhibited proliferative and migratory potentials in ovarian cancer cells with RBBP6 knockdown. In addition, the knockdown of RBBP6 slowed the in vivo growth of ovarian cancer in nude mice, and the alleviated cancer progression was reversed by overexpression of PIK3R6. CONCLUSIONS: RBBP6 is highly expressed in ovarian cancer cases, which stimulates proliferative and migratory potentials by targeting PIK3R6. RBBP6 may be a novel therapeutic target for ovarian cancer.

Identification and characterization of the duck enteritis virus (DEV) US2 gene.

The US2 protein has been reported to contribute to duck enteritis virus (DEV) infection; however, its kinetics and localization during infection, and whether it is a component of virion, have not been previously reported. To elucidate the function of DEV US2, US2 was amplified by polymerase chain reaction (PCR) and inserted into pET-32a(+); this was expressed, the recombinant US2 protein was purified, and a polyclonal antibody generated. In addition, the kinetics and localization of the US2 gene and protein were determined by quantitative real-time fluorescent PCR, ganciclovir (GCV), and cycloheximide (CHX) treatment, western-blot, and indirect immunofluorescence assay. The packaging of US2 into DEV virions was revealed by a protease protection assay. US2 was found to be transcribed 24 h post-infection (pi) and peaked at 72 h pi; the US2 protein was detected 48 h pi, except in the presence of GCV or CHX. US2 was packed into virions and also localized to the plasma membrane and cytoplasm in infected cells. The results showed that the DEV US2 is a late gene, and that its encoding protein could be a tegument component localized mainly in the cytoplasm. This study provides useful data for the further analysis of DEV US2, including an explanation for the genetic conservation among alphaherpesviruses.

Association between ß1 adrenergic receptor gene Arg389Gly polymorphism and risk of heart failure: a meta-analysis.

Numerous studies have evaluated the association between Arg389Gly polymorphism in the ß1 adrenergic receptor gene and heart failure risk. However, the specific association is still controversial. We performed a meta-analysis of all case-control studies that evaluated the association between Arg389Gly polymorphism and heart failure in humans. Studies were identified in the PubMed, Embase, and China National Knowledge Infrastructure databases. Two reviewers independently assessed the studies. Six case-control studies with a total of 1736 participants were included in the meta-analysis, including 882 cases with heart failure and 854 controls, and our results showed no association between the Arg389Gly polymorphism and heart failure [ArgArg vs GlyGly: odds ratio (OR) = 0.84, 95% confidence interval (CI) 0.59-1.20; ArgArg vs ArgGly: OR = 0.95, 95%CI 0.78-1.16; dominant model: OR = 1.08, 95%CI 0.89-1.31; recessive model: OR = 0.96, 95%CI 0.69-1.35]. No publication bias was found in the present study (all P values > 0.05). In conclusion, the ß1 adrenergic receptor gene Arg389Gly polymorphism might not be associated with heart failure risk. Further large and well-designed studies are needed to confirm this conclusion.

Detection, differentiation, and VP1 sequencing of duck hepatitis A virus type 1 and type 3 by a 1-step duplex reverse-transcription PCR assay.

Duck hepatitis A virus (DHAV) is an infectious pathogen causing fatal duck viral hepatitis in ducklings. Although both the inactivated vaccines and live attenuated vaccines have been used to protect ducklings, DHAV-1 and DHAV-3 still cause significant serious damage to the duck industry in China and South Korea. For rapid detection, differentiation, and epidemic investigation of DHAV in China, a genotype-specific 1-step duplex reverse-transcription (RT) PCR assay was established in this study. The sensitivity and specificity of the developed RT-PCR assay was evaluated with nucleic acids extracted from 2 DHAV reference strains, and 9 other infectious viruses and bacteria. The genotype-specific primers amplified different size DNA fragments encompassing the complete VP1 gene of the DHAV-1 or DHAV-3. The assay detected the liver samples collected from experimentally infected ducklings and dead ducklings collected from different regions of China. Sequence analysis of these DNA fragments indicated that VP1 sequences of DHAV-1 can be used to distinguish wild type and vaccine strains. The phylogenetic analysis of VP1 sequences indicated that the developed RT-PCR assay can be used for epidemic investigation of DHAV-1 and DHAV-3. The developed RT-PCR assay can be used as a specific molecular tool for simultaneous detection, differentiation, and sequencing the VP1 gene of DHAV-1 and DHAV-3, which can be used for understanding the epidemiology and evolution of DHAV.

A 90-day subchronic toxicity study of neem oil, a Azadirachta indica oil, in mice.

To determine the no-observed-adverse-effect level (NOAEL) of exposure and target organs of neem oil for establishing safety criteria for human exposure, the subchronic toxicity study with neem oil in mice was evaluated. The mice (10 per sex for each dose) was orally administered with neem oil with the doses of 0 (to serve as a control), 177, 533 and 1600 mg/kg/day for 90 days. After the treatment period, observation of reversibility or persistence of any toxic effects, mice were continuously fed without treatment for the following 30 days. During the two test periods, the serum biochemistry, organ weight and histopathology were examined. The results showed that the serum biochemistry and organ coefficient in experimental groups had no statistical difference compared with those of the control group. At the 90th day, the histopathological examinations showed that the 1600 mg/kg/day dose of neem oil had varying degrees of damage on each organ except heart, uterus and ovarian. After 30-day recovery, the degree of lesions to the tissues was lessened or even restored. The NOAEL of neem oil was 177 mg/kg/day for mice and the target organs of neem oil were determined to be testicle, liver and kidneys.

Adhesion and invasion to duck embryo fibroblast cells by Riemerella anatipestifer.

Here, we investigated adhesion and invasion of Riemerella anatipestifer (RA) to primary duck embryo fibroblast (DEF) cells. The ability of RA to adhere to, and more importantly, to invade DEF cells was demonstrated by using a gentamicin invasion assay and was confirmed by transmission electron microscopy (TEM). Adhesion of RA could be found by TEM after 1 h of inoculation. Both apoptosis and necrocytosis of DEF were indicated by TEM after 10 h of incubation, which suggested a complex mechanism of DEF cell death induced by RA. Our results showed that internalized RA had the ability to leave the DEF cells. Inhibition studies indicated that RA proteins play a role in adhesion. Moreover, invasion of RA to DEF cells was shown to require rearrangement of actin microfilaments and microtubular cytoskeletal elements. Because the adhesion and invasion ability of RA to DEF cells could be demonstrated in vitro, similar processes might occur in vivo, where DEF cells play a crucial role in the diffusion of RA in ducks.

Antiviral activity and mode of action of extracts from neem seed kernel against duck plague virus in vitro1.

Four fractions obtained from alcohol extracts of neem (Azadirachta indica) seed kernel by column chromatography were investigated for antivirus activity against the duck plague virus (DPV) in vitro. Duck embryo fibroblasts (DEF) infected with DPV were treated with the neem seed kernel extracts, and the effect of antivirus was judged by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide colorimetric method assay and direct immunofluorescence assay. The mode of action was tested by the plaque reduction assay. The results showed that fractions 1 to 3 were inactive. The median inhibitory concentration (IC(50)) of fraction 4 was 10.9 µg/mL and inhibited the virus protein expression in the direct immunofluorescence assay. In the plaque reduction assay, fraction 4 could significantly reduce the number of plaques compared with the negative control (P < 0.01) in all modes of action. This study indicated that the fourth fraction obtained from neem seed kernel could improve the viability of infected cells, and reduce the cytopathic effects caused by DPV and the amount of the virus protein expressed in virus-infected cells. The antiviral activity works in the whole process of virus infecting the normal cells.

Development and application of specific polymerase chain reaction assay targeting the gyrB gene for rapid detection of Riemerella anatipestifer.

A pair of PCR primers was designed and synthesized to amplify a gyrB gene sequence from Riemerella anatipestifer (RA). A fragment of 194 bp was detected in RA-positive isolates, whereas other isolates were negative, which confirmed the high specificity of the primers and PCR conditions. As little as 1.6 × 10(4) cfu/mL of cultural liquid was required by this method. We compared a 16S rRNA sequence-based PCR method and a Biolog bacterial identification system used in the detection and identification of suspicious isolates of RA in clinical tests. The results showed that the gyrB-based PCR was consistent with the results of the Biolog identification system and was more specific. By applying the gyrB-PCR to detect RA strains in 56 duck livers, a positive rate of 46% (26/56) was observed, whereas the positive rate of 85 throat swabs from clinically healthy ducks was 11%. Thus, this method could be used for the epidemiological investigation and preliminary isolate identification of RA.

Isolation and characterization of 16 novel microsatellite loci in two inbred strains of the Chinese hamster (Cricetulus griseus).

The Shanyi inbred A and E strains of the Chinese hamster are widely used in biomedical research, but detailed genetic characterization has been lacking. We developed microsatellite markers that could be used for genetic diversity analysis and linkage map construction. We isolated and characterized 16 novel microsatellite loci from a microsatellite-enriched genomic DNA library. These loci were genotyped in 48 animals from the two strains, and the polymorphic information content was determined. In the Shanyi A and E populations, 14 and 15 loci were found to be polymorphic, respectively, with polymorphic information content ranging from 0.1393 to 0.8082 and from 0.1109 to 0.7397, respectively. A total of 115 alleles were found for the 16 microsatellite loci in the two populations; the mean observed heterozygosity (H(O)) was 0.5191 and 0.4333 for the A and E populations, respectively, indicating marked genetic variation within the two populations. Correspondingly, the F(ST) values ranged from 0.002 to 0.9253, with an overall mean of 0.1935, indicating significant genetic difference between the two strains. The population differentiation levels were substantiated by Nei's genetic distance and full Bayesian analyses computed with STRUCTURE. Despite the genetic diversity and differentiation within and between the two inbred populations, the 48 individuals were correctly allocated into their original populations with high statistical confidence based on these 16 microsatellite loci. These novel microsatellite loci should be useful genetic markers for these two Chinese hamster inbred strains.

Efficacy study and field application of an inactivated new type gosling viral enteritis virus vaccine for domestic geese.

New type gosling viral enteritis virus (NGVEV) caused a serious disease in naive juvenile goslings. In the described studies the performance of 2 vaccines was analyzed: a vaccine containing adjuvanted inactivated NGVEV and a vaccine containing adjuvanted inactivated NGVEV and recombinant goose IL-2. Breeder geese were subcutaneously vaccinated at the beginning of the egg production period with the vaccines. Breeder geese sham vaccinated with PBS served as control. The cellular and humoral immune responses of the vaccinated breeder geese, as well as the presence of maternally derived antibody to NGVEV, were investigated by ELISA, virus neutralization test, and lymphocyte proliferation assay, respectively. A significantly higher immunogenicity (P < 0.05) was induced by the inactivated NGVEV-recombinant goose IL-2 adjuvant vaccine compared with the inactivated NGVEV vaccine. The offspring of the vaccinated birds were challenged with virulent NGVEV (100 50% lethal dose) and the protective efficacy of the vaccines was determined. Furthermore, in a field trial the efficacy of the inactivated NGVEV vaccine was recorded from years 2003 to 2007. No clinical signs or abnormal health status were observed in the vaccinated breeder geese and the progeny. After a single application, >80% protection was shown in the progeny of geese vaccinated against NGVEV challenge for approximately 5 mo. The extensive field trials further demonstrated that vaccination of breeder geese with the inactivated NGVEV vaccine could be a safe and efficacious means to control NGVE disease. Moreover, the level of maternally derived NGVEV antibody titer in the egg yolk reflected the level of NGVEV antibodies in the breeder geese, suggesting that the egg yolk could be used to monitor the vaccination efficacy in commercial goose breeder flocks.

Humoral and cellular immune responses in adult geese induced by an inactivated vaccine against new type gosling viral enteritis virus.

To assess the immunogenicity of an inactivated new type gosling viral enteritis virus (NGVEV) vaccine, we investigated 3 different doses of the inactivated vaccine and the inactivated vaccine in conjunction with 3 different doses of recombinant goose interleukin-2 (rGoIL-2) adjuvant. A virus concentration of 10(5) 50% embryo infective dose/mL was subcutaneously inoculated into adult geese divided into 6 groups. The dynamic changes of the humoral and cellular immunity responses elicited by the vaccines in the adult geese postvaccination (PV) were investigated using ELISA, virus neutralization test, and lymphocyte proliferation assay. The clearance of virus from the intestines of geese (175 d PV) was studied by histopathological examination and indirect immunofluorescence assay after virulent NGVEV challenge. This study showed that the inactivated NGVEV vaccine elicits strong humoral and cellular responses in the vaccinated adult geese. The absorbance values of specific anti-NGVEV antibodies, the neutralization antibody titer, and the lymphocyte proliferation index rapidly increased, peaked at about 28 d PV, progressed to the plateau stage, and then decreased slightly. The rGoIL-2 adjuvant enhanced the immune response, and this adjuvant in conjunction with the inactivated NGVEV vaccine induces a significantly higher specific anti-NGVEV antibody absorbance value, neutralization antibody titer, and lymphocyte proliferation index than the non-adjuvant-inactivated NGVEV vaccine (P < 0.05). The inactivated NGVEV vaccine conferred adequate efficient ability to clear NGVEV in vaccinated geese even in the last phase of the vaccination period (175 d PV). The inactivated NGVEV vaccine (0.5 mL/goose) with 1,000 units of rGoIL-2 adjuvant/goose is the most effective dose, thereby eliciting the strongest humoral and cellular immunity responses and providing the most efficacious clearance of NGVEV in vivo.

Development and application of an indirect immunohistochemical method for the detection of duck plague virus vaccine antigens in paraffin sections and localization in the vaccinated duckling tissues.

The objective of the present study was to develop and apply a streptavidin-alkaline phosphatase labeling system of indirect immunohistochemistry (SP-IHC) to detect antigenic distribution and localization regularity of duck plague virus (DPV) vaccine antigens in paraformaldehyde-fixed paraffin-embedded tissues of experimentally vaccinated ducklings. Male New Zealand rabbits were immunized with purified DPV antigens, which were engaged by a combination of differential centrifugation and sucrose-density gradient ultracentrifugation. The rabbit anti-DPV polyclonal antibodies were purified and used as the primary antibodies. Forty-eight 28-d-old DPV-free Pekin ducklings were subcutaneously inoculated with attenuated DPV vaccine in the immunization group and sterile PBS in the control group. The tissues were collected at sequential time points between 4 h and 18 wk postvaccination (PV) and were prepared for SP-IHC observation. The presence of DPV-specific antigens was first observed in the liver and spleen at 12 h PV; in the bursa of Fabricius, thymus, Harderian gland, esophagus, and intestinal tract at 1 d PV; and in the heart, lung, kidney, pancreas, and brain at 3 d PV. The positive staining reaction could be detected in the vaccinated duckling tissues until 18 wk PV, and no positive staining cells could be observed in the controls. The highest levels of positive staining reaction were found in the liver, spleen, bursa of Fabricius, thymus, and intestinal tract, whereas a few DPV vaccine antigens were distributed in the heart, pancreas, and esophagus. The target cells had a ubiquitous distribution, especially in the mucosal epithelial cells, lamina propria cells, macrophages, hepatocytes, and lymphocytes, which served as the principal sites for antigen localization. These findings demonstrated that SP-IHC was a reliable method for detecting antigenic distribution and localization regularity of DPV vaccine antigens in routine paraffin sections. The present study may be useful for describing proliferation and distribution regularity of DPV vaccine in the vaccinated duckling tissues and enhance further studies and clinical application of attenuated DPV vaccine.

Simple and rapid detection of Salmonella serovar Enteritidis under field conditions by loop-mediated isothermal amplification.

AIM: The objective of this study is to develop a serovar-specific loop-mediated isothermal amplification (LAMP) method for sensitive, rapid, and inexpensive detection of Salmonella serovar Enteritidis under field conditions. METHODS: A set of six specific primers was designed with Salmonella Enteritidis DNA as the target. LAMP conditions were optimized by incubating the target DNA with the Bst DNA polymerase large fragment in a simple water bath. The sensitivity and specificity of LAMP was then compared with those of fluorescent quantitative real-time polymerase chain reaction (FQ-PCR). RESULTS: The results were as follows. (1) Serovar-specific Salmonella Enteritidis DNA was amplified at 65°C in as early as 20min in a water bath. (2) A colour change visible to the naked eye indicated a positive amplification reaction. (3) The detection limit of the LAMP assay was 4 copies µl(-1) ; thus, the sensitivity and specificity of this assay is similar to those of the FQ-PCR. CONCLUSIONS: LAMP is a high-throughput detection technique with high sensitivity, specificity, and simplicity; these factors make it suitable for specifically detecting Salmonella Enteritidis under field conditions and in laboratory settings. Thus, LAMP eliminates the need for complicated equipment and technical training in the detection of this specific serovar. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study involving the use of LAMP to detect Salmonella serovar-specific DNA sequences. It is also the first to report an ideal method of distinguishing between Salmonella Enteritidis and other Salmonella under field conditions.

Histopathology, immunohistochemistry, in situ apoptosis, and ultrastructure characterization of the digestive and lymphoid organs of new type gosling viral enteritis virus experimentally infected gosling.

Here, for the first time, to colocalize new type gosling viral enteritis virus (NGVEV) with histological lesions and in situ apoptosis in the digestive organs (esophagus, proventriculus, gizzard, small intestine, cecum, rectum, liver, and pancreas) and the lymphoid organs (bursa of Fabricius, thymus, Harderian gland, and spleen) of experimentally infected goslings, portions of tissues were collected at sequential infection time points and examined by histopathology for histological lesions, immunohistochemical staining for viral antigens, ultrastructural observation by transmission electron microscope (TEM) for virus particles and apoptotic cells, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay for in situ apoptosis. The hyperemia, hemorrhage, infiltration of lymphocytes, progressive lymphoid depletion, apoptosis, and necrosis were readily observed in the lymphoid organs and intestine tract by histopathological examination. The NGVEV particles and viral antigens widely appeared in the small intestine and bursa of Fabricius as early as 2 d postinfection (PI) by TEM and immunohistochemical staining, and the presence and quantity of it reached a maximum during 6 to 12 d PI. The principal sites for NGVEV were endothelial cells, epithelia, mucosal cells, glandular cells, fibrocytes, macrophages, and lymphocytes. A series of apoptotic morphological changes including chromatin condensation and margination, cytoplasmic shrinkage, and formation of apoptotic body were observed by TEM, and the number of apoptotic cells was largely increased from 4 d PI and peaked at 9 d PI by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling analysis. The histological organ lesions and apoptosis in vivo were generally associated with sites of NGVEV localization, which can be regarded as the cause of death. This work may shed light on the pathogenesis of new type gosling viral enteritis and put new insight into the pathogenesis of goose adenovirus.